Cerebral dicarboxylate transport and metabolism studied with isotopically labelled fumarate,malate and malonate

Transport and metabolism of dicarboxylates may be important in the glial-neuronal metabolic interplay. Further, exogenous dicarboxylates have been suggested as cerebral energy substrates. After intrastriatal injection of [(14) C]fumarate or [(14) C]malate, glutamine attained a specific activity 4.1 and 2.6 times higher than that of glutamate, respectively, indicating predominantly glial uptake of these four-carbon dicarboxylates. In contrast, the three-carbon dicarboxylate [(14) C]malonate gave a specific activity in glutamate which was approximately five times higher than that of glutamine, indicating neuronal uptake of malonate. Therefore, neurones and glia take up different types of dicarboxylates, probably by different transport mechanisms. Labelling of alanine from [(14) C]fumarate and [(14) C]malate demonstrated extensive malate decarboxylation, presumably in glia. Intravenous injection of 75 micromol [U-(13) C]fumarate rapidly led to high concentrations of [U-(13) C]fumarate and [U-(13) C]malate in serum, but neither substrate labelled cerebral metabolites as determined by (13) C NMR spectroscopy. Only after conversion of [U-(13) C]fumarate into serum glucose was there (13) C-labelling of cerebral metabolites, and only at <10% of that obtained with 75 micromol [3-(13) C]lactate or [2-(13) C]acetate. These findings suggest a very low transport capacity for four-carbon dicarboxylates across the blood-brain barrier and rule out a role for exogenous fumarate as a cerebral energy substrate.     

Effect of proline on nitrogenase activity in symbiosomes from root nodules of soybean (Glycine max L.) subjected to drought stress

Experiments were carried out to investigate if drought stressaffects the ability of bacteroids from soybean (Glycine maxL.) root nodules to utilize proline and malate to support nitrogenaseactivity. The bacteroids were isolated in sub-ambient oxygenand nitrogenase activity was measured by acetylene reduction.Nitrogenase activity supported by proline was 8-fold higherin bacteroids from drought-stressed nodules than in bacteroidsfrom control nodules. In contrast to the results with prolinethere was no significant response to drought stress in the rateof bacteroid nitrogenase activity supported by malate. The effectof drought stress on transport of proline and malate acrossthe symbiosome membrane was investigated by incubation of symbiosomesisolated in sub-ambient oxygen with radioactive tracers. Droughtstress tended to increase the rate of proline uptake relativeto a minor decrease in malate uptake into symbiosomes in responseto drought. There was no indication of a saturable camer inthe symbiosome membrane for either substrate at concentrationsin the range 0.1-2 mM. The rate of malate uptake into symbiosomeswas twice as high as the rate of proline uptake at all substratelevels tested. The protein composition of the symbiosome membranewas altered in response to drought stress and these changesmay relate .to the permeability of the symbiosome membrane. Key words: Drought stress, nitrogenase activity, proline, soybean nodules, symbiosome membrane, transport     

Nitrogen fixation and carbon metabolism by nodules and bacteroids of pea plants under sodium chloride stress

Acetylene reduction activity (ARA) and leghemoglobin (Lb) content in nodules were sigificantly reduced when pea ( Pisum sativum L. cv. Lincoln) plants were subjected to 50 m M sodium chloride stress for 3 weeks. C₂H₂ reduction activity by bacteriods isolated from pea nodules was drastically inhibited by saline stress, and malate appeared to be a more appropriate substrate than glucose or succinate in maintaining this activity. Salt added directly to the incubation mixture of bacteriods or to the culture medium of plants inhibited O₂ uptake by bacteroids. Nodule cytosolic phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and bacteriod malate dehydrogenase (MDH; EC 1.1.1.37) activities were strongly enhanced by salt stress. Under these conditions, malate concentration was depressed in bacteroids and cytosol, whereas total soluble sugar (TSS)content slightly increased in both fractions. The effect of salt stress on TSS and malate content suggests that the utilization of carbohydrate within nodules could be inhibited during salt stress. The inhibitory effect of NaCl on N₂ fixation activity of bacteroids and to the decrease in bacteroid respiration. The stimulation of fermentative metabolism induced by salinity suggests some reduction in O₂ availability within the nodule. Salt stress was also responsible for a decrease of the cytosolic protein content, specifically of leghemoglobin, in the nodules.     

Changes in the activity and isozyme patterns of malate dehydrogenase in root nodules of yellow lupine

The malate dehydrogenase present in the cytoplasmic fraction of plant origin and bacteroids from yellow lupine root nodules was investigated. The plant enzyme was 14 times more active in nodules than in roots and it contained 6 molecular forms in nodules compared with 3 forms detected in roots. The highest malate dehydrogenase activity in plant fraction and bacteroids was noted in 50-day old plants. Changes in the isoenzymatic patterns of malate dehydrogenase in plant fraction and bacteroids accompanying ageing of the lupine root nodules were observed. Possible physiological role of malate pathway in metabolism of lupine root nodules is discussed.     

Periplasmic metabolism of glutamate and aspartate by intact Bradyrhizobium japonicum bacteroids

In studies on the uptake and metabolism of [14C]glutamate by Bradyrhizobium japonicum bacteroids we found that, in the presence of unlabeled malate, succinate or alpha-ketoglutarate, substantial label was recovered in alpha-ketoglutarate in the reaction mixtures. As much as 30% of the total 14C supplied could be found in alpha-ketoglutarate in the reaction mixtures after 30 min and this occurred in the absence of detectable labeling of alpha-ketoglutarate in the cells. The labeling of alpha-ketoglutarate was almost completely inhibited by aminooxyacetate (aminotransferase inhibitor). Direct assay of aspartate aminotransferase in intact bacteroids was possible in the presence of very dilute Triton X-100 (less than or equal to 0.02%, w/v). The response of the aminotransferase to detergent was similar to the response of phosphodiesterase, a periplasmic marker, and different from malate dehydrogenase and beta-hydroxybutyrate dehydrogenase, cytoplasmic markers. Comparison of maximum enzyme activity assayable with intact bacteroids and maximum activity in sonicated bacteroids indicated that about half of the total cellular aminotransferase activity was accessible to the external medium. The combined labeling and enzyme assay results indicated that B. japonicum bacteroids have a capability for transamination in the periplasmic space. Although this may not be important in the transfer of reducing equivalents from host cytoplasm to bacteroids in nodules, the transamination capability may facilitate the acquisition of metabolites by free-living bacteria.     

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with CO(2)

The aim of the work reported here was to ascertain that the patterns of labeling seen in isolated bacteroids also occurred in bacteroids in intact nodules and to observe early metabolic events following exposure of intact nodules to ¹⁴CO₂. Intact nodules of soybean (Glycine max L. Merr. cv Ripley) inoculated with Bradyrhizobium japonicum USDA 110 and pea (Pisum sativum L. cv Progress 9) inoculated with Rhizobium leguminosarum bv viciae isolate 128C53 were detached and immediately fed ¹⁴CO₂ for 1 to 6 min. Bacteroids were purified from these nodules in 5 to 7 min after the feeding period. In the cytosol from both soybean and pea nodules, malate had the highest radioactivity, followed by citrate and aspartate. In peas, asparagine labeling equaled that of aspartate. In B. japonicum bacteroids, malate was the most rapidly labeled compound, and the rate of glutamate labeling was 67% of the rate of malate labeling. Aspartate and alanine were the next most rapidly labeled compounds. R. leguminosarum bacteroids had very low amounts of ¹⁴C and, after a 1-min feeding, malate contained 90% of the radioactivity in the organic acid fraction. Only a trace of activity was found in aspartate, whereas the rate of glutamate and alanine labeling approached that of malate after 6 min of feeding. Under the conditions studied, malate was the major form of labeled carbon supplied to both types of bacteroids. These results with intact nodules confirm our earlier results with isolated bacteroids, which showed that a significant proportion of provided labeled substrate, such as malate, is diverted to glutamate. This supports the conclusion that microaerobic conditions in nodules influence carbon metabolism in bacteroids.     

14C-glucose utilization byRhizobium lupini bacteroids

Summary The ability of bacteroids, isolated fromLupinus luteus L. nodules at the stage of active nitrogen fixation, to assimilate (1-¹⁴C)-glucose and (2-¹⁴C)-glucose was being studied. The label is incorporated into all the Krebs cycle metabolites, amino acids and sugars after 5 min of glucose insertion into cell suspension. High activity of glucose phosphorylation was found in bacteroidsin vitro, the reaction rate being the highest at a glucose concentration of over 100 mM.In lupine nodules sugars can be essential carbon substrate delivered to the bacteroids from host-plant cells. This point of view is discussed.     

Labelling of lupine nodule metabolites with 14CO2 assimilated from the leaves

On feeding ¹⁴CO₂ to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of ¹⁴C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H(+) -dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of D- and L-[U-(14)C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-D-glucose/D-[U-(14)C]glucose and 3-O-methyl-D-glucose/3-O-methyl-D-[U-(14)C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH(4)Cl inhibited neither the linear component of D- and L-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-D-[U-(14)C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol(-1), respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol(-1)). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Involvement of glutamate in the respiratory metabolism of Bradyrhizobium japonicum bacteroids.

Bradyrhizobium japonicum bacteroids were isolated anaerobically and supplied with 14C-labeled succinate, malate, aspartate, or glutamate for periods of up to 60 min in the presence of myoglobin to control the O2 concentration. Succinate and malate were absorbed about twice as rapidly as glutamate and aspartate. Conversion of substrate to CO2 was most rapid for malate, followed by succinate, glutamate, and aspartate. When CO2 production was expressed as a proportion of total carbon taken up, malate was still the most rapidly respired substrate, with 68% of the label absorbed converted to CO2. The comparable values for succinate, glutamate, and aspartate were 37, 50, and 38%, respectively. Considering the fate of labeled substrate not respired, greater than 95% of absorbed glutamate remained as glutamate in the bacteroids. In contrast, from 39 to 66% of the absorbed succinate, malate, or aspartate was converted to glutamate. An increase in the rate of CO2 formation from labeled substrates after 20 min appeared to coincide with a maximum accumulation of label in glutamate. The results indicate the presence of a substantial glutamate pool in bacteroids and the involvement of glutamate in the respiratory metabolism of bacteroids.     

Hypoxic stress and the transport systems of the peribacteroid membrane of bean root nodules

When the roots of Vicia faba L. beans were subjected to hypoxic stress, the activity of H⁺-ATPase on the peribacteroid membrane, as well as the transport of dicarboxylates (malate and succinate) mediated by this enzyme, decreased. Since malate and succinate are the main carbon-containing metabolites involved in the energy supply to bacteroids, this caused a change of the relation type from mutualism to commensalism, and the domination of the eukaryotes over the prokaryotes consequently increased.     

Alcohol dehydrogenase and its relation to respiratory pathways in lupine root nodules.

Changes in the isoenzymatic patterns of alcohol dehydrogenase (EC 1.1.1.1) accompanying ageing of the lupine root nodules were observed. Ethanol and other products of anaerobic metabolic pathways (lactate and malate) are better respiratory substrates for bacteroids and symbiosomes (peribacteroid units, PBUs) than glucose and pyruvate. It is postulated that fermentative processes in lupine root nodule provide energy and substrates for bacteroids.     

Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent K_m for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.     

Nitrogenase activity of pea bacteroids as affected by carbohydrates and ammonium chloride

Summary Regulation and efficiency of the nitrogen-fixing system of the rhizobium-pea symbiosis were investigated. Acetylene reduction of detached root nodules was measured with various substrates added. Succinate, fumarate and malate were most effective in stimulating nitrogenase activity; glucose, pyruvate and citrate were also active. Acetylene reducing activity of detached nodules was inhibited by the addition of NH₄Cl, irrespective of the substrate present. Nitrogenase activity of isolated bacteroids was not influenced by NH₄Cl.Respiration of detached nodules was not significantly stimulated by the addition of substrates. Ammonium chloride did not influence respiration. With detached nodules and isolated bacteroids a consumption of about 16 g of carbohydrate per g of nitrogen fixed could be calculated. Detached nodules produced more hydrogen relative to the acetylene reduced than did isolated bacteroids and intact plants.Results obtained indicate that the regulation of nitrogenase activity and the efficiency of substrate consumption depend on environmental conditions.     

Gluconeogenesis in the kidney cortex. Flow of malate between compartments

1. Kidney-cortex slices from starved rats were incubated with l-[U-(14)C]lactate or l-[U-(14)C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-(14)C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-(14)C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. (3)H from compounds such as l-[2-(3)H]lactate, which form NAD(3)H in the cytosol, appears in part in water; and (3)H from dl-beta-hydroxy[3-(3)H]butyrate, which forms NAD(3)H in the mitochondria, appears in part in glucose, largely on C-4.     

Transport and metabolism of galactose in rat kidney cortex

1. Analysis of transport of d-galactose was complicated by metabolism of the compound but appeared to have two components: a substrate-saturable component and a diffusion component. At low substrate concentration (<1mm) active transport was observed. Accumulation of galactose was largely independent of Na(+) concentration. The apparent K(m) for this component was 0.2mm. At substrate concentrations above 1mm the active transport system appeared saturated and further increases in substrate concentration resulted in a linear increase in the rate of galactose accumulation, but no concentration gradient was formed. 2. d-[1-(14)C]Galactose (2mm) was metabolized to (14)CO(2) by rat kidney-cortex slices incubated at 37 degrees C, at the rate of 68nmol/h per 100mg of tissue. 3. Intracellular components from such incubations were separated into a neutral fraction, the only major labelled component being galactose, and a phosphorylated fraction. 4. Phosphorylated metabolites found in galactose-incubated slices increased with increasing substrate concentration and achieved a limiting value of 0.42mm after 60min of incubation. 5. Galactose uptake was inhibited by anaerobiosis, dinitrophenol and phlorrhizin. 6. Methyl alpha-d-glucoside and d-glucose partially inhibited galactose uptake only at ratios of 100:1. 7. The presence of pyruvate did not decrease galactose metabolism although it did decrease production of (14)CO(2) from [1-(14)C]galactose. Gluconeogenesis occurred in the presence of pyruvate and (14)C from galactose was found in glucose. 8. Rat kidney-cortex slices metabolized 2mm-[1-(14)C]galactonate to (14)CO(2) at a rate of 20nmol/h per 100mg of tissue.     

Carbon metabolism and bacteroid respiration in nodules of chick-pea (Cicer arietinum L.) plants grown under saline conditions

ABSTRACT

The present work investigates the relationships between nitrogen fixation, carbon metabolism and oxygen consumption by bacteroids of Mesorhizobium ciceri in root nodules of chick-pea plants. Its aim was to establish whether some of the compounds which accumulate under salt stress may be used as respiratory substrates by bacteroids to fuel their own metabolism and nitrogenase activity. Plants were grown in a growth chamber, and salt stress was induced by adding 50 mM NaCl to the nutrient solution at sowing. The data presented here show a rise in fermentative metabolism in nodules of chick-pea plants exposed to high salinity, and suggest that proline, lactate or ethanol, may play an important role as energy-yielding substrates for bacteroids in this plant species. The bacteroids could utilize glucose as a respiratory substrate both under control and saline conditions, while malate did not appear to be the preferred substrate in the presence of salt.     

Metabolism of the dimethyl ester of [2,3-13C]succinic acid in rat hepatocytes

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-13C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-13C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.     

Effects of Potassium and Glutamine on Metabolism of Glucose in Astrocytes

The metabolic effects of extracellular glutamine (2.5 mM) or high potassium (25 mM) on glucose metabolism were studied in cultured cerebellar astrocytes. High potassium caused an increased glycolytic flux and an increase in glutamine release. Exposure to glutamine increased glycolytic flux and alanine formation, indicating that glutamine uptake is an energy requiring process. The effects of glutamine and high potassium on glycolytic flux were additive. Formation of metabolites from [1-¹³C]glucose and [2-¹³C]acetate confirmed the effects of glutamine and high potassium on glycolytic metabolism. In the presence of extracellular glutamine, analysis of the ¹³C labeling patterns of citrate and glutamine indicated a decrease in the cycling ratio and/or pyruvate carboxylation and glutamine synthesis from [1-¹³C]glucose did occur, but was decreased. Exposure to high potassium led to extracellular accumulation of acetate, presumably through non-enzymatic decarboxylation of pyruvate.