Neuronal Tricarboxylic Acid Cycle Research Articles

A Novel Biomarker of Neuronal Glutamate Metabolism in Nonhuman Primates Using Localized 1H-Magnetic Resonance Spectroscopy: Development and Effects of BNC375, an α7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulator

BackgroundThe development of treatments for cognitive deficits associated with central nervous system disorders is currently a significant medical need. Despite the great need for such therapeutics, a significant challenge in the drug development process is the paucity of robust biomarkers to assess target modulation and guide clinical decisions. We developed a novel, translatable biomarker of neuronal glutamate metabolism, the 13C-glutamate+glutamine (Glx) H3:H4 labeling ratio, in nonhuman primates using localized 1H-magnetic resonance spectroscopy combined with 13C-glucose infusions. MethodsWe began with numerical simulations in an established model of brain glutamate metabolism, showing that the 13C-Glx H3:H4 ratio should be a sensitive biomarker of neuronal tricarboxylic acid cycle activity, a key measure of overall neuronal metabolism. We showed that this biomarker can be measured reliably using a standard 1H-magnetic resonance spectroscopy method (point-resolved spectroscopy sequence/echo time = 20 ms), obviating the need for specialized hardware and pulse sequences typically used with 13C-magnetic resonance spectroscopy, thus improving overall clinical translatability. Finally, we used this biomarker in 8 male rhesus macaques before and after administration of the compound BNC375, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor that enhances glutamate signaling ex vivo and elicits procognitive effects in preclinical species. ResultsThe 13C-Glx H3:H4 ratios in the monkeys showed that BNC375 increases neuronal metabolism in nonhuman primates in vivo, detectable on an individual basis. ConclusionsThis study demonstrates that the ratio of 13C-Glx H3:H4 labeling is a biomarker that may provide an objective readout of compounds affecting glutamatergic neurotransmission and could improve decision making for the development of therapeutic agents.

Cortical Substrate Oxidation during Hyperketonemia in the Fasted Anesthetized Rat in Vivo

Ketone bodies are important alternate brain fuels, but their capacity to replace glucose and support neural function is unclear. In this study, the contributions of ketone bodies and glucose to cerebral cortical metabolism were measured in vivo in halothane-anesthetized rats fasted for 36 hours (n=6) and receiving intravenous [2,4-(13)C(2)]-D-β-hydroxybutyrate (BHB). Time courses of (13)C-enriched brain amino acids (glutamate-C4, glutamine-C4, and glutamate and glutamine-C3) were measured at 9.4 Tesla using spatially localized (1)H-[(13)C]-nuclear magnetic resonance spectroscopy. Metabolic rates were estimated by fitting a constrained, two-compartment (neuron-astrocyte) metabolic model to the (13)C time-course data. We found that ketone body oxidation was substantial, accounting for 40% of total substrate oxidation (glucose plus ketone bodies) by neurons and astrocytes. D-β-Hydroxybutyrate was oxidized to a greater extent in neurons than in astrocytes (≈ 70:30), and followed a pattern closely similar to the metabolism of [1-(13)C]glucose reported in previous studies. Total neuronal tricarboxylic acid cycle (TCA) flux in hyperketonemic rats was similar to values reported for normal (nonketotic) anesthetized rats infused with [1-(13)C]glucose, but neuronal glucose oxidation was 40% to 50% lower, indicating that ketone bodies had compensated for the reduction in glucose use.

Synthesis of neurotransmitter GABA via the neuronal tricarboxylic acid cycle is elevated in rats with liver cirrhosis consistent with a high GABAergic tone in chronic hepatic encephalopathy

Hepatic encephalopathy (HE) is a neuropsychiatric complication to liver disease. It is known that ammonia plays a role in the pathogenesis of HE and disturbances in the GABAergic system have been related to HE. Synthesis of GABA occurs by decarboxylation of glutamate formed by deamidation of astrocyte-derived glutamine. It is known that a fraction of glutamate is decarboxylated directly to GABA (referred to as the direct pathway) and that a fraction undergoes transamination with formation of alpha-ketoglutarate. The latter fraction is cycled through the neuronal tricarboxylic acid cycle, an energy-generating pathway, prior to being employed for GABA synthesis (the indirect pathway). We have previously shown that ammonia induces an elevation of the neuronal tricarboxylic acid cycle activity. Thus, the aims of the present study were to determine if increased levels of ammonia increase GABA synthesis via the indirect pathway in a rat model of HE induced by bile-duct ligation and in co-cultures of neurons and astrocytes exposed to ammonia. Employing (13) C-labeled precursors and subsequent analysis by mass spectrometry, we demonstrated that more GABA was synthesized via the indirect pathway in bile duct-ligated rats and in co-cultures subjected to elevated ammonia levels. Since the indirect pathway is associated with synthesis of vesicular GABA, this might explain the increased GABAergic tone in HE.

Compartmentalized Cerebral Metabolism of [1,6-13C]Glucose Determined by in vivo13C NMR Spectroscopy at 14.1 T

Cerebral metabolism is compartmentalized between neurons and glia. Although glial glycolysis is thought to largely sustain the energetic requirements of neurotransmission while oxidative metabolism takes place mainly in neurons, this hypothesis is matter of debate. The compartmentalization of cerebral metabolic fluxes can be determined by 13C nuclear magnetic resonance (NMR) spectroscopy upon infusion of 13C-enriched compounds, especially glucose. Rats under light α-chloralose anesthesia were infused with [1,6-13C]glucose and 13C enrichment in the brain metabolites was measured by 13C NMR spectroscopy with high sensitivity and spectral resolution at 14.1 T. This allowed determining 13C enrichment curves of amino acid carbons with high reproducibility and to reliably estimate cerebral metabolic fluxes (mean error of 8%). We further found that TCA cycle intermediates are not required for flux determination in mathematical models of brain metabolism. Neuronal tricarboxylic acid cycle rate (VTCA) and neurotransmission rate (VNT) were 0.45 ± 0.01 and 0.11 ± 0.01 μmol/g/min, respectively. Glial VTCA was found to be 38 ± 3% of total cerebral oxidative metabolism, accounting for more than half of neuronal oxidative metabolism. Furthermore, glial anaplerotic pyruvate carboxylation rate (VPC) was 0.069 ± 0.004 μmol/g/min, i.e., 25 ± 1% of the glial TCA cycle rate. These results support a role of glial cells as active partners of neurons during synaptic transmission beyond glycolytic metabolism.

Evaluation of Cerebral Acetate Transport and Metabolic Rates in the Rat Brain in vivo Using 1H-[13C]-NMR

Acetate is a well-known astrocyte-specific substrate that has been used extensively to probe astrocytic function in vitro and in vivo. Analysis of amino acid turnover curves from (13)C-acetate has been limited mainly for estimation of first-order rate constants from exponential fitting or calculation of relative rates from steady-state (13)C enrichments. In this study, we used (1)H-[(13)C]-Nuclear Magnetic Resonance spectroscopy with intravenous infusion of [2-(13)C]acetate-Na(+) in vivo to measure the cerebral kinetics of acetate transport and utilization in anesthetized rats. Kinetics were assessed using a two-compartment (neuron/astrocyte) analysis of the (13)C turnover curves of glutamate-C4 and glutamine-C4 from [2-(13)C]acetate-Na(+), brain acetate levels, and the dependence of steady-state glutamine-C4 enrichment on blood acetate levels. The steady-state enrichment of glutamine-C4 increased with blood acetate concentration until 90% of plateau for plasma acetate of 4 to 5 mmol/L. Analysis assuming reversible, symmetric Michaelis-Menten kinetics for transport yielded 27+/-2 mmol/L and 1.3+/-0.3 micromol/g/min for K(t) and T(max), respectively, and for utilization, 0.17+/-0.24 mmol/L and 0.14+/-0.02 micromol/g/min for K(M_util) and V(max_util), respectively. The distribution space for acetate was only 0.32+/-0.12 mL/g, indicative of a large excluded volume. The astrocytic and neuronal tricarboxylic acid cycle fluxes were 0.37+/-0.03 micromol/g/min and 1.41+/-0.11 micromol/g/min, respectively; astrocytes thus comprised approximately 21%+/-3% of total oxidative metabolism.

The redox switch/redox coupling hypothesis

We provide an integrative interpretation of neuroglial metabolic coupling including the presence of subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells. The subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates, depending on their relative extracellular concentration and the operation of a redox switch. This mechanism is based on the inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase by NAD + limitation, under sufficiently reduced cytosolic NAD +/NADH redox conditions. Lactate and pyruvate recycling through the plasma membrane allows the return to the extracellular medium of cytosolic monocarboxylates enabling their transcellular, reversible, exchange between neurons and astrocytes. Together, intracellular pyruvate compartmentation and monocarboxylate recycling result in an effective transcellular coupling between the cytosolic NAD +/NADH redox states of both neurons and glial cells. Following glutamatergic neurotransmission, increased glutamate uptake by the astrocytes is proposed to augment glycolysis and tricarboxylic acid cycle activity, balancing to a reduced cytosolic NAD +/NADH in the glia. Reducing equivalents are transferred then to the neuron resulting in a reduced neuronal NAD +/NADH redox state. This may eventually switch off neuronal glycolysis, favoring the oxidation of extracellular lactate in the lactate dehydrogenase (LDH) equilibrium and in the neuronal tricarboxylic acid cycles. Finally, pyruvate derived from neuronal lactate oxidation, may return to the extracellular space and to the astrocyte, restoring the basal redox state and beginning a new loop of the lactate/pyruvate transcellular coupling cycle. Transcellular redox coupling operates through the plasma membrane transporters of monocarboxylates, similarly to the intracellular redox shuttles coupling the cytosolic and mitochondrial redox states through the transporters of the inner mitochondrial membrane. Finally, transcellular redox coupling mechanisms may couple glycolytic and oxidative zones in other heterogeneous tissues including muscle and tumors.

The contribution of GABA to glutamate/glutamine cycling and energy metabolism in the rat cortexin vivo

Previous studies have shown that the glutamate/glutamine (Glu/Gln) neurotransmitter cycle and neuronal glucose oxidation are proportional (1:1), with increasing neuronal activity above isoelectricity. GABA, a product of Glu metabolism, is synthesized from astroglial Gln and contributes to total Glu/Gln neurotransmitter cycling, although the fraction contributed by GABA is unknown. In the present study, we used (13)C NMR spectroscopy together with i.v. infusions of [1,6-(13)C(2)]glucose and [2-(13)C]acetate to separately determine rates of Glu/Gln and GABA/Gln cycling and their respective tricarboxylic acid cycles in the rat cortex under conditions of halothane anesthesia and pentobarbital-induced isoelectricity. Under 1% halothane anesthesia, GABA/Gln cycle flux comprised 23% of total (Glu plus GABA) neurotransmitter cycling and 18% of total neuronal tricarboxylic acid cycle flux. In isoelectric cortex, glucose oxidation was reduced >3-fold in glutamatergic and GABAergic neurons, and neurotransmitter cycling was below detection. Hence, in both cell types, the primary energetic costs are associated with neurotransmission, which increase together as cortical activity is increased. The contribution of GABAergic neurons and inhibition to cortical energy metabolism has broad implications for the interpretation of functional imaging signals.

Regional glucose metabolism and glutamatergic neurotransmission in rat brain in vivo.

Multivolume (1)H-[(13)C] NMR spectroscopy in combination with i.v. [1,6-(13)C(2)]glucose infusion was used to detect regional glucose metabolism and glutamatergic neurotransmission in the halothane-anesthetized rat brain at 7 T. The regional information was decomposed into pure cerebral gray matter, white matter, and subcortical structures by means of tissue segmentation based on quantitative T(1) relaxation mapping. The (13)C turnover curves of [4-(13)C]glutamate, [4-(13)C]glutamine, and [3-(13)C]glutamate + glutamine were fitted with a two-compartment neuronal-astroglial metabolic model. The neuronal tricarboxylic acid cycle fluxes in cerebral gray matter, white matter, and subcortex were 0.79 +/- 0.15, 0.20 +/- 0.11, and 0.42 +/- 0.09 micromol/min per g, respectively. The glutamate-glutamine neurotransmitter cycle fluxes in cerebral gray matter, white matter, and subcortex were 0.31 +/- 0.07, 0.02 +/- 0.04, and 0.18 +/- 0.12 micromol/min per g, respectively. The exchange rate between the mitochondrial and cytosolic metabolite pools was fast relative to the neuronal tricarboxylic acid cycle flux for all cerebral tissue types.

Glial–neuronal interactions following kainate injection in rats

Limbic seizures were induced in rats by intraperitoneal injection of the glutamate receptor agonist kainic acid, followed, 24h later by injection of [1-13C]glucose and [1,2-13C]acetate. Analyses of forebrain extracts were performed using 13C magnetic resonance spectroscopy and HPLC. A significant increase in label derived from [1,2-13C]acetate was observed in glutamine and glutamate. Label in most metabolites derived from [1-13C]glucose was unchanged, however, a decrease was observed in [2-13C]GABA, possibly due to reduced GABA release, 24h after kainic acid injection. It should be noted that only astrocytes are able to utilize acetate as a substrate efficiently, whereas acetyl CoA derived from glucose is metabolized predominantly in the neuronal tricarboxylic acid cycle. No significant differences were found in total amounts of amino acids between the two groups. Thus, these results indicate that turnover of metabolites was increased predominantly in astrocytes whereas glutamatergic neurons were not affected. Previous results obtained using the same model [Neurosci. Lett. 279 (2000) 169] showed an increased turnover in both glutamatergic and GABAergic neurons 2 weeks after kainic acid injection. Combining the results from the two studies, it can be suggested that increased astrocytic activity 1 day after epileptic seizures results, subsequently, in an increased amino acid turnover in neurons.

Alterations in cerebral metabolism by the neurotoxin kainic acid studied by 13C MRS

Kainic acid is a potent agonist at the kainate subclass of ionotropic glutamate receptors, and functional kainate receptors have not only been demonstrated on neurons but also on glial cells in culture. Kainic acid injections are used to induce limbic seizures in rodents. When combined with injections of [1‐13C]glucose and [1,2‐13C]acetate followed by analyses of forebrain extracts using 13C magnetic resonance spectroscopy (MRS) and HPLC information about glial neuronal interaction can be obtained. Using kainic acid treatment and 24 h later injection of 13C label a significant increase in label derived from [1,2‐13C]acetate was observed in glutamine and glutamate. Label derived from [1‐13C]glucose was unchanged in most metabolites, however, a decrease was observed in [2‐13C]GABA. It should be noted that only astrocytes are able to utilize acetate as a substrate, whereas acetyl CoA derived from glucose is metabolized predominantly in the neuronal tricarboxylic acid cycle. These results indicate that turnover of metabolites was increased predominantly in astrocytes whereas glutamatergic neurons were not affected. However, GABAergic neurons showed decreased GABA labelling, possibly due to reduced GABA release 24 h after kainic acid injection. Taken together with results obtained 2 weeks after kainic acid injection, it can be suggested that increased astrocytic activity one day after epileptic seizures results, subsequently, in an increased amino acid turnover in neurons. Cell culture work was also performed, results will be presented at the meeting.

Study of tricarboxylic acid cycle flux changes in human visual cortex during hemifield visual stimulation using (1)H-[(13)C] MRS and fMRI.

The relationships between brain activity and accompanying hemodynamic and metabolic alterations, particularly between the cerebral metabolic rate of oxygen utilization (CMR(O2)) and cerebral blood flow (CBF), are not thoroughly established. CMR(O2) is closely coupled to the rate of tricarboxylic acid (TCA) cycle flux. In this study, the changes in glutamate labeling during (13)C labeled glucose administration were determined in the human brain as an index of alterations in neuronal TCA cycle turnover during increased neuronal activity. Two-volume (1)H-[(13)C] MR spectroscopy (MRS) of the visual cortex was combined with functional MRI (fMRI) at 4 Tesla. Hemifield visual stimulation was employed to obtain data simultaneously from activated and control regions located symmetrically in the two hemispheres of the brain. The results showed that the fractional change in the turnover rate of C4 carbon of glutamate was less than that of CBF during visual stimulation. The fractional changes in CMR(O2) (Delta CMR(O2)) induced by activation must be equal to or less than the fractional change in glutamate labeling kinetics. Therefore, the results impose an upper limit of approximately 30% for Delta CMR(O2) and demonstrate: 1) that fractional CBF increases exceed Delta CMR(O2) during elevated activity in the visual cortex, and 2) that such an unequal change would explain the observed positive blood oxygenation level dependent (BOLD) effect in fMRI. Magn Reson Med 45:349-355, 2001.

Amino acid neurotransmitter metabolism in neurones and glia following kainate injection in rats

Limbic seizure was induced in rats by intraperitoneal injection of the glutamate receptor agonist kainic acid. After 14 days [1– 13C]glucose and [1,2– 13C]acetate were injected subcutaneously and the rats killed 15 min later. Analysis of brain extracts was performed using 13C-magnetic resonance spectroscopy and high performance liquid chromatography. No significant differences between the two groups of rats were found for label concentration in blood or total metabolite tissue levels. Only astrocytes are able to utilize acetate as a substrate, whereas glucose is thought to be metabolized predominantly in the neuronal tricarboxylic acid cycle. Thus information about neuronal and astrocytic metabolism could be obtained in the same animal. A significant increase in label derived from [1– 13C]glucose was observed in metabolites such as glutamate, γ-aminobutyric acid, aspartate, and succinate (all of which are mainly labelled in neurones). The increased labelling of glutamine in epileptic rats might be due to transfer of labelled glutamate from neurones to astrocytes. Astrocytic metabolism of acetate and transfer of glutamine to neurones were not affected. The results suggest that increased neuronal activity 2 weeks following epileptic seizures produces increased amino acid turnover in neurones. Changes in astrocytic metabolism were not detected.

Effects of ammonia on the anaplerotic pathway and amino acid metabolism in the brain: an ex vivo [formula omitted] NMR spectroscopic study of rats after administering [ [formula omitted]] glucose with or without ammonium acetate

The 13 C -label incorporation into glutamate, glutamine, aspartate and γ-aminobutyric acid (GABA) from [ 2- 13 C ] glucose was measured by 13 C nuclear magnetic resonance (NMR) spectroscopy to directly examine the effects of ammonia on the activity of pyruvate carboxylase (i.e., the anaplerotic pathway) and the amino acid metabolism in the rat brain in vivo. Rats were sacrificed by exposure to microwaves at 7.5, 15, 30, and 60 min after an i.v. injection of [ 2- 13 C ] glucose with or without ammonium acetate. After the injection of ammonium acetate, the brain contents of glutamate, aspartate and GABA had decreased, however, the percentage of 13 C enrichment of C3 of glutamine, glutamate and GABA, and C2 and C3 of aspartate had increased. The 13 C entered the TCA cycle via pyruvate carboxylase from [ 2- 13 C ] glucose, labeling the C2 or C3 positions of aspartate, the C2 or C3 positions of glutamate and glutamine, and the C3 or C4 positions of GABA first and second turns of the tricarboxylic acid (TCA) cycle. The C4/C3 labeling ratio in GABA was lower than the analogous ratio in glutamate (C2/C3) and higher than that of glutamine (C2/C3). The order of these ratios (glutamate>GABA>glutamine) was not altered by the injection of ammonium acetate. These findings directly indicate that ammonia increases the anaplerotic pathway and that the 13 C -skeletons entered glial glutamine through the anaplerotic pathway flow from glia to neuron. A fraction of the glutamine is used in the direct synthesis of GABA via glutamate, whereas the remaining fraction of glutamine passed through the neuronal TCA cycle before synthesizing GABA.

Trafficking of Amino Acids between Neurons and Glia In Vivo. Effects of Inhibition of Glial Metabolism by Fluoroacetate

Glial-neuronal interchange of amino acids was studied by 13C nuclear magnetic resonance spectroscopy of brain extracts from fluoroacetate-treated mice that received [1,2-(13)C]acetate and [1-(13)C]glucose simultaneously. [13C]Acetate was found to be a specific marker for glial metabolism even with the large doses necessary for nuclear magnetic resonance spectroscopy. Fluoroacetate, 100 mg/kg, blocked the glial, but not the neuronal tricarboxylic acid cycles as seen from the 13C labeling of glutamine, glutamate, and gamma-aminobutyric acid. Glutamine, but not citrate, was the only glial metabolite that could account for the transfer of 13C from glia to neurons. Massive glial uptake of transmitter glutamate was indicated by the labeling of glutamine from [1-(13)C]glucose in fluoroacetate-treated mice. The C-3/C-4 enrichment ratio, which indicates the degree of cycling of label, was higher in glutamine than in glutamate in the presence of fluoroacetate, suggesting that transmitter glutamate (which was converted to glutamine after release) is associated with a tricarboxylic acid cycle that turns more rapidly than the overall cerebral tricarboxylic acid cycle.

Adult-onset hypothyroidism and the cerebral metabolism of (1,2-13C2) acetate as detected by 13C nuclear magnetic resonance.

The effects of adult-onset hypothyroidism on the metabolic compartmentation of the cerebral tricarboxylic acid cycle and the gamma-aminobutyric acid (GABA) shunt have been investigated by 13C nuclear magnetic resonance spectroscopy. Rats thyroidectomized as adults and age-matched controls were infused in the right jugular vein with unlabeled or (1,2-13C2) acetate solutions for 60 min. At the end of the infusion, the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by 13C nuclear magnetic resonance and reverse-phase HPLC. Thyroidectomized animals showed a decrease in the incorporation of 13C from (1,2-13C2) acetate in cerebral metabolites and an increase in the concentrations of unlabeled glutamate and GABA. Computer-assisted interpretation of the 13C multiplets observed for the carbons of glutamate, glutamine, and GABA indicated that adult-onset hypothyroidism produced 1) a decrease in the contribution of infused (1,2-13C2) acetate to the glial tricarboxylic acid cycle; 2) an increase in the contribution of unlabeled acetyl-CoA to the neuronal tricarboxylic acid cycle; and 3) impairments in the exchange of glutamate, glutamine, and GABA between the neuronal and glial compartments. Despite the fact that the adult brain has often been considered metabolically unresponsive to thyroid hormone status, present results show metabolic alterations in the neuronal and glial compartments that are reversible with substitution therapy.