Intramolecular labelling of sucrose made by leaves from [14C)carbon dioxide or [3-14C]serine.

Pea leaves were illuminated in air containing 150 or 1000p.p.m. of 14CO2 for various times. Alternatively, segments of wheat leaves were supplied with [3-14C]serine for 40 min in the light in air with 145, 326 or 944p.p.m. of 12CO2. Sucrose was extracted from the leaf material, hydrolysed with invertase, and 14C in the pairs of carbon atoms C-3+C-4, C-2+C-5 and C-1+C-6 in the glucose moiety was measured. The results obtained after metabolism of 14CO2 were consistent with the operation of the photosynthetic carbon-reduction cycle; the effects of CO2 concentration on distribution of 14C in the carbon chain of glucose after metabolism of [3-14C]serine is more easily explained by metabolism through the glycollate pathway than by the carbon-reduction cycle.     

The pentose cycle and insulin release in isolated mouse pancreatic islets during starvation.

When islets from mice were incubated with 16.7 mM-glucose, previous starvation for 48 h decreased the rate of insulin release by approx. 50% and glucose utilization was decreased by approx. 35%. The maximally extractable activity of glucose 6-phosphate dehydrogenase was diminished by 28% after starvation. The formation of 14CO2 from both [1-14C]glucose was, however, higher than the rate of oxidation of [6-14C]-glucose in islets from both fed and starved mice. The fraction of glucose utilized that was oxidized (specific 14CO2 yield) ranged from one-fifth to one-third and was higher in islets from starved mice with both [1-14C]glucose and [6-14C]glucose as substrate. The contribution of pentose-cycle oxidation to total glucose metabolism was small (3% in the fed state and 4% in the starved state). The absolute rates of glucose carbon metabolism via the pentose-cycle oxidation to total glucose metabolism was small (3% in the fed state and 4% in the starved state). The absolute rates of glucose carbon metabolism via the pentose cycle and the turnover of NADPH in this pathway were identical in islets from fed and starved animals. After incubation at 16.7 mM-glucose for 30 min the contents of glucose (6-phosphate and 6-phosphogluconate were both unchanged by starvation. It is concluded that there is no correlation between the decreased sensitivity of the insulin secretory mechanism during starvation and the metabolism of glucose via the pentose cycle, the islet content of glucose 6-phosphate or 6-phosphogluconate.     

Oxidation of glucose, ribose, alanine, and glutamate by Leishmania braziliensis panamensis

The metabolism of [1-14C]- and [6-14C]glucose, [1-14C]ribose, [1-14C]- and [U-14C]alanine, and [1-14C]- and [5-14C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of 14CO2 produced from [1-14C]glucose to that from [6-14C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of 14CO2 from [1-14C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of 14CO2 production from [1-14C]glucose was almost linear with time of incubation, whereas that of [6-14C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26 degrees C to 34 degrees C had no appreciable effect on the rates or time courses of oxidation of either [1-14C]- or [6-14C]glucose or of [1-14C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of 14CO2 produced from [1-14C]- to [U-14C]alanine and from [1-14C]- to [5-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiorespirometry evidence for the discrimination between 13C-enriched glucose and unlabelled glucose molecules by Paracoccus denitrificans

Paracoccus denitrificans was grown on either unlabelled glucose, [1-13C]glucose or [6-13C]glucose as the sole carbon source for growth. The cells were then incubated with a range of 14C-glucose substrates to compare the 14CO2-evolution rates between cells grown on the glucose and the 13C-labelled glucose. Cells grown on 13C-glucose had significantly faster rates of 14CO2-evolution than those grown on unlabelled glucose. The % yields of 14CO2, per [1-14C]-, [6-14C]- and [U-14C]glucose supplied were also substantially greater than those measured for cells grown on unlabelled glucose. The data indicated that growth of Paracoccus on 13C-enriched glucose substrates resulted in cells with notably different 14C-glucose oxidation metabolism compared to that observed in cells grown on unlabelled glucose.     

Glucose and glutamine metabolism in rat thymocytes.

The metabolism of glucose and glutamine in freshly prepared resting and concanavalin A-stimulated rat thymocytes was studied. Concanavalin A addition enhanced uptake of both glucose and glutamine and led to an increase in oxidative degradation of both substrates to CO2. With variously labelled [14C]glucose, it was shown that the pathways of glucose dissimilation were equally stimulated by the mitogen. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused an increase in the oxidation of pyruvate as judged by the enhanced release of 14CO2 from [2-14C]-, [3,4-14C]- and [6-14C]-glucose. Addition of glutamine did not affect glucose metabolism. The major end products of glutamine metabolism by resting and mitogen-stimulated rat thymocytes were glutamate, aspartate, CO2 and NH3. Virtually no lactate was formed from glutamine. Concanavalin A enhanced the formation of all end products except glutamate, indicating that more glutamine was metabolized beyond the stage of glutamate in the mitogen-activated cells. Addition of glucose caused a significant decrease in the rates of glutamine utilization and conversion into aspartate and CO2 in the absence and in the presence of concanavalin A. In the presence of glucose, almost all nitrogen of the metabolized glutamine was accounted for as NH3 released via the glutaminase and/or glutamate dehydrogenase reactions. In the absence of glucose, part (18%) of the glutamine nitrogen was metabolized by the resting and to a larger extent (38%) by the mitogen-stimulated thymocytes via a transaminase or amidotransferase reaction.     

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)     

Effects of Ammonia and β-Methylene-dl-Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture

Both ammonia and beta-methylene-DL-aspartate (beta-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and beta-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mM beta-MA resulted in a decreased production of 14CO2 from [U-14C]glucose, but did not affect 14CO2 production from [2-14C]pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2-14C]pyruvate, but 14CO2 production from [U-14C]glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C]glucose or [2-14]pyruvate by neurons. However, incubation of neurons with beta-MA or beta-MA plus ammonium chloride resulted in a approximately 45% decrease of 14CO2 production from both [U-14C]glucose and [2-14C]pyruvate. A 2-h incubation of astrocytes with beta-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in greater than 50% decrease in ATP, but had little effect on phosphocreatine. beta-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards beta-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by approximately 45% in astrocytes and by approximately 55% in neurons.     

Metabolism of [2-14C]acetate and its use in assessing hepatic Krebs cycle activity and gluconeogenesis

To examine the fate of the carbons of acetate and to evaluate the usefulness of labeled acetate in assessing intrahepatic metabolic processes during gluconeogenesis, [2-14C]acetate, [2-14C]ethanol, and [1-14C]ethanol were infused into normal subjects fasted 60 h and given phenyl acetate. Distributions of 14C in the carbons of blood glucose and glutamate from urinary phenylacetylglutamine were determined. With [2-14C]acetate and [2-14C]ethanol, carbon 1 of glucose had about twice as much 14C as carbon 3. Carbon 2 of glutamate had about twice as much 14C as carbon 1 and one-half to one-third as much as carbon 4. There was only a small amount in carbon 5. These distributions are incompatible with the metabolism of [2-14C]acetate being primarily in liver. Therefore, [2-14C]acetate cannot be used to study Krebs cycle metabolism in liver and in relationship to gluconeogenesis, as has been done. The distributions can be explained by: (a) fixation of 14CO2 from [2-14C]acetate in the formation of the 14C-labeled glucose and glutamate in liver and (b) the formation of 14C-labeled glutamate in a second site, proposed to be muscle. [1,3-14C]Acetone formation from the [2-14C]acetate does not contribute to the distributions, as evidenced by the absence of 14C in carbons 2-4 of glutamate after [1-14C]ethanol administration.     

Monitoring flux through the oxidative pentose phosphate pathway using [1-14C]gluconate

The aim of this work was to examine the metabolism of exogenous gluconate by a 4-day-old cell suspension culture of Arabidopsis thaliana (L.) Heynh. Release of (14)CO(2) from [1-(14)C]gluconate was dependent on the concentration in the medium and could be resolved into a substrate-saturable component (apparent K(m) of approximately 0.4 mM) and an unsaturable component. At an external concentration of 0.3 mM, the rate of decarboxylation of applied gluconate was 0.2% of the rate of oxygen consumption by the cells. There was no effect of 0.3 mM gluconate on the rate of oxygen consumption, or on the rate of (14)CO(2) release from either [1-(14)C]glucose or [6-(14)C]glucose by the culture. The following observations argue that gluconate taken up by the cells is metabolised by direct phosphorylation to 6-phosphogluconate and subsequent decarboxylation through 6-phosphogluconate dehydrogenase. First, more than 95% of the label released from [1-(14)C]gluconate during metabolism by the cell culture was recovered as (14)CO(2). Secondly, inhibition of the oxidative pentose phosphate pathway (OPPP) by treatment with 6-aminonicotinamide preferentially inhibited release of (14)CO(2) from [1-(14)C]gluconate relative to that from [1-(14)C]glucose. Thirdly, perturbation of glucose metabolism by glucosamine did not affect (14)CO(2) from [1-(14)C]gluconate. Fourth, stimulation of the OPPP by phenazine methosulphate stimulated release of (14)CO(2) from [1-(14)C]gluconate to a far greater extent than that from [1-(14)C]glucose. It is proposed that measurement of (14)CO(2) from [1-(14)C]gluconate provides a simple and sensitive technique for monitoring flux through the OPPP pathway in plants.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Carbon dioxide fixation and mixotrophic metabolism by strain DCB-1, a dehalogenating anaerobic bacterium

Fixation by strain DCB-1 of CO2 carbon into cell material and organic acids occurred during growth on pyruvate both with and without thiosulfate. By using sodium [14C]bicarbonate and sodium [2-14C]pyruvate, the isotopic composition of products and cells was investigated. Up to 70% of cell carbon was derived from CO2. CO2 carbon was also incorporated into succinate, formate, and acetate. Both carbons of acetate underwent exchange reactions with CO2, although the carboxyl-group exchange was twice as fast. Because strain DCB-1 uses CO2 as its major but not sole carbon source while deriving energy from pyruvate metabolism, we describe its metabolism as mixotrophic. Other mixotrophic conditions also supported growth. Lactate or butyrate, which could not support growth in mineral medium, could replace pyruvate as the oxidizable substrate only when acetate was added to the medium.     

Carbon dioxide fixation and mixotrophic metabolism by strain DCB-1, a dehalogenating anaerobic bacterium.

Fixation by strain DCB-1 of CO2 carbon into cell material and organic acids occurred during growth on pyruvate both with and without thiosulfate. By using sodium [14C]bicarbonate and sodium [2-14C]pyruvate, the isotopic composition of products and cells was investigated. Up to 70% of cell carbon was derived from CO2. CO2 carbon was also incorporated into succinate, formate, and acetate. Both carbons of acetate underwent exchange reactions with CO2, although the carboxyl-group exchange was twice as fast. Because strain DCB-1 uses CO2 as its major but not sole carbon source while deriving energy from pyruvate metabolism, we describe its metabolism as mixotrophic. Other mixotrophic conditions also supported growth. Lactate or butyrate, which could not support growth in mineral medium, could replace pyruvate as the oxidizable substrate only when acetate was added to the medium.     

Measurement of the metabolism of energy substrates in individual bovine blastocysts

Individual blastocysts from cows were cultured for 3 h under 5% CO2 in air, in 4 microliters droplets of Ham's F-10 medium containing D-[5-3H]glucose, D-[1-14C]-glucose, D-[6-14C]glucose, [2-14C]pyruvate, or L-[U-14C]glutamine, and with or without 2,4-dinitrophenol (DNP) or phenazine ethosulphate (PES). The 14CO2 or 3H2O produced were collected by exchange with an outer bath of 400 microliter 25 mM-NaHCO3. All combinations of substrate and treatment (control, DNP or PES) produced measurable quantities of labelled product except for D-[6-14C]glucose in the presence of PES. Untreated and DNP-treated embryos developed normally during a subsequent 48-h culture period in fresh medium, but PES-treated embryos degenerated. Pyruvate and glutamine metabolism both increased markedly in the presence of DNP, indicating that the Krebs' cycle is active, and that glutamine can be used as an energy substrate. Conversely, DNP has no significant effect on glucose metabolism, indicating that glycolysis is blocked in the bovine blastocyst due to a lack or inhibition of pyruvate kinase. The production of 14CO2 from D-[1-14C]glucose increased significantly in the presence of PES, indicating that the activity of the pentose shunt is less than maximal.     

Competition of glycerol with other oxidizable substrates in rat brain.

The rate of conversion of [1,3-14C]glycerol into 14CO2 was measured in the presence and absence of unlabelled alternative substrates in whole homogenates from the brains of young (4-6 and 18-20 days old) and adult rats. Unlabelled glucose decreased 14CO2 production from [1,3-14C]glycerol by about 40% at all ages studied. Unlabelled 3-hydroxybutyrate significantly decreased the 14CO2 production from both low (0.2 mM) and high (2.0 mM) concentrations of glycerol in 4-6- and 18-20-day-old rat pups. However, the addition of 3-hydroxybutyrate had no effect on the rate of 14CO2 production from 2.0 mM-glycerol in adult rats, suggesting that the interaction of 3-hydroxybutyrate with glycerol in adult rat brain is complex and may be related to the biphasic kinetics previously reported for glycerol oxidation. Unlabelled glutamine decreased the production of 14CO2 by brain homogenates from 18-20-day-old and adult rats, but not in 4-6-day-old rat pups. In the converse situation, the addition of unlabelled glycerol to whole brain homogenates had little effect on the rate of 14CO2 production from [6-14C]glucose, 3-hydroxy[3-14C]butyrate and [U-14C]glutamine, although some significant differences were noted. Collectively these results suggest that glycerol and these other substrates may be metabolized in separate subcellular compartments in brain such that the products of glucose, 3-hydroxybutyrate and glutamine metabolism can dilute the oxidation of glycerol, but the converse cannot occur. The data also demonstrate that there are complex age-related changes in the interaction of glycerol with 3-hydroxybutyrate and glutamine. The fact that glycerol oxidation was only partially suppressed by the addition of 1-5 mM-glucose, -3-hydroxybutyrate or -glutamine could also suggest that glycerol may be selectively utilized as an energy substrate in some discrete brain region.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

The pentose cycle and insulin release in mouse pancreatic islets

1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.     

The pentose phosphate pathway in rabbit liver. Studies on the metabolic sequence and quantitative role of the pentose phosphate cycle by using a system in situ

1. The reactions of the pentose phosphate cycle were investigated by the intraportal infusion of specifically labelled [(14)C]glucose or [(14)C]ribose into the liver of the anaesthetized rabbit. The sugars were confined in the liver by haemostasis and metabolism was allowed to proceed for periods up to 5min. Metabolism was assessed by measuring the rate of change of the specific radioactivity of CO(2), the carbon atoms of glucose 6-phosphate, fructose 6-phosphate and tissue glucose. 2. The quotient oxidation of [1-(14)C]glucose/oxidation of [6-(14)C]glucose as measured by the incorporation into respiratory CO(2) was greater than 1.0 during most of the time-course and increased to a maximum of 3.1 but was found to decrease markedly upon application of a glucose load. 3. The estimate of the pentose phosphate cycle from C-1/C-2 ratios generally increased during the time-course, whereas the estimate of the pentose phosphate cycle from C-3/C-2 ratios varied depending on whether the ratios were measured in glucose or hexose 6-phosphates. 4. The distribution of (14)C in hexose 6-phosphate after the metabolism of [1-(14)C]ribose showed that 65-95% of the label was in C-1 and was concluded to have been the result of a rapidly acting transketolase exchange reaction. 5. Transaldolase exchange reactions catalysed extensive transfer of (14)C from [2-(14)C]glucose into C-5 of the hexose 6-phosphates during the entire time-course. The high concentration of label in C-4, C-5 and C-6 of the hexose 6-phosphates was not seen in tissue glucose in spite of an unchanging rate of glucose production during the time-course. 6. It is concluded that the reaction sequences catalysed by the pentose phosphate pathway enzymes do not constitute a formal metabolic cycle in intact liver, neither do they allow the definition of a fixed stoicheiometry for the dissimilation of glucose.     

Anomeric specificity of glucose metabolism in the pentose cycle

The production of 3H2O from alpha- and beta-D-[5-3H]glucose and that of 14CO2 from either alpha- and beta-D-[1-14C] or alpha- and beta-D-[6-14C]glucose were measured in rat pancreatic islets and tumoral insulin-producing cells incubated at 7 degrees C. The ratio in 14CO2 output from D-[1-14C]glucose/D-[6-14C]glucose, the fraction of glucose metabolism occurring through the pentose cycle, and the flow rate through such a cycle were always higher in the presence of beta- than alpha-D-glucose. This indicates that the anomeric specificity of glucose-6-phosphate dehydrogenase is operative in intact islet cells.     

Metabolism of radiolabeled glucose by mouse oocytes and oocyte-cumulus cell complexes.

This study was carried out to examine the metabolism of [1-14C]-, [6-14C]-, and [5-3H]glucose by oocyte-cumulus cell complexes (OCC) and denuded oocytes (DO) and to test the hypothesis that metabolism of glucose through the pentose phosphate pathway is associated with meiotic induction. OCC or DO were cultured in hanging drops suspended from the cap of a microfuge tube, with NaOH serving as a trap to collect released 3H2O or 14CO2. Preliminary experiments established that this culture system supports both spontaneous and ligand-induced meiotic maturation. An initial time course experiment (1.5-6 h) showed that hypoxanthine-treated OCC from eCG-primed animals metabolized glucose principally via glycolysis, with an increase to 2.7-fold in response to FSH. Though more [1-14C]glucose was oxidized than [6-14C]glucose, its metabolism was about two orders of magnitude less than that of [5-3H]glucose. Also, FSH significantly increased oxidation of [1-14C]glucose but not [6-14C]glucose, indicating a preferential activation of the pentose phosphate pathway. Pyrroline carboxylate, an activator of the pentose phosphate pathway, increased the activity of this pathway to over 2-fold but failed to affect glucose oxidation through the tricarboxylic acid cycle. Glycolytic metabolism was increased by 25%. The addition of pyruvate to pyruvate-free medium resulted in significant reduction in the metabolism of all three glucose analogues. In OCC retrieved from hCG-injected, primed mice and cultured under hormone-free conditions, metabolic responses were similar to those in FSH-treated complexes cultured in hypoxanthine. DO metabolized glucose, but at a much reduced rate when compared to OCC. Pyruvate reduced the consumption of all three glucose analogues by DO. Pyrroline carboxylate reduced [5-3H]glucose metabolism by DO but had little effect on [1-14C]- and [6-14C]glucose oxidation. These data demonstrate metabolism of glucose by both DO and OCC, but reveal that cumulus cells are more active than the oocyte in this regard. In addition, induction of maturation by FSH, hCG, or pyrroline carboxylate was accompanied by a significant increase in the oxidation of [1-14C]glucose but not [6-14C]glucose by OCC, supporting a proposed role for the pentose phosphate pathway in meiotic induction.     

Effect of alloxan-diabetes and treatment with anti-insulin serum on pathways of glucose metabolism in lactating rat mammary gland

1. The overall metabolic changes in lactating mammary gland in alloxan-diabetic and anti-insulin-serum-treated rats were assessed by measurement of the incorporation of (14)C from specifically labelled glucose, pyruvate and acetate into carbon dioxide and lipid, together with measurements of enzymes concerned with the pentose phosphate pathway and with citrate metabolism. 2. Alloxan-diabetes depressed the rate of formation of (14)CO(2) from [1-(14)C]glucose and [2-(14)C]glucose to approx. 10% of the control rate; this was partially reversed by addition of insulin in vitro. The quotient Oxidation of [1-(14)C]glucose/Oxidation of [6-(14)C]glucose fell from a value of 17.6 in the control group to 3.9 in the diabetic group and was restored to 14.3 in the presence of insulin in vitro. In keeping with these results it was shown that glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were significantly decreased in alloxan-diabetic rats. 3. Alloxan-diabetes depressed the decarboxylation and the oxidation of labelled pyruvate, but not the oxidation of labelled acetate. 4. The synthesis of lipid from specifically labelled glucose was greatly decreased, that from [2-(14)C]pyruvate was almost unchanged and that from [1-(14)C]acetate alone was increased in alloxandiabetic rats. However, the stimulation of lipid synthesis from acetate by glucose was small in the alloxan-diabetic rats compared with the controls. Insulin in vitro partially reversed all these effects. Both citrate-cleavage enzyme and acetate thiokinase activities were decreased in alloxan-diabetic rats. 5. Treatment of rats with anti-insulin serum depressed the formation of (14)CO(2) from [1-(14)C]glucose and [2-(14)C]glucose, but increased that from [6-(14)C]glucose. This was completely restored by the presence of insulin in vitro. The quotient Oxidation of [1-(14)C]glucose/Oxidation of [6-(14)C]glucose fell from a value of 17.6 in the control group to 3.8 in the anti-insulin-serum-treated group. There were no changes in the activity of glucose 6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but the hexokinase distribution changed and the content of the soluble fraction increased significantly. 6. The synthesis of lipid from specifically labelled glucose was depressed in anti-insulin-serum-treated rats; this effect was completely reversed by addition of insulin in vitro to the tissue slices.