Quantification of the GABA Shunt and the Importance of the GABA Shunt Versus the 2-Oxoglutarate Dehydrogenase Pathway in GABAergic Neurons

Abstract: We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-¹³C]glucose, and brain extracts were analyzed by ¹³C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 ± 1.6 and 5.1 ± 0.2%, respectively (mean ± SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the ¹³C labeling of aspartate from [1-¹³C]glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [¹³C]aspartate during inhibition of the GABA shunt by γ-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with γ-vinyl-GABA, which increased GABA levels dramatically, caused changes in the ¹³C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.     

PERMEABILITY OF MITOCHONDRIA FROM RAT BRAIN AND RAT LIVER TO GABA

Abstract— For the GABA shunt to operate in rivo , GABA must be able to enter brain mitochondria. GABA causes reduction of intra-mitochondrial NAD⁺; glutamate or 2-oxoglutarate stimulate this reduction at concentrations at which they do not themselves cause reduction. This stimulation is not abolished by Triton X-100. The rates of swelling of brain and liver mitochondria are similar in iso-osmotic GABA and in several analogues. The rate of swelling is proportional to the concentration of GABA in the iso-osmotic suspension medium. GABA penetrates 60% of the mitochondrial matrix volume, this value is unaffected by energizing the mitochondria. The activity of GABA-oxoglutarate aminotransferase is not latent. We conclude that GABA diffuses into both brain and liver mitochondria as a species with no net charge at rates which are able to sustain maximum activity of the GABA shunt.     

Mitochondrial and cytosolic NADPH systems and isocitrate dehydrogenase indicator metabolites during ureogensis from ammonia in isolated rat hepatocytes.

1. Citrate isocitrate and 2-oxoglutarate levels were determined in isolated rat hepatocytes and in particulate and soluble fractions, thereof, obtained by the digitonin and silicone oil fractionation technique. 2. Caculated from isocitrate/2-oxoglutarate ratios ("indicator metabolite method"), the redox potential of mitochondrial free NADPH is -402 mV, whereas that of the extramitochondrial (cytosolic) space is about 10 mV more positive, -392 mV. 3; Addition of ammonia (either as ammonium chloride or from urea plus urease) to isolated hepatocytes causes preferential oxidation of mitochondrial NADPH, is demonstrated by spectrophotometry of the dihydro band and by the changes in the isocitrate/2-oxoglutarate ratios. The redox potential difference of free NADPH between mitochondria and cytosol is abolished or even reserved. 4. It is concluded that during urogenesis from ammonia mitochondrial isocitrate oxidation is shifted largely in favor of the NADP-linked as opposed to the NAD-linked enzyme; isocitrate concentration under these conditions is less than 10 muM, below the Km (isocitrate) of the NAD-linked enzyme but in the range of that for the NADP-linked enzyme. 5. Both in the absence and in the presence of ammonia there is a concentration gradient across the mitochondrial inner membrane (from mitochondria to cytosol) for citrate, isocitrate, and also, to a smaller extent, for 2-oxoglutarate. 6. These results and data in the literature on enzyme activity are in agreement with the assumption of near-equilibrium of NADP-dependent isocitrate dehydrogenases in the mitochondrial matrix and cytosolic spaces in the absence of ammonia; accordingly, during urea formation from added ammonia the redox potential of mitochondrial free NADPH is increased to -391 mV or possibly even higher if there exists an indicator error under this condition.     

Control of glutamate oxidation in brain and liver mitochondrial systems

1. Glutamate oxidation in brain and liver mitochondrial systems proceeds mainly through transamination with oxaloacetate followed by oxidation of the α-oxoglutarate formed. Both in the presence and absence of dinitrophenol in liver mitochondria this pathway accounted for almost 80% of the uptake of glutamate. In brain preparations the transamination pathway accounted for about 90% of the glutamate uptake. 2. The oxidation of [1-¹⁴C]- and [5-¹⁴C]-glutamate in brain preparations is compatible with utilization through the tricarboxylic acid cycle, either after the formation of α-oxoglutarate or after decarboxylation to form γ-aminobutyrate. There is no indication of γ-decarboxylation of glutamate. 3. The high respiratory control ratio obtained with glutamate as substrate in brain mitochondrial preparations is due to the low respiration rate in the absence of ADP: this results from the low rate of formation of oxaloacetate under these conditions. When oxaloacetate is made available by the addition of malate or of NAD⁺, the respiration rate is increased to the level obtained with other substrates. 4. When the transamination pathway of glutamate oxidation was blocked with malonate, the uptake of glutamate was inhibited in the presence of ADP or ADP plus dinitrophenol by about 70 and 80% respectively in brain mitochondrial systems, whereas the inhibition was only about 50% in dinitrophenol-stimulated liver preparations. In unstimulated liver mitochondria in the presence of malonate there was a sixfold increase in the oxidation of glutamate by the glutamate-dehydrogenase pathway. Thus the operating activity of glutamate dehydrogenase is much less than the `free' (non-latent) activity. 5. The following explanation is put forward for the control of glutamate metabolism in liver and brain mitochondrial preparations. The oxidation of glutamate by either pathway yields α-oxoglutarate, which is further metabolized. Since aspartate aminotransferase is present in great excess compared with the respiration rate, the oxaloacetate formed is continuously removed by the transamination reaction. Thus α-oxoglutarate is formed independently of glutamate dehydrogenation, and the question is how the dehydrogenation of glutamate is influenced by the continuous formation of α-oxoglutarate. The results indicate that a competition takes place between the α-oxoglutarate-dehydrogenase complex and glutamate dehydrogenase, probably for NAD⁺, resulting in preferential oxidation of α-oxoglutarate.     

Comparative aspects of aminooxyacetate inhibition of glycine oxidation and aminotransferase activity of pea leaf mitochondria

Aminooxyacetate (AOA) was found to inhibit glycine oxidation by pea leaf mitochondria at micromolar levels. The inhibition resulted from an inhibition of both glycine decarboxylase and serine hydroxymethyltransferase (SHMT) activity. Aspartate: 2-oxoglutarate aminotransferase (AsAT) and alanine: 2-oxoglutarate aminotransferase activities of pea leaf mitochondria were also very sensitive to AOA inhibition. Inhibition of both glycine oxidation and aminotransferase activity was likely competitive with respect to the amino group substrate, but also displayed a time-dependent increase in inhibition at constant AOA concentration. In the case of glycine oxidation, this time-dependent component may be related to the rate of penetration of AOA across the inner mitochondrial membrane. Furthermore, the AOA-inhibition of glycine oxidation could be reversed by pyridoxal 5-phosphate (PLP), whereas AOA-inhibited aminotransferase activity was not reversed. The results indicate that the pyridoxal 5-phosphate antagonist, AOA, results in varying types of inhibition depending on the type of enzyme involved.     

Glucose inhibits GABA release by pancreatic beta-cells through an increase in GABA shunt activity

GABA is the major inhibitory neurotransmitter in the nervous system. It is also released by the insulin-producing beta-cells, providing them with a potential paracrine regulator. Because glucose was found to inhibit GABA release, we investigated whether extracellular GABA can serve as a marker for glucose-induced mitochondrial activity and thus for the functional state of beta-cells. GABA release by rat and human beta-cells was shown to reflect net GABA production, varying with the functional state of the cells. Net GABA production is the result of GABA formation through glutamate decarboxylase (GAD) and GABA catabolism involving a GABA-transferase (GABA-T)-mediated shunt to the TCA cycle. GABA-T exhibits K(m) values for GABA (1.25 mM) and for alpha-ketoglutarate (alpha-KG; 0.49 mM) that are, respectively, similar to and lower than those in brain. The GABA-T inhibitor gamma-vinyl GABA was used to assess the relative contribution of GABA formation and catabolism to net production and release. The nutrient status of the beta-cells was found to regulate both processes. Glutamine dose-dependently increased GAD-mediated formation of GABA, whereas glucose metabolism shunts part of this GABA to mitochondrial catabolism, involving alpha-KG-induced activation of GABA-T. In absence of extracellular glutamine, glucose also contributed to GABA formation through aminotransferase generation of glutamate from alpha-KG; this stimulatory effect increased GABA release only when GABA-T activity was suppressed. We conclude that GABA release from beta-cells is regulated by glutamine and glucose. Glucose inhibits glutamine-driven GABA formation and release through increasing GABA-T shunt activity by its cellular metabolism. Our data indicate that GABA release by beta-cells can be used to monitor their metabolic responsiveness to glucose irrespective of their insulin-secretory activity.     

A method of determining 2-oxoglutarate dehydrogenase activity in intact mitochondria

A rather simple method is suggested for measuring the activity of 2-oxoglutarate dehydrogenase of intact mitochondria. The method is based on the determination of the rate of exogenic 2-oxoglutarate decrease in the mitochondrial suspension. Experiments with sodium arsenite and comparison of kinetic parameters of the 2-oxoglutarate, dehydrogenase reaction and transport of 2-oxoglutarate to mitochondria have shown that the measurable exogenic 2-oxoglutarate oxidation rate corresponds to the 2-oxoglutarate dehydrogenase activity in intact mitochondria. The method made it possible to establish the stimulating effect of ADP on the 2-oxoglutarate dehydrogenase activity of intact mitochondria and the absence of such an effect in destructed mitochondria.     

A novel role for fatty acid transport protein 1 in the regulation of tricarboxylic acid cycle and mitochondrial function in 3T3-L1 adipocytes

Fatty acid transport proteins (FATPs) are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. Whereas some FATP1 translocates to the plasma membrane in response to insulin, the majority of FATP1 remains within intracellular structures and bioinformatic and immunofluorescence analysis of FATP1 suggests the protein primarily resides in the mitochondrion. To evaluate potential roles for FATP1 in mitochondrial metabolism, we used a proteomic approach following immunoprecipitation of endogenous FATP1 from 3T3-L1 adipocytes and identified mitochondrial 2-oxoglutarate dehydrogenase. To assess the functional consequence of the interaction, purified FATP1 was reconstituted into phospholipid-containing vesicles and its effect on 2-oxoglutarate dehydrogenase activity evaluated. FATP1 enhanced the activity of 2-oxoglutarate dehydrogenase independently of its acyl-CoA synthetase activity whereas silencing of FATP1 in 3T3-L1 adipocytes resulted in decreased activity of 2-oxoglutarate dehydrogenase. FATP1 silenced 3T3-L1 adipocytes exhibited decreased tricarboxylic acid cycle activity, increased cellular NAD⁺/NADH, increased fatty acid oxidation, and increased lactate production indicative of altered mitochondrial energy metabolism. These results reveal a novel role for FATP1 as a regulator of tricarboxylic acid cycle activity and mitochondrial function.     

Tetraphenylphosphonium inhibits oxidation of physiological substrates in heart mitochondria

We show that tetraphenylphosphonium inhibits oxidation of palmitoylcarnitine, pyruvate, malate, 2-oxoglutarate and glutamate in heart mitochondria in the range of concentration (1–5 µM) commonly used for the determination of mitochondrial membrane potential. The inhibition of 2-oxoglutarate (but not other substrate) oxidation by tetraphenylphosphonium is dependent on the concentration of 2-oxoglutarate and on extramitochondrial free calcium, and the kinetic plots are consistent with a mixed type of inhibition. Our results indicate that tetraphenylphosphonium interacts with enzymes, specifically involved in the oxidation of 2-oxoglutarate, most possibly, 2-oxoglutarate dehydrogenase.     

Ghrelin Causes a Decline in GABA Release by Reducing Fatty Acid Oxidation in Cortex

Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and γ-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and α-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system.     

REGIONAL AND SUBCELLULAR DISTRIBUTION OF AMINOTRANSFERASES IN RAT BRAIN

Abstract— Aminotransferase activity was measured in various areas of the nervous system of the rat (cortical grey matter, midbrain, corpus callosum, spinal cord and sciatic nerve) and in subcellular fractions of rat brain (nuclei, mitochondria and cytosol). Activity was low or absent in the sciatic nerve relative to that in the other areas, with the exception of incubation of glutamate with oxaloacetate (25 per cent of the activity found in brain) and of asparagine with 2-oxoglutarate (65 per cent of the activity found in brain). The distribution of enzymic activity was not homogeneous; alanine-2-oxoglutarate aminotransferase was highest in cortical grey matter; leucine- and GABA-2-oxoglutarate aminotransferases were highest in midbrain. Incubation of phenylalanine or tyrosine with 2-oxoglutarate gave similar activities in grey matter and midbrain. Activity generally was higher in the grey matter than in corpus callosum or spinal cord. However, incubations of methionine with 2-oxoglutarate, or glutamine with glyoxylate, gave similar activities in the three areas studied from the brain, whereas incubations of glutamate with glyoxylate gave highest activity in the corpus callosum. Only incubations of asparagine with 2-oxoglutarate, and glutamate with glyoxylate, gave significant activity in the nuclear subcellular fraction. Aminotransferase activity of phenylalanine, tyrosine or GABA with 2-oxoglutarate, or ornithine or glutamine with glyoxylate, was localized to mitochondria. The remaining reactions studied (glutamate with oxaloacetate; leucine, alanine, methionine or asparagine with 2-oxoglutarate and glutamate with glyoxylate) demonstrated activity in both the mitochondrial fraction and the soluble supernatant fraction.     

Effect of L-cycloserine on brain GABA metabolism

The administration of L-cycloserine to mice resulted in a dramatic decrease in the activities of 4-aminobutyrate:2-oxoglutarate aminotransferase (GABA-T) and L-alanine:2-oxoglutarate aminotransferase (ALA-T) in both brain and liver. L-Aspartate:2-oxoglutarate aminotransferase was inhibited only slightly, and brain glutamic acid decarboxylase not at all. Liver ALA-T activity returned to near normal levels within 24 h of L-cycloserine administration whereas liver GABA-T and brain ALA-T activities had returned only halfway to normal levels in the same time period. The recovery in the activity of brain GABA-T was even slower. A consequence of the inhibition of brain GABA-T activity was an elevation in the GABA content of the tissue which was maximal 3 h after L-cycloserine administration and which was still noticeable 8 h after the drug treatment. L-Cycloserine was also a potent in vitro inhibitor of brain GABA-T activity. The inhibition was competitive with respect to GABA, the Ki value being 3.1 X 10(-5) M. The prior administration of L-cycloserine to mice significantly delayed the onset of isonicotinic acid hydrazide induced convulsions.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

Effect of thiamine deprivation and thiamine antagonists on the level of gamma-aminobutyric acid and on 2-oxoglutarate metabolism in rat brain

Brain levels of y-aminobutyric acid (GABA), glutamate and 2-oxoglutarate, activities of glutamate decarboxylase GABA-transaminase plus succinic semiaidehyde dehydrogenase and blood levels of glutamate and 2-oxoglutarate were determined in normal, thiamine-deprived, oxythiamine-treated and pyrithiamine-treated rats. Brain GABA levels were significantly reduced in thiamine-deprived and pyrithiamine-treated rats, but the activities of the enzymes of the GABA shunt pathway were not affected. Brain levels of glutamate were decreased and of 2-oxoglutarate increased in all three types of deficiency. This was associated with similar decreases in glutamate and increases in 2-oxoglutarate in the blood in all three deficient groups. Intraventricular injections of 2-[U-¹⁴C] oxoglutarate into the brain in these four groups of rats resulted in some significant differences in distribution of ¹⁴C in various TCA-pathway intermediates and satellite compounds in the brain. Increases in ¹⁴C-label were observed for glutamine and 2-oxoglutarate in all three deficient groups as compared to controls. The ¹⁴C content of succinate, fumarate and aspartate was decreased in the thiamine deprived and PTh-treated groups and [¹⁴C]glutamate was decreased in all three deficient groups. The ¹⁴C content of GABA was not significantly affected.     

The adaptive behaviour of isoenzyme forms of rat liver alanine amino transferases during development

1. The activities of the mitochondrial and cytosol isoenzyme forms of l-alanine–glyoxylate and l-alanine–2-oxoglutarate aminotransferases were determined in rat liver during foetal and neonatal development. 2. The mitochondrial glyoxylate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. The cytosol glyoxylate aminotransferase and the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities first appear prenatally, increase further after birth and then rise to the adult values during weaning. 4. In foetal liver the mitochondrial glyoxylate aminotransferase and the cytosol 2-oxoglutarate aminotransferase activities are increased after injection in utero of glucagon, dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) or thyroxine. The cytosol glyoxylate aminotransferase and the mitochondrial 2-oxoglutarate aminotransferase activities are increased after injection in utero of cortisol or thyroxine. 5. After birth the further normal increases in the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities can be hastened by cortisol injection, whereas the increase in cytosol glyoxylate aminotransferase activity requires cortisol treatment together with the intragastric administration of casein. 6. The results are discussed with reference to the metabolic patterns and the changes in regulatory stimuli (hormonal and dietary) that occur during the period of development.     

Functioning of the γ-Aminobutyrate Pathway in Wheat Seedlings Affected by Osmotic Stress

γ-Aminobutyrate (GABA) was the only amino acid out of three amino acid intermediates of GABA shunt that increased significantly after 28 h from the beginning of osmotic stress induced by 20 % polyethylene glycol 6000 in wheat seedlings. At the same time specific activities of glutamate decarboxylase (GAD) and GABA aminotransferase (GABA-T) two enzymes of GABA pathway did not change as compared with the control plants. The response of two GABA-T activities (with pyruvate or 2-oxoglutarate as amino acid acceptor) to aminooxyacetate, 3-chloro-L-alanine and p-hydroxymercuribenzoate prompted us to suggest that at least two isoforms of GABA-T showing different substrate specificity do exist in wheat leaves. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebro-cortical mitochondria

The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxyburyrate on the flux of PDHC (as determined by [1-¹⁴C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-¹⁴C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-¹⁴C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861–1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.     

1-Piperideine as an In Vivo Precursor of the γ-Aminobutyric Acid Homologue 5-Aminopentanoic Acid

Intraperitoneal injection of the cyclic imine 1-piperideine in mice resulted in measurable quantities of 5-aminopentanoic acid in brain. 5-Aminopentanoic acid is a methylene homologue of gamma-aminobutyric acid (GABA) that is a weak GABA agonist. 5-Aminopentanoic acid formed in the periphery was ruled out as the source of brain 5-aminopentanoic acid based on the absence of detection in brain following injection of 100 mg/kg of 5-aminopentanoic acid. Deuterium-labeled 1-piperideine was prepared by exchange in deuterated phosphate buffer. Injection of [3.3-2H2]1-piperideine yielded [2.2-2H2]5-aminopentanoic acid in brain. The results are consistent with uptake of 1-piperideine into brain and oxidation of the precursor to 5-aminopentanoic acid. Inhibition of GABA catabolism by pretreatment with aminooxyacetic acid increased brain concentrations of 5-aminopentanoic acid formed from 1-piperideine, suggesting that 5-aminopentanoic acid is an in vivo substrate of 4-aminobutyrate:2-oxoglutarate aminotransferase.     

Role of 2-oxoglutarate dehydrogenase in brain pathologies involving glutamate neurotoxicity

Decreased activity of the mitochondrial thiamin-dependent 2-oxoglutarate dehydrogenase complex (OGDHC) is associated with a number of inborn and acquired neuropathologies. We hypothesized that perturbation in flux through the complex influences brain development and function, in particular, because the OGDHC reaction is linked to the synthesis/degradation of neurotransmitters glutamate and GABA. Developmental impact of this metabolic knot was studied by characterizing the brain OGDHC activity in offspring of rats exposed to acute hypobaric hypoxia at a critical organogenesis period of pregnancy. In this model, we detected the hypoxia-induced changes in the brain OGDHC activity and in certain physiologic and morphometric parameters. The changes were mostly abrogated by application of specific effector of cellular OGDHC, the phosphonate analog of 2-oxoglutarate (succinyl phosphonate), shortly before hypoxia. The glutamate excitotoxicity known to greatly contribute to hypoxic damage was alleviated by succinyl phosphonate in situ. That is, the delayed calcium deregulation, mitochondrial depolarization and reactive oxygen species (ROS) production became less pronounced in cultivated neurons loaded with succinyl phosphonate. In vitro, succinyl phosphonate protected OGDHC from the catalysis-induced inactivation. Thus, the protective effects of the phosphonate upon hypoxic insult in vivo may result from the preservation of mitochondrial function and Ca²⁺ homeostasis due to the phosphonate inhibition of both the OGDHC-dependent ROS production and associated OGDHC inactivation. As a result, we showed for the first time that the hypoxia- and glutamate-induced cerebral damage is linked to the function of OGDHC, introducing the phosphonate analogs of 2-oxoglutarate as promising diagnostic tools to reveal the role of OGDHC in brain function and development.     

2-Oxoglutarate dehydrogenase and pyruvate dehydrogenase activities in plant mitochondria: interaction via a common coenzyme a pool

2-Oxoglutarate (2-OG)-dependent O2 uptake by washed or purified turnip (Brassica rapa L.) and pea (Pisum sativum L. cv. Massey Gem) leaf mitochondria, in the presence of malonate, was inhibited between 65 and 90% by micromolar levels of pyruvate. The inhibition was not observed in the absence of malonate and was reversed by alpha-cyano-4-hydroxycinnamic acid. The inhibition was also reversed by oxaloacetate or by malate, but not by any other tricarboxylic acid cycle intermediates. The stimulation of O2 uptake by oxaloacetate was half maximal at 8-9 microM and was transient, indicating its action was not mediated through the complete metabolic removal of pyruvate. Pyruvate had not effect on 2-OG oxidation under conditions in which pyruvate dehydrogenase was not active, indicating that pyruvate metabolism, rather than pyruvate itself, was responsible for producing the inhibition of 2-OG oxidation. Similar results were obtained with detergent-treated mitochondrial extracts with the exception that the inhibition of 2-OG oxidation by pyruvate could also be reversed by coenzyme A. The results suggest that pyruvate inhibits 2-oxoglutarate oxidation, in intact plant mitochondria, by sequestering intramitochondrial CoA as acetyl-CoA and, in the absence of citrate synthase activity, reduces the amount of free coenzyme A available for 2-oxoglutarate dehydrogenase. These results indicate that pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase share a common CoA pool within plant mitochondria and that the turnover of the acyl-CoA product of one enzyme will dramatically influence the activity of the other.