Role of Branched-Chain Aminotransferase Isoenzymes and Gabapentin in Neurotransmitter Metabolism

Abstract: Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with α-ketoglutarate to form the branched-chain α-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-¹⁴C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to α-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an α-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from α-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.     

Distribution of key enzymes of branched-chain amino acid metabolism in glial and neuronal cells in culture.

Transamination of branched-chain amino acids (BCAAs) catalyzed by the branched chain aminotransferase isoenzymes (BCATs) is believed to play an important role in nitrogen shuttling and excitatory neurotransmitter glutamate metabolism in brain. Recently, we have shown that the mitochondrial isoenzyme (BCATm) is the predominant form found in cultured astrocytes. In this study we used immunocytochemistry to examine the distribution of BCAT isoenzymes in cultured rat neurons and microglial cells. The cytoplasm of neurons displayed intense staining for the cytosolic isoenzyme (BCATc), whereas BCATm staining was not detectable in neurons. In contrast, microglial cells expressed BCATm in high concentration. BCATc appeared to be absent in this cell type. The second and committed step in the BCAA catabolic pathway is oxidative decarboxylation of the alpha-keto acid products of BCAT catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Because the presence of BCKD should provide an index of the ability of a cell to oxidize BCAA, we have also immunocytochemically localized BCKD in neuron and glial cell cultures from rat brain. Our results suggest ubiquitous expression of this BCKD enzyme complex in cultured brain cells. BCKD immunoreactivity was detected in neurons and in astroglial and microglial cells. Therefore, the expression of BCAT isoenzymes shows cell-specific localization, which is consistent with the operation of an intercellular nitrogen shuttle between neurons and astroglia. On the other hand, the ubiquitous expression of BCKD suggests that BCAA oxidation can probably take place in all types of brain cells and is most likely regulated by the activity state of BCKD rather than by its cell-specific localization.     

Immunocytochemical localization of 3-methylcrotonyl-CoA carboxylase in cultured ependymal, microglial and oligodendroglial cells

To evaluate the ability of ependymal, microglial and oligodendroglial cells to degrade leucine, the presence of 3-methylcrotonyl-CoA carboxylase (MCC) was investigated in cultures of these cells. MCC is a biotin-containing heterodimeric enzyme that is specific for the irreversible part of the leucine catabolic pathway. It has been reported previously that in cell culture MCC is expressed in astrocytes and a subpopulation of neurones. In the present study ependymal, microglial and oligodendroglial cell cultures, derived from the brains of newborn rats, were examined for the expression of MCC by RT-PCR, western blotting and immunocytochemistry. The results of RT-PCR and western blotting showed the presence of mRNA as well as protein of both subunits of MCC in ependymal, microglial and oligodendroglial cell cultures. Immunocytochemical investigation of the cellular and subcellular distribution of MCC demonstrated a mitochondrial location of MCC in all neuroglial cell types investigated. The ubiquitous expression of MCC in glial cells demonstrates the ability of the cells to engage in the catabolism of leucine transported into the brain, mainly for the generation of energy.     

Generation of Ketone Bodies from Leucine by Cultured Astroglial Cells

Abstract: To elucidate the significance of branched-chain amino acids (BCAAs) for brain energy metabolism, the capacity to use BCAAs for oxidative metabolism was investigated in astroglia-rich primary cultures derived from newborn rat brain. The cells selectively removed BCAAs from the culture medium, the disappearance following first-order kinetics. The BCAAs disappeared rapidly in spite of the presence of sufficient glucose as substrate for the generation of energy. Taking into consideration that the ketogenic amino acid leucine could be degraded only to acetyl-CoA and acetoacetate, and with the knowledge that astroglial cells have the capacity to secrete ketone bodies, this amino acid was chosen for further metabolic studies. After incubation of the cells with leucine, acetoacetate, d -β-hydroxybutyrate, and α-ketoisocaproate were found to have accumulated in the culture medium. Identification of the radioactive metabolites generated from [4,5-³H]leucine established that the source of the substances released was indeed leucine. These results indicate that, at least in culture, astroglial cells degrade leucine via the known metabolite α-ketoisocaproate, to acetoacetate, which can be further reduced to d -β-hydroxybutyrate. It is hypothesized that upon release from brain astrocytes, the ketone bodies could serve as fuel molecules for neighboring cells such as neurons and oligodendrocytes. In view of these and other results, astrocytes may be considered the brain's fuel processing plants.     

Mitochondrial branched chain aminotransferase gene expression in AS-30D hepatoma rat cells and during liver regeneration after partial hepatectomy in rat

Branched chain aminotransferase (BCAT) is the first enzyme in the catabolism of branched chain amino acids (BCAA). Unlike other amino acid degrading enzymes present in liver, BCAT is only expressed in extrahepatic tissues, and is not regulated by dietary protein, glucagon or glucocorticoids. However, the mitochondrial (m) isoform of BCAT is highly expressed in the fetal liver and rapidly decays after birth. The purpose of the present work was to establish if liver cells under conditions of rapid cell proliferation such as in hepatoma AS30D cells or during liver regeneration after partial hepatectomy were associated with an increase in the activity and expression of BCATm. BCAT activity in mitochondria of AS30D cells was 18.6 mU/mg protein. Western, Northern blot, and immunohistochemical analysis revealed that AS30D hepatoma cells expressed only BCATm. The apparent Km of BCATm in isolated AS30D cells mitochondria for leucine, isoleucine and valine was 1.0+/-0.02, 1.3+/-0.1 and 2.1+/-0.1 mM, respectively. The regenerated liver showed BCAT activity from day 3 to day 6, and the maximal BCAT activity (7.0 mU/mg protein) was on day 5. By day 14 after partial hepatectomy BCAT activity and expression was almost undetectable. Interestingly, there was a relationship between BCAT activity and the Mr. of the immunoreactive band of BCATm. The presence of a 41 kDa band was associated with BCAT activity, whereas the 43 kDa band with undetectable activity. The results of this study indicate that BCATm activity is required in liver cells under conditions of rapid cell proliferation.     

Cytosolic and mitochondrial isoforms of NADP+-dependent isocitrate dehydrogenases are expressed in cultured rat neurons,astrocytes, oligodendrocytes and microglial cells

NADP+-dependent isocitrate dehydrogenases (ICDHs) are enzymes that reduce NADP+ to NADPH using isocitrate as electron donor. Cytosolic and mitochondrial isoforms of ICDH have been described. Little is known on the expression of ICDHs in brain cells. We have cloned the rat mitochondrial ICDH (mICDH) in order to obtain the sequence information necessary to study the expression of ICDHs in brain cells by RT-PCR. The cDNA sequence of rat mICDH was highly homologous to that of mICDH cDNAs from other species. By RT-PCR the presence of mRNAs for both the cytosolic and the mitochondrial ICDHs was demonstrated for cultured rat neurons, astrocytes, oligodendrocytes and microglia. The expression of both ICDH isoenzymes was confirmed by western blot analysis using ICDH-isoenzyme specific antibodies as well as by determination of ICDH activities in cytosolic and mitochondrial fractions of the neural cell cultures. In astroglial and microglial cultures, the total ICDH activity was almost equally distributed between cytosolic and mitochondrial fractions. In contrast, in cultures of neurons and oligodendrocytes about 75% of total ICDH activity was present in the cytosolic fractions. Putative functions of ICDHs in cytosol and mitochondria of brain cells are discussed.     

Mitochondrial Malic Enzyme: Purification from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization in Neurons of Rat Brain

Abstract: To elucidate the cellular location of mitochondrial malic enzyme in brain, immunocytochemical studies were performed. For this purpose, mitochondrial malic enzyme was purified to apparent homogeneity from bovine brain and used for the immunization of rabbits. Subjecting the antiserum to affinity purification on immobilized antigen as an absorbent yielded a purified immunoreactive antibody preparation, which was characterized by probing cytosolic and mitochondrial fractions of bovine and rat brain in western blotting. As neither crossreactivity with cytosolic malic enzyme nor immunoreactivity against other proteins could be observed, the antibody preparation was found suitable for immunocytochemistry. By using sections of perfusion-fixed rat brain, considerable resolution was achieved at the light-microscopic level. Distinct and specific staining of neurons was observed; in contrast, no staining of astrocytes and possibly unspecific staining within the nuclei of oligodendrocytes were obtained. From these data, it is concluded that mitochondrial malic enzyme is located in neurons; however, in astrocytes, the enzyme appears to be either lacking or present at a much lower level. A protective role against oxidative stress in neurons is proposed for mitochondrial malic enzyme.     

Hormonal Regulation of the mRNA Encoding the Ketogenic Enzyme Mitochondrial 3-Hydroxy-3-Methylglutaryl-CoA Synthase in Neonatal Primary Cultures of Cortical Astrocytes and Meningeal Fibroblasts

Abstract: We have previously identified cerebellum to contain significantly higher levels, compared with other brain regions, of the mRNA encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). In this report, we extend these observations, using primary cultures of cerebellar astrocytes and cerebellar granule neurons, and show that mHS mRNA was not readily detected in these cell types, suggesting that other cerebellar cell types account for mHS mRNA abundances observed in cerebellum. In contrast, we report, for the first time, the ready detection of mHS mRNA together with the mRNAs encoding the remaining enzymes of the 3-hydroxy-3-methylglutaryl-CoA cycle, namely, mitochondrial acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA lyase, in primary cultures of neonatal meningeal fibroblasts. Based on observations of the effects of fetal calf serum in the culture medium and the documented effects of various hormones on mHS mRNA levels in liver, we show that the glucocorticoid hydrocortisone effects a selective fourfold increase in mHS mRNA abundances in both neonatal meningeal fibroblasts and neonatal cortical astrocytes cultured in a serum-free/hormone-free medium.     

Distribution of the branched chain aminotransferase proteins in the human brain and their role in glutamate regulation

The branched chain aminotransferase enzymes (BCAT) serve as nitrogen donors for the production of 30% of de novo glutamate synthesis in rat brain. Despite the importance of this major metabolite and excitatory neurotransmitter, the distribution of BCAT proteins in the human brain (hBCAT) remains unreported. We have studied this and report, for the first time, that the mitochondrial isoform, hBCATm is largely confined to vascular endothelial cells, whereas the cytosolic hBCATc is restricted to neurons. The majority of hBCATc‐labelled neurons were either GABA‐ergic or glutamatergic showing both cell body and axonal staining indicating a role for hBCATc in both glutamate production and glutamate release during excitation. Strong staining in hormone secreting cells suggests a further role for the transaminases in hormone regulation potentially similar to that proposed for insulin secretion. Expression of hBCATm in the endothelial cells of the vasculature demonstrates for the first time that glutamate could be metabolized by aminotranferases in these cells. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions, where the role of hBCATm in metabolizing excess glutamate may factor more prominently.     

Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid aminotransferase.

Branched chain amino-acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU x mg(-1) protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41-kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43-kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41-kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.     

Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae

The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals.     

Effects of Antimycin, Glucose Deprivation, and Serum on Cultures of Neurons, Astrocytes, and Neuroblastoma Cells

The resistance of cultured mouse neuroblastoma cells, primary cultures of rat cerebellar neurons, and rat brain astrocytes to a block of aerobic metabolism was studied. Parameters such as lactate production and ATP content were measured in the presence of antimycin A and under various conditions of glucose, oxygen, and serum supply. The following conclusions can be drawn: (1) All cell types studied were characterized by an active production of lactate; (2) Incubation of the various cell types in the absence of glucose at normal oxygen tension did not affect ATP levels; (3) Respiration blocked by antimycin led to a Pasteur effect; (4) Neuroblastoma cells, but not the other cell types, were fully resistant to inhibition of respiration provided that sufficient glucose was supplied; (5) In the absence of glucose no stores of energy or utilizable substrate were present in the cell types studied when respiration was blocked; (6) In the presence of fetal calf serum anoxic neurons showed irreversible signs of degeneration.     

Enzyme levels in cultured astrocytes,oligodendrocytes and Schwann cells,and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.Special issue dedicated to Dr. Lewis Sokoloff.     

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°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. -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.These data were presented in part at the meeting of the American Society for Neurochemistry in Richmond, Virginia, March 1993     

Ontogeny and Cellular Localization of the Pyruvate Recycling System in Rat Brain

Abstract: The ontogeny of the cerebral pyruvate recycling pathway and the cellular localization of associated enzymes, malic enzyme (ME) and phosphoenolpyruvate carboxykinase (PEPCK), have been investigated using a combination of ¹³C NMR spectroscopy, enzymatic analysis, and molecular biology approaches. Activity of the pathway, using [1,2-¹³C₂]acetate as a substrate, was detected by ¹³C NMR in brain extracts 3 weeks after birth, increasing progressively up to the third month of age. In whole-brain homogenates, ME activity increased to adult levels with the same time course as the recycling pathway. PEPCK activity was low during the first 2 weeks of life and decreased further toward adulthood. ME and PEPCK activity were found in primary cultures of astrocytes and in synaptosomal fractions of adult brain. Primary cultures of cortical neurons showed PEPCK activity but no detectable ME activity. The cytosolic ME gene was expressed in primary cultures of neurons and in astrocytes as well as in the neonatal and adult brain. The PEPCK gene was expressed both in primary cultures of cortical neurons and in astrocytes, but the level of its expression in the neonatal and adult brain was undetectable.     

Oral Leucine Supplementation Is Sensed by the Brain but neither Reduces Food Intake nor Induces an Anorectic Pattern of Gene Expression in the Hypothalamus

Leucine activates the intracellular mammalian target of the rapamycin (mTOR) pathway, and hypothalamic mTOR signaling regulates food intake. Although central infusion of leucine reduces food intake, it is still uncertain whether oral leucine supplementation is able to affect the hypothalamic circuits that control energy balance. We observed increased phosphorylation of p70s6k in the mouse hypothalamus after an acute oral gavage of leucine. We then assessed whether acute oral gavage of leucine induces the activation of neurons in several hypothalamic nuclei and in the brainstem. Leucine did not induce the expression of Fos in hypothalamic nuclei, but it increased the number of Fos-immunoreactive neurons in the area postrema. In addition, oral gavage of leucine acutely increased the 24 h food intake of mice. Nonetheless, chronic leucine supplementation in the drinking water did not change the food intake and the weight gain of ob/ob mice and of wild-type mice consuming a low- or a high-fat diet. We assessed the hypothalamic gene expression and observed that leucine supplementation increased the expression of enzymes (BCAT1, BCAT2 and BCKDK) that metabolize branched-chain amino acids. Despite these effects, leucine supplementation did not induce an anorectic pattern of gene expression in the hypothalamus. In conclusion, our data show that the brain is able to sense oral leucine intake. However, the food intake is not modified by chronic oral leucine supplementation. These results question the possible efficacy of leucine supplementation as an appetite suppressant to treat obesity.     

Identification of complement 5a-like receptor (C5L2) from astrocytes: characterization of anti-inflammatory properties

Brain inflammation is regulated by endogenous substances, including neurotransmitters such as noradrenaline (NA), which can increase anti-inflammatory genes. To identify NA-regulated, anti-inflammatory genes, we used TOGA (total gene expression analysis) to screen rat astrocyte-derived RNA. NA-inducible cDNA clone DST11 encodes an isoform of the complement C5a receptor (C5aR), with 39% identity at the amino acid level to the rat C5aR, and 56% identity to a recently described human C5aR variant termed C5L2 (complement 5a-like receptor). Quantitative PCR confirmed that in astrocytes, DST11 mRNA expression is increased by NA, whereas in vivo depletion of cortical NA reduced DST11 levels. Western blot analysis demonstrated basal and NA-induced expression of DST11 as a 45 kDa protein in primary astrocytes cultures. Immunocytochemical staining of adult rat brain revealed DST11-immunoreactivity throughout brain, co-localized to neurons and astrocytes. In astrocytes, induction of nitric oxide synthase type 2 was increased by treatment with antisense oligonucleotides to DST11. Reducing DST11 expression also increased nuclear factor kappaB reporter gene, and decreased cAMP response element reporter gene activation. These results demonstrate that DST11 is a C5aR isoform expressed by glia and neurons, which is regulated by NA, and exerts anti-inflammatory functions. Changes in DST11 levels in diseased brain could therefore contribute to the progression of inflammatory damage.