The redistribution of carbon label by the reactions involved in glycolysis, gluconeogenesis and the tricarboxylic acid cycle in rat liver

A scheme is presented that shows how the reactions involved in gluconeogenesis, glycolysis and the tricarboxylic acid cycle are linked in rat liver. Equations are developed that show how label is redistributed in aspartate, glutamate and phosphopyruvate when it is introduced as specifically labelled pyruvate or glucose either at a constant rate (steady-state theory) or at a variable rate (non-steady-state theory). For steady-state theory the fractions of label introduced as specifically labelled pyruvate that are incorporated into glucose and carbon dioxide are also given, and for both theories the specific radioactivities of aspartate and glutamate relative to the specific radioactivity of the substrate. The theories allow for entry of label into the tricarboxylic acid cycle via both oxaloacetate and acetyl-CoA, for (14)CO(2) fixation and for loss of label from the tricarboxylic acid cycle in glutamate, but not for losses in citrate. They also allow for incomplete symmetrization of label in oxaloacetate due to incomplete equilibration with fumarate both in the extramitochondrial part of the cell and in the mitochondrion on entry of oxaloacetate into the tricarboxylic acid cycle. In the latter case failure both of oxaloacetate to equilibrate with malate and of malate to equilibrate with fumarate are considered.     

Disequilibrium in the malate dehydrogenase reaction in rat liver mitochondria in vivo

1. When [2-(14)C]pyruvate is injected into rats the C3-position of liver glutamate becomes more heavily labelled than the C2-position, thus establishing that oxaloacetate and fumarate are not in equilibrium in rat liver mitochondria in vivo. The amount of disequilibrium was shown to be simply related to the value that the C3-label/C2-label ratio would have were no label recycled. This ratio, z, was calculated for post-absorptive rats in environmental temperatures of 20 degrees and 30 degrees C from determinations of the distribution of label within glutamate 1, 3 and 10min after intravenous injection of [2-(14)C]pyruvate. The values of z (best estimate and range) were 1.65 (1.60-1.69) in rats at 20 degrees C and 2.43 (2.23-2.63) in rats at 30 degrees C. These values of z imply the following rates of interconversion in mitochondria of fumarate and oxaloacetate (in terms of the oxaloacetate-->citrate flux, R) in rats at 20 degrees C: [Formula: see text] and in rats at 30 degrees C: [Formula: see text] 2. The kinetic parameters of malate dehydrogenase and fumarate hydratase and the intramitochondrial concentrations of NAD(+) and NADH under (as far as could be judged) conditions in vivo were collated. From them and the best estimates of R now available were calculated the rates of interconversion of fumarate, malate and oxaloacetate required to give the found values of z. These rates showed that the fumarate hydratase reaction was nearly in equilibrium, but that the malate dehydrogenase reaction was considerably out of equilibrium. The calculations also led to the following conclusions. 3. In livers of rats at 20 degrees and 30 degrees C mitochondrial malate concentrations were respectively about 5 and 1.5 times mean cellular concentrations. 4. Mitochondrial oxaloacetate concentrations were less than 0.2 of the mean cellular concentrations. They were also only 0.65 and 0.55 of the equilibrium concentrations for the malate dehydrogenase reaction in rats at 20 degrees and 30 degrees C respectively. 5. Malate dehydrogenase activity was low because of the very low oxaloacetate concentrations in the mitochondria and the very small fraction of the enzyme complexed with NAD(+), i.e. in each direction one substrate concentration was very sub-optimal.     

A 13C-n.m.r. investigation of the metabolism of amino acids in renal proximal convoluted tubules of normal and streptozotocin-treated rats and rabbits.

13C-n.m.r. spectroscopy was used to determine the metabolic fate of alanine and aspartate in rat and rabbit kidney proximal tubules. The main purpose of the present study was to investigate the effect of streptozotocin-induced diabetes on the influx of 13C label from [3-13C]alanine into the tricarboxylic acid cycle and through the fructose-1,6-bisphosphatase pathway. This influx was calculated from the relative enrichment of 13C in the various glutamate and glutamine carbon atoms. The relative proportion of 13C label which entered the tricarboxylic acid cycle via pyruvate carboxylase relative to the proportion that entered via pyruvate dehydrogenase was 1.92 +/- 0.02 in fed control rats and 2.27 +/- 0.04 in streptozotocin-treated rats. However, streptozotocin-induced diabetes did not significantly affect this ratio in rabbit proximal convoluted tubular cells. Only in rat proximal convoluted tubular cells did we observe an increase in flux through the fructose-1,6-bisphosphatase pathway by streptozotocin treatment compared with fed controls. The data suggest that streptozotocin-induced diabetes in rats causes the same metabolic changes as does chronic acidosis.     

Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.     

Influence of oxalate on the rate of the tricarboxylic acid cycle in rat hepatocytes

In hepatocytes isolated from fed rats, physiological concentrations of oxalate lower the flux through the tricarboxylic acid cycle (-48%) and reduce the steady-state levels of oxaloacetate and other Krebs cycle intermediates. All the metabolic modifications observed are explained by pyruvate carboxylase inhibition, since oxalate hardly modifies the flux through pyruvate dehydrogenase.     

The tricarboxylic acid cycle in Dictyostelium discoideum. Metabolite concentrations, oxygen uptake and 14c-labelled amino acid labelling patterns.

Some aspects of tricarboxylic acid-cycle activity during differentiation and aging in Dictyostelium discoideum were examined. The concentrations of glutamate, aspartate, alanine, citrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, pyruvate and acetyl-CoA were determined at four stages over the course of differentiation. The rate of O2 utilization was also determined over differentiation. In addition, experiments are described in which the specific radioactivities of citrate, 2-oxoglutarate, succinate, fumarate and malate were determined during a 30 min labelling of cells from the preculmination stage of development with [14C]glutamate, [14C]aspartate or [14C]alanine. A similar experiment was also performed with cells from the aggregation stage of development using [14C]glutamate.     

Factors affecting the survival of Neisseria sicca

Cultures of Neisseria sicca incubated at 37 degrees C died rapidly (within 36 h) after growth ceased. Re-suspending cells in a brain heart infusion broth and storing at 4 degrees C greatly reduced the rate of decline in viability (decimal reduction time 6 days). An important factor in maintaining viability was apparently the presence of external energy source(s). Survival comparable to that in broth was obtained by incubation in Ringer's solution with pyruvate plus glucose (but not with pyruvate or glucose alone). Medium pH had little effect on survival in the range pH 7.0 to 8.5. Energy sources also promoted survival of cells in Ringer's solution or a buffered salts solution at 37 degrees C. Highest levels of survival (up to 30% at 24 h) were obtained with pyruvate, lactate, proline and glutamate. A number of other amino acids and the tricarboxylic acid cycle intermediates, isocitrate, oxoglutarate, succinate, fumarate, malate and oxaloacetate, enhanced survival to a lesser extent.     

The effects of adenine nucleotides on pyruvate metabolism in rat liver

1. The effects of adenine nucleotides on pyruvate metabolism by isolated liver cells and isolated mitochondria have been investigated. The amount of pyruvate carboxylated has been estimated by determining the tricarboxylic acid-cycle intermediates, glutamate and aspartate accumulating in the incubation medium. The extent of pyruvate oxidation has been assessed by measuring oxygen uptake and the yield of ¹⁴CO₂ from [1-¹⁴C]pyruvate and [2-¹⁴C]pyruvate. 2. When catalytic amounts of adenine nucleotides (1–2mm) were added to suspensions of isolated liver cells incubated with pyruvate an ATP:ADP ratio greater than 6:1 was maintained. Both pyruvate oxidation to acetyl-CoA and the oxidation of acetyl-CoA through the tricarboxylic acid cycle were stimulated but pyruvate carboxylation was not affected. The production of acetyl-CoA exceeded the capacity of the cells for the oxidation of acetyl-CoA and the excess was converted into ketone bodies. 3. If a low ATP:ADP ratio was maintained in isolated cells or mitochondria by incubating them with dinitrophenol or hexokinase, pyruvate carboxylation was grossly inhibited, oxygen uptake depressed and ketone-body formation stimulated. Measurement of oxaloacetate concentrations confirmed that under these conditions oxaloacetate was rate-limiting for the oxidation of acetyl-CoA via the tricarboxylic acid cycle. The inclusion in the incubation medium of fumarate (1·25mm) completely prevented the ketogenic action of dinitrophenol or hexokinase. 4. When ADP (5mm) was added to a suspension of isolated liver cells incubated with pyruvate an actual ADP concentration of about 1mm was attained. This brought about effects on pyruvate metabolism similar to those obtained with dinitrophenol or hexokinase. 5. These results support the concept that the relative concentrations of adenine nucleotides within the liver cell may play a role in governing the rates of pyruvate oxidation and carboxylation. In addition, they provide further evidence that the availability of oxaloacetate in the liver cell can play a key role in determining whether acetyl-CoA arising from pyruvate is oxidized through the tricarboxylic acid cycle or converted into ketone bodies.     

13C Nuclear Magnetic Resonance Evidence for γ-Aminobutyric Acid Formation via Pyruvate Carboxylase in Rat Brain: A Metabolic Basis for Compartmentation0

The compartmentation of amino acid metabolism is an active and important area of brain research. 13C labeling and 13C nuclear magnetic resonance (NMR) are powerful tools for studying metabolic pathways, because information about the metabolic histories of metabolites can be determined from the appearance and position of the label in products. We have used 13C labeling and 13C NMR in order to investigate the metabolic history of gamma-aminobutyric acid (GABA) and glutamate in rat brain. [1-13C]Glucose was infused into anesthetized rats and the 13C labeling patterns in GABA and glutamate examined in brain tissue extracts obtained at various times after infusion of the label. Five minutes after infusion, most of the 13C label in glutamate appeared at the C4 position; at later times, label was also present at C2 and C3. This 13C labeling pattern occurs when [1-13C]glucose is metabolized to pyruvate by glycolysis and enters the pool of tricarboxylic acid (TCA) intermediates via pyruvate dehydrogenase. The label exchanges into glutamate from the TCA cycle pool through glutamate transaminases or dehydrogenase. After 30 min of infusion, approximately 10% of the total 13C in brain extracts appeared in GABA, primarily (greater than 80%) at the amino carbon (C4), indicating that the GABA detected is labeled through pyruvate carboxylase. The different labeling patterns observed for glutamate and GABA show that the large detectable glutamate pool does not serve as the precursor to GABA. Our NMR data support previous experiments suggesting compartmentation of metabolism in brain, and further demonstrate that GABA is formed from a pool of TCA cycle intermediates derived from an anaplerotic pathway involving pyruvate carboxylase.     

Metabolic fate of fumarate, a side product of the purine salvage pathway in the intraerythrocytic stages of Plasmodium falciparum

In aerobic respiration, the tricarboxylic acid cycle is pivotal to the complete oxidation of carbohydrates, proteins, and lipids to carbon dioxide and water. Plasmodium falciparum, the causative agent of human malaria, lacks a conventional tricarboxylic acid cycle and depends exclusively on glycolysis for ATP production. However, all of the constituent enzymes of the tricarboxylic acid cycle are annotated in the genome of P. falciparum, which implies that the pathway might have important, yet unidentified biosynthetic functions. Here we show that fumarate, a side product of the purine salvage pathway and a metabolic intermediate of the tricarboxylic acid cycle, is not a metabolic waste but is converted to aspartate through malate and oxaloacetate. P. falciparum-infected erythrocytes and free parasites incorporated [2,3-(14)C]fumarate into the nucleic acid and protein fractions. (13)C NMR of parasites incubated with [2,3-(13)C]fumarate showed the formation of malate, pyruvate, lactate, and aspartate but not citrate or succinate. Further, treatment of free parasites with atovaquone inhibited the conversion of fumarate to aspartate, thereby indicating this pathway as an electron transport chain-dependent process. This study, therefore, provides a biosynthetic function for fumarate hydratase, malate quinone oxidoreductase, and aspartate aminotransferase of P. falciparum.     

Cataplerotic TCA cycle flux determined as glutamate-sustained oxygen consumption in primary cultures of astrocytes

Utilization of glucose by adult brain as its metabolic substrate does not mean that glutamate cannot be synthesized from glucose and subsequently oxidatively degraded. Between 10 and 20% of total pyruvate metabolism in brain occurs as formation of oxaloacetate (OAA), a tricarboxylic acid (TCA) cycle intermediate, from pyruvate plus CO(2). This anaplerotic ('pool-filling') process occurs in astrocytes, which in contrast to neurons express pyruvate carboxylase (PC) activity. Equivalent amounts of pyruvate are converted to acetylcoenzyme A and condensed with oxaloacetate to form citrate (Cit), which is metabolized to alpha-ketoglutarate (generating oxidatively-derived energy), glutamate and glutamine and transferred to neurons in the glutamate-glutamine cycle and used as precursor for transmitter glutamate. Since the blood-brain barrier is poorly permeable to glutamate and its metabolites, net synthesis of glutamate must be followed by degradation of equivalent amounts of glutamate, a cataplerotic ('pool-emptying') process, in which glutamate is converted in the TCA cycle to malate or oxaloacetate (generating additional energy), which exit the cycle to form one molecule pyruvate. To obtain an estimate of the rate of astrocytic oxidation of glutamate the rate of oxygen consumption was measured in primary cultures of mouse astrocytes metabolizing glutamate in the absence of other metabolic substrates. The observed rate is compatible with complete oxidative degradation of glutamate.     

Biochemical Aspects of Fumaric Acid Accumulation by Rhizopus arrhizus

The accumulation and excretion of fumaric acid, and to a lesser extent malic and succinic acids, by Rhizopus arrhizus occurs under aerobic conditions in a high-glucose medium containing a limiting amount of nitrogen and a neutralizing agent (CaCO₃). An overall four-carbon dicarboxylic acid molar yield of up to 145% (moles of acid produced per mole of glucose utilized) is obtained after incubation for 4 to 5 days. Evidence is presented that fumarate is synthesized from pyruvate via a carboxylation reaction yielding oxaloacetate, which is then converted to malate and further on to fumarate via the reductive reactions of the tricarboxylic acid cycle. The possible formation of fumarate from the normal (oxidative) operation of the tricarboxylic acid cycle was not excluded by the data. Yield, ¹³C nuclear magnetic resonance, and enzymatic activity studies were carried out in a strain of R. arrhizus which produces high levels of fumarate from glucose and carbonate. The observed high fumarate molar yield (greater than 100%) can therefore be explained in terms of the carboxylation of pyruvate and the operation of the reductive reactions of the tricarboxylic acid cycle under aerobic conditions.     

Effects of pentylenetetrazole and glutamate on metabolism of [U-(13)C]glucose in cultured cerebellar granule neurons.

This study was performed to analyze the effects of glutamate and the epileptogenic agent pentylenetetrazole (PTZ) on neuronal glucose metabolism. Cerebellar granule neurons were incubated for 2 h in medium containing 3 mM [U-(13)C]glucose, with and without 0.25 mM glutamate and/or 10 mM PTZ. In the presence of PTZ, decreased glucose consumption with unchanged lactate release was observed, indicating decreased glucose oxidation. PTZ also slowed down tricarboxylic acid (TCA) cycle activity as evidenced by the decreased amounts of labeled aspartate and [1,2-(13)C]glutamate. When glutamate was present, glucose consumption was also decreased. However, the amount of glutamate, derived from [U-(13)C]glucose via the first turn of the TCA cycle, was increased. The decreased amount of [1,2-(13)C]glutamate, derived from the second turn in the TCA cycle, and increased amount of aspartate indicated the dilution of label due to the entrance of unlabeled glutamate into TCA cycle. In the presence of glutamate plus PTZ, the effect of PTZ was enhanced by glutamate. Labeled alanine was detected only in the presence of glutamate plus PTZ, which indicated that oxaloacetate was a better amino acid acceptor than pyruvate. Furthermore, there was also evidence for intracellular compartmentation of oxaloacetate metabolism. Glutamate and PTZ caused similar metabolic changes, however, via different mechanisms. Glutamate substituted for glucose as energy substrate in the TCA cycle, whereas, PTZ appeared to decrease mitochondrial activity.     

The effects of the stress caused by experimental procedures on alanine aspartate, glutamate and glutamine in rat liver

Rats were stressed by intravenous injection, tail-warming or moderate restraint for 30s, i.e. by stresses imposed by normal handling during experiment. Liver glutamate concentrations were greatly affected. The results were substantially the same in two varieties of rat (Wistar and Sprague-Dawley), in two laboratories, in experiments carried out by two sets of workers, and after all three stresses. The following detailed results refer to Wistar rats. 1. In starved rats at 20 degrees C and 30 degrees C and in post-absorptive rats at 20 degrees C stress by injection raised liver glutamate concentrations from 1.54, 1.57 and 1.88mumol/g wet wt. 30s after injection to 3.4, 2.7 and 3.6mumol/g wet wt. respectively a few minutes later. In starved rats at 20 degrees C the concentration then fell slowly to 2.3mumol/g wet wt., in starved rats at 30 degrees C it remained steady, and in post-absorptive rats at 20 degrees C it rose slowly to about 4.3mumol/g wet wt. The final values seemed fairly steady and corresponded to an ;alert' state. 2. In starved rats at 20 degrees C anaesthesia, with or without injection or cannulation during it, raised glutamate concentrations to the ;alert' values, which were maintained for 2-3h. 3. Liver alanine concentration in post-absorptive rats initially fell from 1.5 to 0.8mumol/g, and then stayed fairly constant. 4. Aspartate and glutamine concentrations altered only in starved rats, and proportionately much less than those of glutamate. 5. The necessity for knowing the time-dependence of glutamate concentrations after experimental handling is emphasized. 6. There is no wholly satisfactory explanation of the observations.     

Metabolism of propionate by sheep liver: Pathway of propionate metabolism in aged homogenate and mitochondria

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-¹⁴C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of ¹⁴C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of ¹⁴C in dicarboxylic acids, citrate, α-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-¹⁴C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.     

The metabolic fate of lactate in renal cortical tubules

1. Isolated kidney cortex tubules prepared from fed rats and incubated with near-physiological concentrations of [¹⁴C]lactate decrease the specific radioactivity of the added lactate. This effect may be attributable to at least two mechanisms; formation of lactate from endogenous precursors, or entry of unlabelled carbon into the lactate pool as a result of substrate cycling, via phosphoenolpyruvate, pyruvate and oxaloacetate, together with equilibration of the oxaloacetate pool with malate and fumarate. Such substrate cycling could occur within a single cell, or between two populations of different cells, one glycolytic and the other gluconeogenic. These possibilities have been investigated by using metabolic inhibitors or alternative metabolic substrates. 2. Tubules from fed rats produced a fall in specific radioactivity of 14.4% when incubated for 40min with 2mm-lactate alone. A mathematical treatment of this result is presented, which allows the rate of fall in specific radioactivity to be expressed as the addition of unlabelled lactate to the pool. This corresponds to a rate of formation of unlabelled lactate of 121±22μmol/h per g dry wt., a rate close to that of gluconeogenesis. In tubules from fasting rats, there was no reduction of the specific radioactivity of lactate, indicating that fasting for 24h suppresses production of unlabelled-lactate carbon. 3. Addition of 2mm-fumarate resulted in a significantly greater decrease in the specific radioactivity of lactate, but aspartate (2mm), malate (2mm) and glucose (5mm) were without effect. Total inhibition of gluconeogenesis with 3-mercaptopicolinate did not prevent the fall in specific radioactivity of lactate observed in tubules from fed-rat kidney, thereby excluding significant activity of the substrate cycle pyruvate→oxaloacetate→phosphoenolpyruvate→pyruvate. 4. The capacity of pyruvate kinase under the test conditions in tubules prepared from kidneys of fed or starved rats was at least ten times higher than the observed rate of production of lactate, so that failure to observe recycling of lactate in starved-rat tubules indicates suppression of pyruvate kinase activity. 5. The endogenous glycogen and glucose content of isolated renal cortex tubules is too low to account for the dilution of label of lactate. Endogenous concentrations of glycerol and amino acids were also very low. As for glycogen, the possibility that very rapid turnover of these metabolites, in fed rats but not in starved rats, may account for formation of unlabelled lactate cannot be excluded. 6. It is concluded that substrate cycling via phosphoenolpyruvate does not occur to any significant extent in either fed or starved-rat kidney. In fed rats recycling of lactate carbon does occur and the rate of this reaction is similar to the rate of gluconeogenesis at physiological concentrations of lactate. The present results favour participation of oxaloacetate decarboxylase rather than `malic' enzyme in this cycle.     

13C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver

13C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13C enrichments at the individual carbons of glutamate when [3-13C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13C label into fatty acids was monitored in this liver preparation. Livers were perfused under steady-state conditions with labeled substrates that are converted to either [2-13C]acetyl-CoA or [1-13C]acetyl-CoA, which in the de novo synthesis pathway label alternate carbons in fatty acids. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1-13C]acetyl-CoA (from [2-13C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3-13C]alanine plus [2-13C]ethanol, which are converted to [2-13C]acetyl-CoA. Thus, measurement of 13C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

The regulation and glutamate metabolism by tricarboxylic acid-cycle activity in rat brain mitochondria

1. The interrelationship of metabolism of pyruvate or 3-hydroxybutyrate and glutamate transamination in rat brain mitochondria was studied. 2. If brain mitochondria are incubated in the presence of equimolar concentrations of pyruvate and glutamate and the K(+) concentration is increased from 1 to 20mm, the rate of pyruvate utilization is increased 3-fold, but the rate of production of aspartate and 2-oxoglutarate is decreased by half. 3. Brain mitochondria incubated in the presence of a fixed concentration of glutamate (0.87 or 8.7mm) but different concentrations of pyruvate (0 to 1mm) produce aspartate at rates that decrease as the pyruvate concentration is increased. At 1mm-pyruvate, the rate of aspartate production is decreased to 40% of that when zero pyruvate was present. 4. Brain mitochondria incubated in the presence of glutamate and malate alone produce 2-oxoglutarate at rates stoicheiometric with the rate of aspartate production. Both the 2-oxoglutarate and aspartate accumulate extramitochondrially. 5. Externally added 2-oxoglutarate has little inhibitory effect (K(i) approx. 31mm) on the production of aspartate from glutamate by rat brain mitochondria. 6. It is concluded that the inhibitory effect of increased C(2) flux into the tricarboxylic acid cycle on glutamate transamination is caused by competition for oxaloacetate between the transaminase and citrate synthase. 7. Evidence is provided from a reconstituted malate-aspartate (or Borst) cycle with brain mitochondria that increased C(2) flux into the tricarboxylic acid cycle from pyruvate may inhibit the reoxidation of exogenous NADH. These results are discussed in the light of the relationship between glycolysis and reoxidation of cytosolic NADH by the Borst cycle and the requirement of the brain for a continuous supply of energy.     

Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia

Oxidative decarboxylation of [1-¹⁴C]pyruvate was studied in primary cultures of neurons and of astrocytes. The rate of this process, which is a measure of carbon flow into the tricarboxylic acid (TCA) cycle and which is inhibited by its end product, acetyl CoA, was determined under conditions which would either elevate or reduce the components of the malate-aspartate shuttle (MAS). Addition of aspartate (1 mM) was found to stimulate pyruvate decarboxylation in astrocytes whereas addition of glutamate (or glutamine) had no effect. Since aspartate is a precursor for extramitochondrial malate, and thus intramitochondrial oxaloacetate, whereas glutamate and glutamine are not, this suggests that an increase in oxaloacetate level stimulates TCA cycle activity. Conversely, a reduction of the glutamate content by 3 mM ammonia, which might reduce exchange between glutamate and aspartate across the mitochondrial membrane, suppressed pyruvate decarboxylation. This effect was abolished by addition of glutamate or glutamine or exposure to methionine sulfoximine (MSO). These findings suggest that impairment of MAS activity by removal of MAS constituents decreases TCA cycle activity whereas replenishment of these compounds restores the activity of the TCA cycle. No corresponding effects were observed in neurons.     

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.