Comparative Characterization of Glutamate Decarboxylase in Crude Homogenates of Oviduct, Ovary, and Hypothalamus

Some biochemical characteristics of L-glutamate decarboxylase (GAD) were compared using crude homogenates of the rat oviduct, ovary, and hypothalamus. As estimated by the measurement of CO2 production, the specific activities of oviductal and ovarian GAD were about 10 and 3% of the hypothalamic value, respectively. Stoichiometric formation of gamma-aminobutyric acid (GABA) and CO2 from L-glutamate could be observed in oviduct and hypothalamus, while in ovarian homogenates the production of CO2 was more than nine times that of GABA. Hypothalamic and tubal GAD showed similar time course, temperature dependence, and pH dependence. The dependence on protein concentration and on exogenous cofactor supply was also similar in these two tissues. The kinetic constant for L-glutamate as a substrate was nearly the same in oviduct (1.30 mM) and hypothalamus (1.64 mM). The responsiveness of tubal and hypothalamic GAD to various inhibitors was also similar. The present findings indicate that the oviductal and the hypothalamic GAD may be identical, and they suggest a possible GABAergic innervation of the Fallopian tube.     

Alteration of GABA levels in ovary and fallopian tube of the pregnant rat

The concentration and the total quantity of gamma-aminobutyric acid (GABA) were determined in ovary and Fallopian tube of the rat on day 3, 7, 14 and 21 of pregnancy and the values were compared with a diestrous control group. In ovary, both GABA concentration and total GABA content gradually elevated up to day 14 of gestation but, thereafter, the diminution of the two parameters was observed. The GABA concentrations in Fallopian tube were found to be strongly reduced during pregnancy, and the total amount of the amino acid in this organ also decreased markedly. The significant alterations of GABA levels in both organs suggest a possible correlation between the functional status and GABA content of ovary and Fallopian tube of the pregnant rat.     

Higher GABA Concentrations in Fallopian Tube Than in Brain of the Rat

Abstract: The GABA content was determined simultaneously in two peripheral organs, i.e., ovary and Fallopian tube. Moreover, the effects of inhibitors of glutamate decarboxylase or γ-aminobutyrate transaminase (GABA-T) on the GABA concentrations of the two organs were examined, to point out similarities and differences between central and peripheral pathways of GABA biosynthesis and degradation. In ovary, GABA concentration was found to be about 30% of that in total brain tissue. Furthermore, isoniazid and thiosemicarbazide caused significant reduction of GABA levels in peripheral organs. In contrast to the CNS, aminooxyacetic acid failed to increase, but even produced a significant diminution in peripheral GABA content. Gabaculine did not change GABA levels. In conclusion, it has been demonstrated for the first time that a peripheral organ, i.e. fallopian tube, contained higher GABA concentrations than the CNS. On the other hand, in the organs examined GABA seemed to be synthesized similarly, but metabolized by a pathway different from that in the brian.     

Inhibition of Brain Glutamate Decarboxylase Activity Is Related to Febrile Seizures in Rat Pups

Because previous work showed that in the newborn brain, but not in the adult brain, glutamate decarboxylase (GAD) is notably susceptible to heat, we have studied the possible involvement of GAD inhibition in febrile convulsions and the related changes in gamma-aminobutyric acid (GABA) content. Rats of different ages were subjected to hyperthermia, and GAD activity was determined in brain homogenates by measuring the release of 14CO2 from labeled glutamate and by measuring the formation of GABA. The latter method gave considerably lower values than the former in the youngest rats, and was considered more reliable. With this method, we found a 37-48% inhibition of GAD activity in rat pups 2-5 days old, which showed febrile seizures at progressively higher body temperatures, whereas in 10- and 15-day-old animals, which did not show convulsions, GAD activity was not affected by hyperthermia. Whole-brain GABA levels, however, did not change at any age. In contrast to GAD, choline acetyltransferase and lactic dehydrogenase activities were not altered by hyperthermia at any of the ages studied. These results suggest that a decreased efficiency of the inhibitory neurotransmission mediated by GABA, consequent to the inhibition of GAD activity, may be a factor related to febrile convulsions.     

Increased Intracellular γ-Aminobutyric Acid Selectively Lowers the Level of the Larger of Two Glutamate Decarboxylase Proteins in Cultured GABAergic Neurons from Rat Cerebral Cortex

The regulation of glutamate decarboxylase (GAD; EC 4.1.1.15) was studied by using cultures of cerebral cortical neurons from rat brain grown in serum-free medium. About 50% of the neurons in the cultures were gamma-aminobutyric acid (GABA)ergic as determined by two double-staining procedures. Immunoblotting experiments with four anti-GAD sera that recognize the two forms to varying degrees, demonstrated that the cultures contained the two forms of GAD that are present in rat brain (apparent molecular masses = 63 and 66 kDa). GAD activity was reduced by 60-70% when intracellular GABA levels were increased by incubating the cultures with the GABA-transaminase inhibitor gamma-vinyl-GABA for greater than 5-10 h or with 1 mM GABA itself. Neither baclofen nor muscimol (100 microM) affected GAD activity. Immunoblotting experiments showed that only the larger of the two forms of GAD (66 kDa) was decreased by elevated GABA levels. These results, together with previous results indicating that the smaller form of GAD is more strongly regulated by pyridoxal 5'-phosphate (the cofactor for GAD), suggest that the two forms of GAD are regulated by different mechanisms.     

Distribution of γ-Aminobutyric Acid and Glutamate Decarboxylase in the Layers of Rat Oviduct

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to yield a high sensitivity and a low blank. The 20-microns thick freeze-dried sections (0.2-1.5 micrograms dry weight) were prepared from the oviduct and ovary of rat. The analysis of these microsamples by the improved method showed that, contrary to the previous observations, the rat ovary is devoid of GAD activity and contains a trace amount of GABA. Both are present abundantly in the oviduct. In the oviduct mucosa, significant GAD activity was found in the estrous phase, whereas the activity was nearly null during other phases of the estrous cycle. GABA concentration in the oviduct mucosa was 10-fold higher than in the cerebral cortex; its variation during the estrous cycle was not remarkable. In the muscle layer of oviduct, GAD activity had a low peak in the estrous phase and GABA concentration was almost constant during the estrous cycle. The denervation experiment showed that GAD is present in the nerve terminals innervating the oviduct.     

Correlation between alterations in brain GABA metabolism and seizure excitability following administration of GABA aminotransferase inhibitors and valproic acid—a re-evaluation

The time course of the effects of aminooxyacetic acid, γ-vinyl GABA, γ-acetylenic GABA, gabaculine, ethanolamine-O-sulphate (EOS) and valproic acid (VPA) on brain GABA content and the activities of glutamic acid decarboxylase (GAD) and GABA aminotransferase (GABA-T), the enzymes involved in biosynthesis and degradation of GABA, was re-determined and compared with the action on the electroconvulsive threshold in mice. All drugs caused significant increases in the seizure threshold, and the temporal pattern of this effect correlated rather well with the induced elevation of brain GABA. However, no clear relationship was found between the extent of GABA increase and the relative increase of seizure threshold. Except for VPA, the time course of the increment in brain GABA followed closely the inhibition of GABA-T. The activity of GAD was gradually decreased by γ-acetylenic GABA and a slow decline of GAD activity was also observed after γ-vinyl GABA. EOS and gabaculine suggesting a feedback repression of GAD synthesis by highly elevated GABA concentrations. Concomitant with significant reduction of GAD activity, a decrease in seizure threshold occurred though brain GABA levels remained markedly elevated. On the other hand, following administration of VPA the effect of GABA levels was paralleled by an increase in GAD activity indicating that the GABA-elevating action of this drug can be attributed at least in part to an activation of GABA synthesis. The data suggest that reduction of GAD activity may be an inevitable consequence of increasing brain GABA concentrations over a certain extent and this effect seems to limit the anticonvulsant efficacy of GABA-T inhibitors.     

REDUCTION OF LEVEL OF L-GLUTAMIC ACID DECARBOXYLASE BY γ-AMINOBUTYRIC ACID IN MOUSE BRAIN1

Abstract— The effects of accumulated endogenous GABA on the activity of L-glutamic acid decarboxylase (GAD) were studied in mouse brain. When the content of GABA in the brain was increased after administration in vivo of aminooxyacetic acid (AOAA), there was a reduction of GAD activity which could not be reversed by the addition of pyridoxal-5′-phosphate (PLP). Since inhibition of GAD activity by AOAA could be readily reversed by PLP, the reduction of GAD activity measured in the presence of added PLP indicated a decrease in the level of GAD apoenzyme. Similarly, increase of GABA content by hydrazine was also accompanied by a reduction in the level of GAD. Thiosemicarbazide and hydroxylamine did not affect the content of GABA appreciably, and in both cases levels of GAD remained unchanged when measured in the presence of added PLP. The correlation of the reduction in the levels of GAD with the increases in content of GABA suggests that GABA may regulate its own synthesizing enzyme by feedback repression.     

The rate of turnover of cortical GABA from [1-13C]glucose is reduced in rats treated with the GABA-transaminase inhibitor vigabatrin (γ-vinyl GABA)

Brain GABA levels rise and plateau following prolonged administration of the irreversible GABA-transaminase inhibitor vigabatrin (γ-vinylGABA). Recently it has been shown that increased GABA levels reduces GAD₆₇ protein, one of two major isoforms of glutamic acid decarboxylase (GAD). The effects of GABA elevation on GABA synthesis were assessed in vivo using¹H and¹³C-edited NMR spectroscopy. Rates of turnover of cortical glutamate and GABA from intravenously administered [1-¹³C]glucose were measured in α-chloralose anesthetized rats 24 hours after receiving vigabatrin (500 mg/kg, i.p.) and in non-treated controls. GABA concentration was increased 2-fold at 24 hours (from 1.3±0.4 to 2.7±0.9 μmol/g) and GABA-T activity was inhibited by 60%. Tricarboxylic acid cycle flux was not affected by vigabatrin treatment compared to non-treated rats (0.47±0.19 versus 0.52±0.18 μmol/g, respectively). GABA-C2 fractional enrichment (FE) measured in acid extracts rose more slowly in vigabatrin-treated compared to nontreated rats, reaching >90% of the glutamate FE after 3 hours. In contrast, GABA FE≥glutamate FE in non-treated rats. A metabolic model consisting of a single glutamate pool failed to account for the rapid labeling of GABA from glutamate. Metabolic modelling analysis based on two (non-communicating) glutamate pools revealed a ∼70% decrease in the rate of GABA synthesis following vigabatrin-treatment, from 0.14 (non-treated) to 0.04 μmol/g/min (vigabatrin-treated). These findings, in conjunction with the previously reported differential effects of elevated GABA on the GAD isoforms, suggests that GAD₆₇ may account for a major fraction of cortical GABA synthesis in the α-chloralose anesthetized rat brain in vivo. Special issue dedicated to Dr. Herman Bachelard.     

Bilobalide prevents reduction of gamma-aminobutyric acid levels and glutamic acid decarboxylase activity induced by 4-O-methylpyridoxine in mouse hippocampus

We previously reported that bilobalide, a constituent of Ginkgo biloba L. leaves, protected mice against convulsions induced by 4-O-methylpyridoxine (MPN). To elucidate the mechanism of the anticonvulsant activity of bilobalide, this study examined the effect of bilobalide on MPN-induced changes in the levels of gamma-aminobutyric acid (GABA) and glutamate, and in the activity of glutamic acid decarboxylase (GAD) in the hippocampus, cerebral cortex and striatum of the mouse. GABA levels and GAD activity in the hippocampus and cerebral cortex were significantly enhanced by bilobalide treatment (30 mg/kg, p.o., for 4 days) alone. MPN significantly decreased GABA levels and GAD activity in the three brain regions tested compared with those in the control. Pretreatment with bilobalide effectively suppressed the MPN-induced reduction in GABA levels and GAD activity in the hippocampus and cerebral cortex. On the other hand, there were no significant differences in the glutamate levels in the three regions despite various treatments. These results suggested that bilobalide prevents MPN-induced reduction in GABA levels through potentiation by bilobalide of GAD activity, and this effect of bilobalide contributes to its anticonvulsant effect against MPN-induced convulsions.     

Response of the cockroach brain gamma-aminobutyric acid system to isonicotinic acid hydrazide and mercaptopropionic acid

The presence of gamma-aminobutyric acid (GABA) as well as glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T) enzymes was demonstrated in the cockroach (Periplaneta americana) brain. Isonicotinic acid hydrazide (INH) in vivo (2.19 mumol/g) inhibited brain GAD activity, the inhibition lasted for about 2 hours and the normal activity levels reappeared at 4 h after INH administration. Brain GABA levels increased initially but then declined and were restored to normal levels at 4 h after INH administration. GABA-T activity was strongly inhibited by INH and a total 100% inhibition was observed at 2-3 h following INH treatment. The GABA-T activity, however, began to recover after 3 h but only 37% of the total enzyme activity was released from inhibition. Mercaptopropionic acid (MPA) in vivo (32 micrograms/g) inhibited brain GAD activity and depleted GABA level also. Results indicate that INH response of the cockroach brain GABA system is similar to that reported for the chick brain but differs from that of the mammalian brain.     

Stimulation of Synaptosomal γ-Aminobutyric Acid Synthesis by Glutamate and Glutamine

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.     

THE DISTRIBUTION OF GLUTAMATE DECARBOXYLASE IN RAT TISSUES; ISOTOPIC VS FLUORIMETRIC ASSAYS

—The activity of glutamate decarboxylase (GAD, EC 4.1.1.15) in normal and neoplastic rat tissues was determined by two assay methods, one based on the production of ¹⁴CO₂ from [¹⁴C]glutamic acid and the other on the fluorimetric measurement of γ-aminobutyric acid (GABA) formation. Activities obtained with the isotopic assay were high in every tissue (ranging from over 800 in liver and brain to 107nmol CO₂/min/g in lung). They were drastically diminished by Triton X-100, by an oxygen-free atmosphere or by the mitochondrial electron transport inhibitors, rotenone and antimycin A. Activities measured fluorimetrically were significant in only a few tissues and were stimulated by Triton (e.g. from 299 to 569 nmol GABA/min/g brain) but were unaffected by rotenone. For several tissues after Triton treatment the fluorimetric and isotopic assays (in air) gave the same results (i.e. the two end products, CO₂ and GABA were in stoichiometric agreement); however, the fluorimetric assay remains the more reliable measure of GAD activity since Triton may not inhibit completely the non-GAD dependent decarboxylation of glutamate in all types of tissue preparations. The hepatic, renal and mammary tumours tested were devoid of GAD; among non-neural normal tissues, kidney, liver and, possibly, adrenal gland contained significant GAD activity. In kidney and liver the activity was 15 and 10 per cent of that in brain.     

Acute and short term effects of ethanol on the metabolism of glutamic acid and GABA in rat brain

Although alcoholic intoxication is attributed to its pharmacological effects on the cell membranes in brain, the rapid metabolic utilisation of the same alters the metabolism of brain affecting the metabolism of glutamate and GABA which have varied metabolic roles besides serving a major proportion of synaptic activity. A study on the effects of ethanol, both acute and short-term, on glutamate (glu) and GABA metabolism in various regions of rat brain was carried out. Increased activities of glutamic acid decarboxylase (GAD) and aspartic acid aminotransferase (AST) in all brain regions, but decreased activity of glutamic acid dehydrogenase (GDH) in cerebral cortex (CC) and cerebellum (CB) following ethanol administration in brain was observed. Differential effects of ethanol were also obtained on the contents of glu and aspartate (asp), which were increased in CC, CB, and brain stem (BS) regions, as opposed to GABA content, which, although found to increase in acute toxicity, showed a decrease in all of the above brain regions in short-term toxicity. It is concluded that the above changes in glu, asp and GABA represent the consequences of metabolic utilization of alcohol in the brain, probably more a state of cerebral excitation than depression, and the changes may be a compensatory phenomenon.     

γ-VINYL GABA: EFFECTS OF CHRONIC ADMINISTRATION ON THE METABOLISM OF GABA AND OTHER AMINO COMPOUNDS IN RAT BRAIN

Rats were given γ-vinyl GABA (4-amino-hex-5-enoic acid), a new irreversible inhibitor of GABA aminotransferase (GABA-T), by daily subcutaneous injection (100mgkg) for 11 days. Amino acids were quantitated in the brains of the γ-vinyl GABA-treated and control animals 24 h after the last injection, and enzyme activities of GABA-T and glutamic acid decarboxylase (GAD) were measured. Chronic administration of γ-vinyl GABA produced a 150% increase in brain GABA content, along with marked increases in the contents of B-alanine and homocarnosine. Brain GABA-T activity was reduced by 26%, and GAD activity was reduced by 22%. In addition, γ-vinyl GABA caused a marked increase in hypotaurine content in rat brain, suggesting that it acts as an inhibitor of hypotaurine dehydrogenase, and it produced significant decreases in brain contents of glutamine and threonine. Although it is an effective GABA-T inhibitor, γ-vinyl GABA apparently affects several other brain enzymes as well, and it may not be an ideal drug for elevating brain GABA levels in man.     

Taurine Biosynthesis in Rat Brain In Vivo: Lack of Relationship with Cysteine Sulfinate Decarboxylase Glutamate Decarboxylase-Associated Activity (GAD/CSDII)

Two distinct forms of cysteine sulfinate decarboxylase (CSD), respectively, CSDI and CSDII, have already been separated in rat brain. One of them, CSDII, appeared to be closely associated with glutamate decarboxylase (GAD). We have investigated whether the taurine concentration in brain was dependent on CSDII activity in vivo. CSDI and CSDII activities were specifically measured in crude brain extracts after selective immunotrapping. After 4 days of chronic treatment of mice with gamma-acetylenic gamma-aminobutyric acid, a drastic and identical decrease in CSDII and GAD activities was observed in the brain. Taurine concentration and CSDI activities were not significantly altered. Following striato-nigral pathway lesioning in the rat brain, GAD and CSDII show an identical 80% decrease in the substantia nigra. In contrast, CSDI activity and taurine concentration in the substantia nigra were similarly but only slightly affected with an about 30% decrease. Our results provide further evidence that GAD and CSDII are indeed the same enzyme. They show that CSDII does not play any role in the biosynthesis of taurine in vivo. Our findings suggest that CSDI might be the biosynthetic enzyme for taurine in vivo and that there might be some endings projecting into the substantia nigra that contain CSDI and taurine.     

Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

β-N-Oxalylamino-l-Alanine: Action on High-Affinity Transport of Neurotransmitters in Rat Brain and Spinal Cord Synaptosomes

beta-N-Oxalylamino-L-alanine (BOAA) is a dicarboxylic diamino acid present in Lathyrus sativus (chickling pea). Excessive oral intake of this legume in remote areas of the world causes humans and animals to develop a type of spastic paraparesis known as lathyrism. BOAA is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal vacuolation and necrosis. The present study investigates the action of BOAA in vitro on CNS high-affinity transport systems for glutamate, gamma-aminobutyric acid (GABA), aspartate, glycine, and choline and in the activity of glutamate decarboxylase (GAD), the rate-limiting enzyme in the decarboxylation of glutamate to GABA. Crude synaptosomal fractions (P2) from rat brain and spinal cord were used for all studies. [3H]Aspartate transport in brain and spinal cord synaptosomes was reduced as a function of BOAA concentration, with reductions to 40 and 30% of control values, respectively, after 15-min preincubation with 1 mM BOAA. Under similar conditions, transport of [3H]glutamate was reduced to 74% (brain) and 60% (spinal cord) of control values. High-affinity transport of [3H]GABA, [3H]glycine, and [3H]choline, and the enzyme activity of GAD, were unaffected by 1 mM BOAA. While these data are consistent with the excitotoxic (convulsant) activity of BOAA, their relationship to the pathogenesis of lathyrism is unknown.     

Correlation between GABA release from rat islet beta-cells and their metabolic state

Pancreatic beta-cells express glutamate decarboxylase (GAD), which is responsible for the production and release of gamma-aminobutyric acid (GABA). Over a 24-h culture period, total GABA release by purified rat beta-cells is eightfold higher than the cellular GABA content and can thus be used as an index of cellular GAD activity. GABA release is 40% reduced by glucose (58 pmol/10(3) cells at 10 mM glucose vs. 94 pmol at 3 mM glucose, P < 0.05). This suppressive effect of glucose was not observed when glucose metabolism was blocked by mannoheptulose or 2,4-dinitrophenol; it was amplified when ATP-dependent beta-cell activities were inhibited by addition of diazoxide, verapamil, or cycloheximide or by reduction of extracellular calcium levels; it was counteracted when beta-cell functions were activated by nonmetabolized agents, such as glibenclamide, IBMX, glucagon, or glucacon-like peptide-1 (GLP-1), which are known to stimulate calcium-dependent activities, such as hormone release and calcium-dependent ATPases. These observations suggest that GABA release from beta-cells varies with the balance between ATP-producing and ATP-consuming activities in the cells. Less GABA is released in conditions of elevated glucose metabolism, and hence ATP production, but this effect is counteracted by ATP-dependent activities. The notion that increased cytoplasmic ATP levels can suppress GAD activity in beta-cells, and hence GABA production and release, is compatible with previous findings on ATP suppression of brain GAD activity.     

Regulation of GABA Level in Rat Brain Synaptosomes: Fluxes Through Enzymes of the GABA Shunt and Effects of Glutamate, Calcium, and Ketone Bodies

Abstract: Stable isotopes were used to measure both the rate of GABA formation by glutamic acid decarboxylase (GAD) and the rate of utilization by GABA-transaminase (GABA-T). The initial rate of GABA accumulation, determined with either [2-¹⁵N]glutamine or [²H₅]glutamine as precursor, was 0.3–0.4 nmol/min/mg of protein. Addition of the calcium ionophore A23187 enhanced GAD activity, whereas changes in levels of inorganic phosphate and H⁺ were without influence. Flux through GABA-T (GABA → glutamate), measured with [¹⁵N]GABA as precursor, was 0.82 nmol/min/mg of protein, whereas the reamination of succinic acid semialdehyde (reverse flux through GABA-T) was almost sixfold faster, 4.8 nmol/min/mg of protein. The rate of GABA metabolism via the tricarboxylic acid cycle was very slow, with the upper limit on flux being 0.03 nmol/min/mg of protein. Addition of either acetoacetate or β-hydroxybutyrate raised the internal content of glutamate and reduced that of aspartate; the GABA concentration and the rate of its formation increased. It is concluded that in synaptosomes (a) GABA-T is a primary factor in regulating the turnover of GABA, (b) a major regulator of GAD activity is the concentration of internal calcium, (c) GAD in nerve endings may not be saturated with its substrate, glutamate, and the concentration of the latter is a determinant of flux through this pathway, and (d) levels of ketone bodies increase, and maintain at a higher value, the synaptosomal content of GABA, a phenomenon that may contribute to the beneficial effect of a ketogenic diet in the treatment of epilepsy.