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.     

Studies on brain-cortex slices: The influence of various inhibitors on the retention of potassium ions and amino acids with glucose or pyruvate as substrate

1. The rate of appearance of (14)CO(2) from [6-(14)C]glucose and [3-(14)C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30mum-2,4-dinitrophenol increases the output of (14)CO(2) from [6-(14)C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0.1m-potassium chloride. The stimulating action of these two agents on the output of (14)CO(2) from [3-(14)C]pyruvate is much less than on that from [6-(14)C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1.3mum) inhibited the oxidation of [6-(14)C]glucose more than 70%, but did not inhibit the oxidation of[3-(14)C]pyruvate. [3-(14)C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH(2) produced during glycolysis.     

Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria

1. The fixation of CO(2) by pyruvate carboxylase in isolated rat brain mitochondria was investigated. 2. In the presence of pyruvate, ATP, inorganic phosphate and magnesium, rat brain mitochondria fixed H(14)CO(3) (-) into tricarboxylic acid-cycle intermediates at a rate of about 250nmol/30min per mg of protein. 3. Citrate and malate were the main radioactive products with citrate containing most of the radioactivity fixed. The observed rates of H(14)CO(3) (-) fixation and citrate formation correlated with the measured activities of pyruvate carboxylase and citrate synthase in the mitochondria. 4. The carboxylation of pyruvate by the mitochondria had an apparent K(m) for pyruvate of about 0.5mm. 5. Pyruvate carboxylation was inhibited by ADP and dinitrophenol. 6. Malate, succinate, fumarate and oxaloacetate inhibited the carboxylation of pyruvate whereas glutamate stimulated it. 7. The results suggest that the metabolism of pyruvate via pyruvate carboxylase in brain mitochondria is regulated, in part, by the intramitochondrial concentrations of pyruvate, oxaloacetate and the ATP:ADP ratio.     

Regulation of gluconeogenesis from pyruvate and lactate in the isolated perfused rat liver

The effects of glucagon and the alpha-adrenergic agonist, phenylephrine, on the rate of 14CO2 production and gluconeogenesis from [1-14C]lactate and [1-14C]pyruvate were investigated in isolated perfused livers of 24-h-fasted rats. Both glucagon and phenylephrine stimulated the rate of 14CO2 production from [1-14C]lactate but not from [1-14C]pyruvate. Neither glucagon nor phenylephrine affected the activation state of the pyruvate dehydrogenase complex in perfused livers derived from 24-h-fasted rats. 3-Mercaptopicolinate, an inhibitor of the phosphoenolpyruvate carboxykinase reaction, inhibited the rates of 14CO2 production and glucose production from [1-14C]lactate by 50% and 100%, respectively. Furthermore, 3-mercaptopicolinate blocked the glucagon- and phenylephrine-stimulated 14CO2 production from [1-14C]lactate. Additionally, measurements of the specific radioactivity of glucose synthesized from [1-14C]lactate, [1-14C]pyruvate and [2-14C]pyruvate indicated that the 14C-labeled carboxyl groups of oxaloacetate synthesized from 1-14C-labeled precursors were completely randomized and pyruvate----oxaloacetate----pyruvate substrate cycle activity was minimal. The present study also demonstrates that glucagon and phenylephrine stimulation of the rate of 14CO2 production from [1-14C]lactate is a result of increased metabolic flux through the phosphoenolpyruvate carboxykinase reaction, and phenylephrine-stimulated gluconeogenesis from pyruvate is regulated at step(s) between phosphoenolpyruvate and glucose.     

Effect of phenylephrine on pyruvate dehydrogenase in fasting rat livers

Previous estimates of flux through the pyruvate-dehydrogenase complex were made by measuring 14CO2 generated from oxidation of [1-14C]pyruvate, assuming a 1:1 stoichiometry. However, this method fails to discriminate between 14CO2 produced from pyruvate dehydrogenase and 14CO2 generated from phospho-enolpyruvate carboxykinase and citric-acid-cycle dehydrogenases. While some previous reports have attempted to correct for the additional 14CO2 production by comparing 14CO2 generated by [1-14C]pyruvate with [2-14C]pyruvate or [3-14C]pyruvate, the estimates are flawed by failure to determine the radioactivity and distribution of the 14C label in the oxalacetate pool. The present method circumvents these problems by utilizing [1,4-14C]succinate to radiolabel the oxalacetate pool and by directly measuring the specific radioactivity of malate. The results demonstrate that flux through the pyruvate-dehydrogenase complex is negligible compared to the other reactions which generate 14CO2 from [1-14C]lactate in the fasted state. Phenylephrine did not significantly alter this result in the fasted state. However, 14CO2 production via the pyruvate-dehydrogenase complex is large (approximately 11.5 nmol.min-1.mg mitochondrial protein-1) compared to 14CO2 production via phosphoenolpyruvate carboxykinase and citric-acid-cycle dehydrogenases (approximately 6.4 nmol.min-1.mg-1) when the pyruvate-dehydrogenase complex is activated, in the fed state with 1 mM dichloroacetate.     

The role of phosphoenolpyruvate carboxykinase in muscle alanine synthesis.

3-Mercaptopicolinic acid (3-MPA) is reportedly a specific inhibitor of phosphoenolpyruvate (PEP) carboxykinase and has hitherto been used accordingly to elucidate the metabolic role of PEP carboxykinase in vitro and in vivo. We show that 3-MPA has multiple effects on intermediary metabolism in hemidiaphragms from 40 h-starved rats. It decreases the release of lactate + pyruvate and alanine in hemidiaphragms provided with no added substrate or with valine, leucine or isoleucine. Moreover, irrespective of the substrate provided (none, valine, leucine, isoleucine, glucose, acetate, oleate), 3-MPA decreases the [lactate]/[pyruvate] ratio. 3-MPA is without effect on 14CO2 production from [U-14C]valine, [1-14C]valine, [1-14C]leucine, [U-14C]isoleucine or [1-14C]oleate, but stimulates 14CO2 production from [U-14C]glucose and [1-14C]pyruvate and inhibits 14CO2 production from [1-14C]acetate. Glycolytic flux (measured as 3H2O formation from [5-3H]glucose) is stimulated by 3-MPA. It is concluded that 3-MPA has site(s) of actions other than PEP carboxykinase and that the putative role of PEP carboxykinase in alanine synthesis de novo in skeletal muscle from tricarboxylic acid-cycle intermediates and related amino acids requires reappraisal.     

Effects of fructose concentration on carbohydrate metabolism, heat production and substrate cycling in isolated rat hepatocytes

1. Hepatocytes from starved rats were incubated with 5mm-glucose, labelled uniformly with (14)C and specifically with (3)H at positions 1, 2, 3 or 6, and with fructose at concentrations of 2.5, 7.5 or 25mm. 2. In the absence of other substrates only 1% of the radioactivity initially present in [U-(14)C]glucose appeared in the metabolic products, CO(2), lactate, pyruvate, amino acids and glycogen. 3. Fructose at 2.5mm caused a 30% increase in the glucose concentration and a 4-fold increase in the apparent oxidation of [U-(14)C]-glucose. 4. The formation of (3)H(2)O from [1-(3)H]-, [2-(3)H]-, [3-(3)H]- or [6-(3)H]-glucose was 2.4, 4.3, 2.15 or 1.6% respectively in the control incubations and 4.1, 10.4, 7.7 or 5.1% with 2.5mm-fructose. 5. Fructose at 7.5 and 25mm decreased the (3)H(2)O yields to less than the control values, but had no apparent effect on the amount of [U-(14)C]glucose metabolized. 6. In the incubations with 5mm-glucose and 25mm-fructose there were significant decreases in heat production, O(2) consumption and in the ratio of O(2) uptake to heat output. 7. Fructose at 2.5mm caused a 64% increase in heat output, but only a 43% increase in O(2) uptake. 8. The radioisotopic and calorimetric data demonstrate that physiological concentrations of fructose greatly increase metabolism in hepatocytes from starved rats. These data also indicate increased cycling at glucose/glucose 6-phosphate and at fructose 6-phosphate/fructose 1,6-bisphosphate in the presence of 2.5mm-fructose, although the rates of cycling were actually decreased relative to the amount of glucose catabolized. 9. At concentrations of 2.5, 7.5 and 25mm, fructose depressed hepatocyte ATP concentrations by 20, 65 and 80% respectively. Although fructose at 7.5 and 25mm increased glucose and lactate release, O(2) consumption, production of heat and formation of(3)H(2)O from [1-(3)H]-, [2-(3)H]-, [3-(3)H]- or [6-(3)H]-glucose were lowered to values equal to, or less than, controls. These effects probably reflect a severe derangement of hepatic metabolism due to excess phosphorylation of fructose when present at high concentrations.     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

Metabolic interactions between glucose, glycerol, alanine and acetate in Leishmania braziliensis panamensis promastigotes

13C-nuclear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of 14CO2 production from 14C-labeled substrate. Cells incubated with [2-13C]glycerol released acetate, succinate and D-lactate in addition to CO2. Cells incubated with acetate released only CO2. More succinate C-2/C-3 than C-1/C-4 was released from both [2-13C]glycerol and [2-13C]glucose, indicating that succinate was formed predominantly by CO2 fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of 14CO2 production from [1,(3)-14C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased 14CO2 production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of 14CO2 production from [U-14C]- and [l-14C]alanine by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-hydroxytryptamine, glucose uptake, glycogen utilization and carbon dioxide fixation in Hymenolepis microstoma (Cestoda)

1. In vitro glucose uptake and glycogen utilization by Hymenolepis microstoma decreased under high oxygen concentrations. 2. 5-Hydroxytryptamine did not stimulate in vitro glucose uptake but did increase glycogen utilizations by H. microstoma. 3. The reduced glucose uptake under high oxygen concentrations (21 and 95%) resulted in a reduction in excretory products. 4. 14CO2-incorporation studies confirmed that, under both 95% O2:5% CO2 and air-minus-CO2 (identical to 21% O2). CO2-fixation by phosphoenolpyruvate carboxykinase (EC 4.1.1.32) was inhibited. 5. The specific activity of hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) was not stimulated by 5-HT. 6. The concentration of ATP required for optimal stimulation of phosphofructokinase activity was 0.67 mM. Activity was further significantly increased by the addition of cAMP and even greater by AMP.     

Changes in the growth and enzyme level of Zymomonas mobilis under oxygen-limited conditions at low glucose concentration

Zymomonas mobilis growing aerobically with 20 g glucose^–1 (carbon-limited) in a chemostat exhibited an increase in both the molar growth yield (Y_x/s) and the maximum molar growth yield (Y_x/s ^max) and a decrease in both the specific substrate consumption rate (q_s) and the maintenance energy consumption rate (m_e). Stepwise increase in the input oxygen partial pressure showed that anaerobic-to-aerobic transitional adaptation occurred in four stages: anaerobic (0 mm HgO₂), oxygen-limited (7.6– 230 mm HgO₂), intermediate (273 mm HgO₂), and oxygen excess (290 mm HgO₂). The steady-state biomass concentration, Y_x/s, and intracellular ATP content increased between oxygen partial pressures of 7.6 and 120 mm HgO₂, accompanied by a decrease in the q_s and the specific acid production rate. The membrane ATPase activity decreased with increasing oxygen partial pressure and reached its lowest levels at 273 mm HgO₂, which was the highest input oxygen partial pressure where steady-state conditions were possible. Glucokinase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase activities also decreased when the oxygen partial pressure was increased above 15 mm Hg, whereas pyruvate decarboxylase was unaffected by aeration. Growth inhibition at 290 mm HgO₂ was characterised by a drastic reduction in the pyruvate kinase activity and a collapse in the intracellular ATP pool. The growth and enzyme data suggest that at low glucose concentrations and oxygen-limited conditions, the increase in biomass yields is a reflection of a redirection of ATP usage rather than a net increase in energy production. Received: 14 August 1996 / Accepted: 31 January 1997     

Decarboxylation and carboxylation of pyruvate in the living mice.

Mice received intravenously [1- or 2-14C]acetate, [1-, 2- or 3-14C] or [2-14C]pyruvate and were killed 1, 3, 5 or 15 min later. The radioactivity of CO2 or HCO3- of liver or carcass as well as the radioactivity of blood glucose were measured. The ratio of the radioactivity found in these compounds after [3-14C] or [2-14C-A1pyruvate injection suggests that in the fed aminals: 1. the decarboxylation of the pyruvate was more rapid than its carboxylation, 2. most of the neosynthesized glucose was derived from pyruvate molecules which had undergone a decarboxylation followed by a condensation to citrate, 3. 1/4 to 1/3 of the pyruvate was carboxylated and 2/3 to 3/4 was decarboxylated in animals receiving a diet poor in fats.     

Evidence for the compartmentation of pyruvate metabolism in perfused rat skeletal muscle.

In rat hindlimbs perfused with [1-14C]pyruvate and 5 mM-dichloroacetate, the calculated apparent rate of pyruvate decarboxylation was decreased with increasing perfusate pyruvate concentrations. However, in the absence of dichloroacetate the apparent rate of decarboxylation increased under these conditions. Dichloroacetate enhanced [1-14C]pyruvate uptake, but decreased the specific radioactivity of effluent lactate. Glycogen metabolism remained unaffected. The results were not consistent with a common pyruvate pool, but provide evidence for the compartmentation of pyruvate metabolism.     

Anabolic regulation of gluconeogenesis by insulin in isolated rat hepatocytes

The role of substrate availability in the regulation of gluconeogenesis in isolated rat hepatocytes was studied using [U-14C]alanine as a tracer in the presence of different concentrations of L-alanine in the incubation medium. At low alanine concentrations (0.5 mM) insulin decreased the 14C incorporation into the glucose pool and increased the incorporation of tracer carbons into the protein and lipid pools and into CO2. The net radioactivity lost from the glucose pool was only a small percentage of the total increase in the activity of the protein, lipid, CO2, or glycogen pools, supporting the notion that the effect of insulin in diminishing gluconeogenesis is secondary to its effects on pathways using pyruvate. At higher concentrations of alanine (2.5, 5.0, and 10.0 mM) in the incubation medium insulin increased the movement of alanine carbons into protein and glucose. This suggests that at higher substrate concentrations the ability of the liver to synthesize proteins is overwhelmed and the pyruvate carbons are forced into the gluconeogenesis pathway. These results were further confirmed by using [U-14C]lactate. The increases in observed specific activity of glucose following insulin administration would not be possible if insulin acted by affecting the activity of any enzyme directly involved in the formation or utilization of pyruvate, most of which have been proposed as sites of insulin action. Data presented show that insulin "inhibits" gluconeogenesis by affecting a change in substrate availability.     

The biosynthesis of intestinal mucins. The effect of salicylate on glycoprotein biosynthesis by sheep colonic and human gastric mucosal tissues in vitro

1. Incubation of sheep colonic mucosal scrapings in Krebs-Ringer buffer for 2(1/2)hr. in the presence of salicylate (15mm) resulted in decreased incorporation of radioactivity into the epithelial glycoprotein from the following labelled precursors: 16.6mum-d-[2-(14)C]glucose (83.9% inhibition), 20mum-l-[U-(14)C]threonine (82%) and (35)SO(4) (2-)(79%). Oxygen uptake measured simultaneously was diminished to 41% of the control value. 2. At lower concentrations of salicylate (e.g. 3.75mm), incorporation of 20mum-l-[U-(14)C]threonine was little affected (3-6% inhibition), whereas utilization of 4mum-d-[U-(14)C]glucose and (35)SO(4) (2-) was inhibited (41-48% and 40-59% of the control values respectively). 3. Analysis of the papain-digested glycoprotein from tissue incubations with 16.6mum-d-[2-(14)C]glucose in the presence of salicylate (3.75mm) showed large decreases in labelling of N-acetylneuraminic acid and N-glycollylneuraminic acid residues (57% and 34% of the control values respectively) and of hexosamine constituents (glucosamine, 55% inhibition; galactosamine, 33% inhibition). Labelling of neutral sugars (galactose and fucose) was relatively little affected (9 and 11% inhibition respectively). 4. Glucose 6-phosphate transaminase and glucosamine 6-phosphate acetylase in particle-free enzyme preparations of the sheep tissue were unaffected by salicylate at the above concentrations. Acetyl-CoA synthetase was markedly inhibited. 5. Human gastric mucosa (from operation), on incubation as above, had in one experiment an oxygen consumption of 9.9mul./hr./mg. dry wt. of tissue and incorporated 5mum-d-[U-(14)C]glucose (15.8% of the total radioactivity added) into bound hexosamine (20.6% of the total radioactivity incorporated), hexoses (glucose and galactose, 5.7%) and fucose (14.2%). The presence of salicylate (15mm) decreased the incorporation of 5mum-d-[U-(14)C]glucose into the glycoprotein by 74%, all sugar constituents being affected, without influence on the rate of oxygen consumption. 6. The results suggest an inhibitory effect of salicylate on glycoprotein biosynthesis at the level of the amino sugar intermediates.     

Rates of flux through the pentose cycle in perfused rat liver. A procedure for the calculation of rates of substrate flux from 14CO2 production from [1-14C]glucose

A method for the determination of substrate flux through the pentose cycle was developed employing [1-14C]glucose in experiments with perfused rat livers. The method consists first of a kinetic analysis which differentiates between the production of 14CO2 from [1-14C]glucose via the pentose cycle and via the citrate cycle and, second of a calculation of the specific radioactivity of the hexose monophosphate pool from measured rates of glycolysis and the specific radioactivity of lactate released into the perfusate. The method was validated by experiments comparing the results of tracer infusions with [1-14C]glucose, [6-14C]glucose and [3-14C]pyruvate. In livers from fed rats perfused with 10 mM glucose, the rate of substrate flux through the pentose cycle was around 0.2 mumol X min-1 X g-1; it was about 20% of the substrate flux via glycolysis. The kinetic data were inconsistent with the existence of an L-type pentose cycle in liver.     

A 14CO2 ratios method for detecting pyruvate carboxylation

The pattern of oxidative metabolism of pyruvate may be assessed by comparing the steady-state 14CO2 production from four isotopes in identical samples. The assay requires measuring the ratios of steady-state 14CO2 production from two isotope pairs, [2-14C]pyruvate:[3-14C]pyruvate and [1-14C]acetate:[2-14C]acetate. These ratios are defined as the "pyruvate 14CO2 ratio" and the "acetate 14CO2 ratio," respectively. If pyruvate is metabolized exclusively via pyruvate dehydrogenase (PDH), the two ratios will be identical. Alternatively, if any pyruvate enters the tricarboxylic acid (TCA) cycle via pyruvate carboxylation (PC), the pyruvate 14CO2 ratio will be less than the acetate 14CO2 ratio. If pyruvate enters the TCA cycle only through PC (with oxaloacetate and fumarate in equilibrium) the pyruvate 14CO2 ratio will approach a value of 1.0. An equation is presented for the quantitative evaluation of pyruvate oxidation by these two pathways. We have used this method to detect relative changes in the pattern of pyruvate metabolism in rat liver mitochondria produced by exposure to 1 mM octanoyl carnitine, a compound known to alter the PC:PDH activity ratio. The major advantages of the method are (i) that it provides a sensitive method for detecting pyruvate carboxylation at physiological pyruvate concentrations and (ii) that it provides a method for distinguishing between effects on pyruvate transport and effects on pyruvate oxidation.     

A study on the precursors of the acetyl moiety of acetylcholine in brain slices. Observations on the compartmentalization of the acetyl-coenzyme A pool

1. A method was devised for the determination of the specific radioactivity of the acetyl moiety of acetylcholine synthesized from various (14)C-labelled substrates. 2. The precursor for the acetyl moiety of acetylcholine was studied in slices of striatum and cerebral cortex from rat and guinea-pig brain. Incorporation of radioactivity into acetylcholine was determined after incubating the slices in the presence of [2-(14)C]acetate, [(14)C]bicarbonate, [1,5-(14)C]citrate, dl-[1- or 5-(14)C]glutamate or [1- or 2-(14)C]pyruvate. 3. After incubation for 1h, acetylcholine was accumulated significantly in both striatum slices (4.1nmol/mg of protein) and cerebral-cortex slices (0.57nmol/mg of protein) from the rat. Final concentrations were about 11 and 5 times respectively the initial values. 4. With slices from rat striatum, rat cerebral cortex and guinea-pig cerebral cortex, the specific radioactivity of acetylcholine derived from [2-(14)C]pyruvate was very high, reaching approx. 30, 20 and 6% respectively of the initial specific radioactivity of added pyruvate in the medium. With the striatum slices this high value was reached after incubation for 15min. Incorporation of radioactivity from [2-(14)C]acetate was only 1.25, 5.3 and 19.7% of that from [2-(14)C]pyruvate in rat striatum, rat cerebral-cortex and guinea-pig cerebral-cortex slices respectively. A small but definite incorporation was found from [5-(14)C]glutamate. No incorporation was found from the other substrates. The findings suggest that pyruvate is the most important precursor for the synthesis of the acetyl moiety of acetylcholine in brain slices. 5. The specific radioactivity of acetylcholine relative to that of citrate when [2-(14)C]pyruvate was used compared with that obtained when [2-(14)C]acetate was used. A marked difference was found in all slices, suggesting metabolic compartmentation of the acetyl-CoA pool.     

Lipogenesis from glucose and pyruvate in fat cells from genetically obese rats

Subcutaneous fat cells were isolated from genetically obese rats and from rats with obesity produced by hypothalamic lesions. Insulin did not augment the oxidation of fatty acids or their synthesis from glucose-1-(14)C or glucose-1-(3)H by fat cells from either group. Radioactivity from pyruvate-3-(14)C was incorporated into fatty acids to the same degree by fat cells from these two groups. The presence of 5 mm glucose in the incubation medium containing fat cells and pyruvate-3-(14)C or aspartate-3-(14)C stimulated the synthesis of fatty acids to a greater extent in cells of genetically obese rats. Fasting, in contrast, reduced the incorporation of radioactivity from pyruvate and glucose into fatty acids by fat cells from the genetically obese animals. In all experiments the fat cells from genetically obese rats converted more radioactivity into glyceride-glycerol relative to CO(2) than did fat cells from hypothalamic obese rats. Parabiosis between one thin and one genetically obese litter mate was performed in three pairs of rats without influencing growth of either rat. Thus in the present studies fat cells from genetically obese rats showed two differences from normal fat cells: they channeled more radioactivity from pyruvate into fatty acids in the presence of glucose, and they uniformly converted more radioactivity into glyceride-glycerol.     

Inhibitory effect of high oxygen pressure on potassium- induced activation of pyruvate dehydrogenase and glucose metabolism in rat brain slices

The effects of high oxygen pressure on pyruvate dehydrogenase (pyruvate: lipoate oxidoreductase (decarboxylating and acceptor-acylating), EC 1.2.4.1) activity, tissue concentration of ATP, and CO2 production from glucose were studied in rat brain cortical slices. The increase in pyruvate dehydrogenase activity and the lowering of cellular ATP, occurring during potassium-induced depolarization at 1 atm of oxygen, were reversed by increasing the oxygen pressure to 5 atm. When brain slices were incubated at 1 atm oxygen with [U-14C]glucose, a high potassium medium approximately doubled the production of 14CO2. Oxygen at 5 atm abolished this potassium-dependent increase in 14CO2 production with no significant effect on glucose oxidation in normal Krebs-Ringer phosphate medium. Adding 4 atm helium to 1 atm oxygen did not interfere with the ability of potassium ions to activate pyruvate dehydrogenase, lower ATP, or increase glucose oxidation. The results show that toxic effects of hyperbaric oxygen, not manifest in "resting" tissue, may be revealed during stress such as potassium depolarization. The site of the toxic effects of oxygen is probably the cell membrane where excess oxygen appears to interfere with the action of the sodium pump, calcium transport or other processes stimulated by increased concentrations of extracellular potassium.