Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells.

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.

Gastrointestinal abnormalities in children with autistic disorder.

OBJECTIVES: Our aim was to evaluate the structure and function of the upper gastrointestinal tract in a group of patients with autism who had gastrointestinal symptoms. STUDY DESIGN: Thirty-six children (age: 5.7 +/- 2 years, mean +/- SD) with autistic disorder underwent upper gastrointestinal endoscopy with biopsies, intestinal and pancreatic enzyme analyses, and bacterial and fungal cultures. The most frequent gastrointestinal complaints were chronic diarrhea, gaseousness, and abdominal discomfort and distension. RESULTS: Histologic examination in these 36 children revealed grade I or II reflux esophagitis in 25 (69.4%), chronic gastritis in 15, and chronic duodenitis in 24. The number of Paneth's cells in the duodenal crypts was significantly elevated in autistic children compared with non-autistic control subjects. Low intestinal carbohydrate digestive enzyme activity was reported in 21 children (58.3%), although there was no abnormality found in pancreatic function. Seventy-five percent of the autistic children (27/36) had an increased pancreatico-biliary fluid output after intravenous secretin administration. Nineteen of the 21 patients with diarrhea had significantly higher fluid output than those without diarrhea. CONCLUSIONS: Unrecognized gastrointestinal disorders, especially reflux esophagitis and disaccharide malabsorption, may contribute to the behavioral problems of the non-verbal autistic patients. The observed increase in pancreatico-biliary secretion after secretin infusion suggests an upregulation of secretin receptors in the pancreas and liver. Further studies are required to determine the possible association between the brain and gastrointestinal dysfunctions in children with autistic disorder.

Lactate transport by cortical synaptosomes from adult rat brain: characterization of kinetics and inhibitor specificity.

Since lactate released by glial cells may be a key substrate for energy in neurons, the kinetics for the uptake of L-[U-14C]lactate by cortical synaptic terminals from 7- to 8-week-old rat brain were determined. Lactate uptake was temperature-dependent, and increased by 64.9% at pH 6.2, and decreased by 43.4% at pH 8.2 relative to uptake at pH 7.3. Uptake of monocarboxylic acids was saturable with increasing substrate concentration. Eadie-Hofstee plots of the data gave evidence of two carrier-mediated uptake mechanisms with a high-affinity Km of 0.66 mM and Vmax of 3.66 mM for pyruvate, and a low-affinity system with a Km of 9.9 mM for both lactate and pyruvate and Vmax values of 16.6 and 23.1 nmol/30 s/mg protein for lactate and pyruvate, respectively. Saturable uptake was seen in the presence of 10 mM alpha-cyano-4-hydroxycinnamate. Lactate transport by synaptic terminals was much more sensitive to inhibition by sulfhydryl reagents than transport in astrocytes. Addition of 0.5 and 2 mM mersalyl decreased the uptake of 1 mM lactate by synaptic terminals by 59.3 and 66.37%, respectively. Pyruvate moderately decreased lactate transport, whereas 3-hydroxybutyrate had little effect. Quercetin, an inhibitor of lactate release, had little effect on the content of 14C lactate in synaptic terminals, supporting the concept that the majority of lactate produced within brain is from glial cells. Oxidation of L-[U-14C]lactate by synaptosomes was saturable, and yielded a Km of 1.23 mM and a Vmax of 116 nmol/h/mg protein. Overall the studies show that synaptic terminals from adult brain have a high capacity for transport and oxidation of lactate, consistent with the proposed role for this compound in metabolic trafficking in brain. Furthermore, the data provide kinetic evidence of two carrier-mediated mechanisms for monocarboxylic acid transport by synaptosomes and demonstrate that uptake of lactate by synaptic terminals is regulated differently than transport by astrocytes. Uptake of lactate by synaptic terminals also has differences from the systems described for neurons.

Compartmentation of [14C]glutamate and [14C]glutamine oxidative metabolism in the rat hippocampus as determined by microdialysis.

Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 +/- 18 and 2.1 +/- 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-14C]glutamate and [U-14C]glutamine were 408 +/- 8 and 387 +/- 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.

Improved social and language skills after secretin administration in patients with autistic spectrum disorders.

We report three children with autistic spectrum disorders who underwent upper gastrointestinal endoscopy and intravenous administration of secretin to stimulate pancreaticobiliary secretion. All three had an increased pancreaticobiliary secretory response when compared with nonautistic patients (7.5 to 10 mL/min versus 1 to 2 mL/min). Within 5 weeks of the secretin infusion, a significant amelioration of the children's gastrointestinal symptoms was observed, as was a dramatic improvement in their behavior, manifested by improved eye contact, alertness, and expansion of expressive language. These clinical observations suggest an association between gastrointestinal and brain function in patients with autistic behavior.

Effect of alpha-ketoisocaproate and leucine on the in vivo oxidation of glutamate and glutamine in the rat brain.

Leucine and alpha-ketoisocaproate (alpha-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or alpha-KIC on the oxidation of L-[U-14C]glutamate and L-[U-14C]glutamine in the brain were determined in the non-anesthetized rat. 14CO2 generated by the metabolic oxidation of [14C]glutamate and [14C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and alpha-KIC exhibited differential effects on 14CO2 generation from radioactive glutamate on glutamine. Infusion of 0.5 mM alpha-KIC increased [14C]glutamate oxidation approximately 2-fold; higher concentrations of alpha-KIC did not further stimulate [14C]glutamate oxidation. The enhanced oxidation of [14C]glutamate may be attributed to the function of alpha-KIC as a nitrogen acceptor from [14C]glutamate yielding [14C]alpha-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [14C]glutamine oxidation was not stimulated as much as [14C]glutamate oxidation and only increased at 10 mM alpha-KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [14C]glutamate or [14C]glutamine. In maple syrup urine disease elevated alpha-KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.

The role of glutamine and other alternate substrates as energy sources in the fetal rat lung type II cell.

Glucose has been thought to be the primary substrate for energy metabolism in the developing lung; however, alternate substrates are used for energy metabolism in other organs. To examine the role of alternate substrates in the lung, we measured rates of oxidation of glutamine, glucose, lactate, and 3-hydroxybutyrate in type II pneumocytes isolated from d 19 fetal rat lungs by measuring the production of 14CO2 from labeled substrates. Glutamine had a rate of 24.36 +/- 4.51 nmol 14CO2 produced/ h/mg of protein (mean +/- SEM), whereas lactate had a significantly higher rate, 40.29 +/- 4.42. 3-Hydroxybutyrate had a rate of 14.91 +/- 1.93. The rate of glucose oxidation was 2.13 +/- 0.36, significantly lower than that of glutamine. To examine the interactions of substrates normally found in the intracellular milieu, we measured the effect of unlabeled substrates as competitors on labeled substrate. This identifies multiple metabolic compartments of energy metabolism. Glucose, but not lactate, inhibited the oxidation of glutamine, suggesting a compartmentation of tricarboxylic acid cycle activity, rather than simple dilution by glucose. Glucose and lactate had reciprocal inhibition. Our data suggest at least two separate compartments in the type II cells for substrate oxidation, one for glutamine metabolism and a second for glucose metabolism. In summary, we have documented that glutamine and other alternate substrates are oxidized preferentially over glucose for energy metabolism in the d 19 fetal rat lung type II pneumocyte. In addition, we have delineated some of the compartmentation that occurs within the developing type II cell, which may determine how these substrates are used.

The role of glutamine as an energy source in the developing rat lung.

Because multiple substrates have been shown to play a role in the metabolic homeostasis of different tissues, a series of studies were initiated to examine the role of alternate substrates in the lung. In these studies, we measured rates of oxidation of glutamine, glucose, lactate and 3-hydroxybutyrate in fibroblasts isolated from d 19 fetal rat lungs by measuring the production of 14CO2 from labeled substrates and compared them with earlier studies of isolated Type II cells. The rate of glutamine oxidation was 16.04 nmol 14CO2 x mg protein(-1) x hr(-1) in the fibroblasts compared with 24.36 in Type II cells. Three-hydroxybutyrate had a rate of 10.75 in the fibroblasts and 14.9 in the Type II cells. Lactate oxidation in fibroblasts was similar to that of glutamine, with a rate of 18.49; however, in Type II cells the rate of lactate oxidation was significantly higher at 40.29. Glucose was oxidized at a rate significantly lower than the other three substrates. In the fibroblasts, that rate was 1.22 and in Type II cells it was 2.13. To examine the interactions of substrates normally found in the intracellular milieu, we measured the effect of unlabeled substrates as competitors on labeled substrate in the fibroblasts, similar to our studies with Type II cells that identified multiple metabolic compartments of energy metabolism in these cell populations. Glucose, but not lactate, inhibited the oxidation of glutamine, suggesting a compartmentation of tricarboxylic acid cycle activity rather than simple dilution by glucose. Glucose and lactate had reciprocal inhibition in the Type II cells. Our data suggest at least two separate compartments in developing lung cells for substrate oxidation: one for glutamine metabolism and a second for glucose metabolism. In summary, we have documented that glutamine and other alternate substrates are oxidized preferentially over glucose for energy metabolism in the d 19 fetal rat lung.

Use of intracellular versus extracellular specific activities in calculation of glutamine metabolism in astrocytes: effect of dibutyryl cyclic AMP.

The rate of glutaminase-dependent metabolism of glutamine in intact astrocytes was determined under conditions in which the extracellular concentration of glutamine was varied between 0.2 and 3.2 mM glutamine for control and dibutyryl cyclic AMP (dBcAMP)-treated cells. Glutamine metabolism by intact cells increased with increasing extracellular glutamine when calculations were based on the extracellular specific activity of glutamine. However, when the rate was based on the intracellular specific activity of glutamine, the rate of glutamine metabolism was independent of the media glutamine concentration. Similar results were obtained when cells were treated with dBcAMP, although the rates were approximately twice as high compared to untreated cells. The rate of formation of 14CO2 from [1-14C]glutamine and [1-14C]glutamate, based on the extracellular specific activities, were 93 +/- 5 and 40 +/- 4 nmol/mg protein/h, respectively. Oxidation rates based on the experimentally determined intracellular specific activity of glutamine and glutamate were 144 +/- 8 and 209 +/- 18 nmol/mg protein/h, respectively. In dBcAMP-treated astrocytes, the oxidation rates were higher than in untreated cells. These studies demonstrate that determination of the specific activity of compounds inside the cell aids in the interpretation of metabolic studies with intact cells and that both the initial steps of glutamine metabolism and the rate of 14CO2 formation from 14C-glutamine via the TCA cycle were increased in dBcAMP-treated astrocytes.

New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes.

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.

Elevation of amino acids in the interstitial space of the rat brain following infusion of large neutral amino and keto acids by microdialysis: alpha-ketoisocaproate infusion.

alpha-Ketoisocaproate was infused into the brain of free-moving, awake rats by microdialysis to create a microenvironment similar to that found in maple syrup urine disease. The eluate of the probe was analyzed for amino acids to determine if alpha-ketoisocaproate was transaminated to leucine and if the amino acid homeostasis was altered. The interstitial levels of leucine were increased up to 11-fold and other large neutral amino acids were increased 2- to 3-fold indicating an active branched chain keto acid transaminase activity and enhanced hetero-exchange across cell membranes. The elevation of large neutral amino acids in the interstitial space is discussed in terms of the synthesis of leucine and neurotransmitters in maple syrup urine disease.

Elevation of amino acids in the interstitial space of the rat brain following infusion of large neutral amino and keto acids by microdialysis: leucine infusion.

A microenvironment similar to that found in maple syrup urine disease was created in the brain of free-moving, awake rats by the infusion of leucine into the brain using microdialysis. To determine the effects on amino acid homeostasis the eluate of the probe was analyzed. Perfusion with leucine elevated the interstitial levels of large neutral amino acids suggesting hetero exchange of large neutral amino acids from neuronal cells into the interstitial space. The data also demonstrated the inter relationship of leucine and glutamine, both of which may be nitrogen sinks in the brain. Elevation of large neutral amino acids in the interstitial space suggests a decreased concentration in neurons which might have an effect on the synthesis of serotonin and catecholamines and suggests a mechanism by which elevated leucine may affect neuronal function in maple syrup urine disease.

Modulation of central nervous system metabolism by macromolecules: effects of albumin and histones on glucose oxidation by synaptosomes.

Since increasing evidence suggests that several proteins play a significant role in the regulation of glucose oxidation in the central nervous system, a series of experiments was designed to determine the specific proteins involved and to delineate their possible mode of action. In these studies, the rate of substrate oxidation by isolated synaptosomes in vitro was determined by measuring the production of [14C]carbon dioxide from labeled compounds in the presence and absence of the added protein. In the initial experiments, an examination of a broad selection of pure proteins revealed that only albumin (bovine serum albumin [BSA]) or histones (at concentrations of 100 micrograms/mL or less) exhibited an inhibitory effect of greater than 60% on the rate of glucose oxidation. Furthermore, isolated cell fractions P1 (nuclei and cellular debris), P2 (mitochondria, synaptosomes, and myelin), and other membrane proteins had little or no effect on the rate of [14C]carbon dioxide production from [6(14)C]glucose. When either BSA or histones were treated with trypsin, the inhibitory effects were eliminated. To determine whether these effects were related to changes in substrate transport, we measured the rate of glucose uptake by synaptosomes using [6(14)C]glucose, [1,2-3H]2-deoxyglucose, and [3H]3-O-methylglucose in the presence of 5% serum protein. These experiments revealed that the rate of glucose transport was not affected by serum proteins. Collectively, these results indicate that albumin and histones attenuate the rate of glucose oxidation by synaptosomes. The results also support the conclusion that the intact protein molecule is required for this inhibition, since treatment with trypsin abolished this effect. It can also be concluded that this effect is not at the site of transport and that the protein(s) are acting either directly at intercellular site(s) or indirectly via specific messengers.

Regulation of mitochondrial and cytosolic malic enzymes from cultured rat brain astrocytes.

Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is converted to pyruvate and CO2 via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37 degrees C in the presence of 0.5 mM malate was 4.15 +/- 0.47 and 11.61 +/- 0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean +/- SEM, n = 18-19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. alpha-Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.

Transport of 3-hydroxybutyrate by cultured rat brain astrocytes.

It is well established that 3-hydroxybutyrate can serve as an energy source for the brain. Since substrate utilization may be regulated in part by transport across the cellular membrane, we investigated the uptake of 3-hydroxybutyrate by primary cultures of rat brain astrocytes. Measurement of the net uptake indicated a saturable system and a Lineweaver-Burke type plot was consistent with a single carrier-mediated mechanism with a Km of 6.03 mM and a Vmax of 32.7 nmol/30 seconds/mg protein. The rate of uptake at pH 6.2 was more than ten times the rate at pH 8.2, with the rate at pH 7.4 being intermediate between these values, suggesting the possibility of cotransport with H+ or exchange with OH- (antiport). Mersalyl had only a slight effect on the transport of 3-hydroxybutyrate, suggesting that sulfhydryl groups are not involved in the transport of this monocarboxylic acid. Phenylpyruvate and alpha-ketoisocaproate also attenuated the transport, but lactate had only a marginal effect. These results suggest that the utilization of 3-hydroxybutyrate as an energy source by astrocytes is regulated in part by carrier-mediated transport and that the uptake system is different from the lactate transport system.

Malic enzyme activity in adult and newborn rat lung.

Using a sensitive technique measuring 14CO2 production from radiolabeled malate, we examined malic enzyme activity in both adult and newborn rat lung tissue and in L2 cells, a cell culture line of type II pneumocytes. Malic enzyme was present in both cytosolic and mitochondrial fractions. Time course experiments demonstrated a linear rate after the initial 10 min, up to 30 min. The optimal pH in the cytosolic fraction was 8.0, whereas maximal mitochondrial malic enzyme activity occurred at pH 7.0. The mitochondrial fraction exhibited biphasic kinetics over the 200-fold range of concentrations examined. The high-affinity Km was 0.16 mmol with Vmax of 7.11 nmol/mg protein/min. The low-affinity Km was 6.95 mmol, with Vmax of 31.82 nmol/mg protein/min. In the cytosol there was a single Km of 0.30 mmol and Vmax of 5.95 nmol/mg protein/min. In paired experiments examining differences between 1-d-old and adult rat lung, significantly higher total and mitochondrial malic enzyme activity occurred in the newborn as compared with the adult. Malic enzyme activity was also present in the L2 cells. The finding of malic enzyme activity in the lung suggests that cytosolic malic enzyme may play a role in generating NADPH needed in the lung for fatty acid synthesis. These findings of developmental differences in malic enzyme activity suggest that alternate substrates such as anaplerotic amino acids may be used in the young animal as energy substrates by way of the tricarboxylic acid cycle.

Energy metabolism in cortical synaptic terminals from weanling and mature rat brain: evidence for multiple compartments of tricarboxylic acid cycle activity.

It is well documented that the brain preferentially utilizes alternative substrates for energy during brain development; however, less is known about the use of these substrates by synaptic terminals. The present study compared the rates of 14CO2 production from 1 mM D-[6-14C]glucose, L-[U-14C]glutamine, D-3-hydroxy[3-14C]butyrate, L-[U-14C]lactate and L-[U-14C]malate by synaptic terminals isolated from 17- to 18-day-old and 7- to 8-week-old rat brain. The rates of 14CO2 production from glucose, glutamine, 3-hydroxybutyrate, lactate and malate were 8.55 +/- 0.78, 25.90 +/- 4.58, 42.28 +/- 3.54, 48.42 +/- 2.09, and 9.31 +/- 1.61 nmol/h/mg protein (mean +/- SEM), respectively, in synaptic terminals isolated from 17- to 18-day-old rat brain and 12.95 +/- 1.64, 30.62 +/- 4.19, 16.09 +/- 2.62, 40.33 +/- 6.77, and 8.25 +/- 1.69 nmol/h/mg protein (mean +/- SEM), respectively, in synaptic terminals isolated from 7- to 8-week-old rat brain. In competition studies using unlabelled added substrates, the addition of 3-hydroxybutyrate, lactate or glutamine greatly decreased the rate of 14CO2 production from labelled glucose. Added unlabelled glucose increased the rate of 14CO2 production from 3-hydroxybutyrate in synaptic terminals from 7- to 8-week-old rat brain, but had no effect on 14CO2 production from any other substrates. Lactate also increased 14CO2 production from 3-hydroxybutyrate at 7-8 weeks, whereas the addition of 3-hydroxybutyrate decreased 14CO2 production from lactate only in synaptic terminals from 17- to 18-day-old rat brain. None of the added substrates altered the rate of 14CO2 production from labelled glutamine or malate suggesting that these substrates are metabolized in relatively distinct compartments within synaptic terminals. Overall the data demonstrate that synaptic terminals from both weanling and adult rat brain can utilize a variety of substrates for energy. In addition, the competition studies demonstrate that the interactions of substrates change with age and suggest that there are multiple compartments of energy metabolism (or tricarboxylic acid cycle activity) in isolated synaptic terminals.

A glutamatergic mechanism for aluminum toxicity in astrocytes.

The effect of aluminum on the metabolism of glutamate and glutamine in astrocytes was studied to provide information about a possible biochemical mechanism for aluminum neurotoxicity and its potential contribution to neurodegenerative disease. Exposure of cultured rat brain astrocytes for 3-4 d to 5-7.5 mM aluminum lactate increased glutamine synthetase activity by 100-300% and diminished glutaminase activity by 50-85%. Increased glutamine synthetase enzyme activity was accompanied by an elevated level of glutamine synthetase mRNA. Alterations in glutaminase and glutamine synthetase following aluminum exposure caused increased intracellular glutamine levels, decreased intracellular glutamate levels, and increased conversion of glutamate to glutamine and the release of the latter into the extracellular space. The results of these changes may alter the availability of neurotransmitter glutamate in vivo and may be a mechanism for the aluminum neurotoxicity observed in individuals exposed to the metal during dialysis procedures and other situations.

Regulation of malate dehydrogenases from neonatal, adolescent, and mature rat brain.

Since the malate-aspartate shuttle in brain has been shown to be closely linked to brain energy metabolism and neurotransmitter synthesis, the activity of MDH, one of the enzymes of the malate-aspartate shuttle, was studied in cortical non-synaptic mitochondria (mMDH) and cytosol (cMDH) in 1-4 day, 18-20 day and 7-8 week old rats. The mean mMDH activity (nmol/min/mg protein) was 10,517 +/- 734 (mean +/- SEM), 8,882 +/- 241 and 10,323 +/- 561 and cMDH activity was 2,453 +/- 99, 4,673 +/- 152 and 6,821 +/- 205 in 1-4 day, 18-20 day and 7-8 week old rats, respectively. While cMDH activity increased with age (p < 0.0001), mMDH activity showed no change. This study also determined if endogenous compounds, previously shown to alter malate metabolism, affected MDH activities. Lactate inhibited only cMDH activity, by a competitive mechanism. Oxaloacetate inhibited mMDH by partial non-competitive inhibition and cMDH by competitive inhibition. Alpha-ketoglutarate competitively inhibited both enzymes; however, the inhibition of mMDH activity was more pronounced than that of cMDH activity. Citrate inhibited mMDH via an uncompetitive mechanism and cMDH via a noncompetitive mechanism. The mechanisms of inhibition of mMDH and cMDH by each of the effectors were the same over the three ages. The results suggest mMDH and cMDH activities show a dissimilar developmental pattern and may be regulated differently by endogenous effectors. The greater sensitivity of mMDH, compared to cMDH, to certain effectors may be related to the dual role of mMDH in the tricarboxylic acid cycle and the malate-aspartate shuttle.

Transport of L-lactate by cultured rat brain astrocytes.

Several reports indicate that lactate can serve as an energy substrate for the brain. The rate of oxidation of this substrate by cultured rat brain astrocytes was 3-fold higher than the rate with glucose, suggesting that lactate can serve as an energy source for these cells. Since transport into the astrocytes may play an important role in regulating nutrient use by individuals types of brain cells, we investigated the uptake of L-[U-14C]lactate by primary cultures of rat brain astrocytes. Measurement of the net uptake suggested two carrier-mediated mechanisms and an Eadie-Hofstee type plot of the data supported this conclusion revealing 2 Km values of 0.49 and 11.38 mM and Vmax values of 16.55 and 173.84 nmol/min/mg protein, respectively. The rate of uptake was temperature dependent and was 3-fold higher at pH 6.2 than at 7.4, but was 50% less at pH 8.2. Although the lactate uptake carrier systems in astrocytes appeared to be labile when incubated in phosphate buffered saline for 20 minutes, the uptake process exhibited an accelerative exchange mechanism. In addition, lactate uptake was altered by several metabolic inhibitors and effectors. Potassium cyanide and alpha-cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, alpha-ketoisocaproate, and 3-hydroxybutyrate at 5 and 10 mM greatly attenuated the rate of lactate uptake. These results suggest that the availability of lactate as an energy source is regulated in part by a biphasic transport system in primary astrocytes.