Keiko Kanamori

Severity of Hyperammonemic Encephalopathy Correlates with Brain Ammonia Level and Saturation of Glutamine Synthetase In Vivo

Abstract: Correlation among in vivo glutamine synthetase (GS) activity, brain ammonia and glutamine concentrations, and severity of encephalopathy was examined in hyperammonemic rats to obtain quantitative information on the capacity of GS to control these metabolites implicated in the etiology of hepatic encephalopathy. Awake rats were observed for neurobehavioral impairments after ammonium acetate infusion to attain a steady‐state blood ammonia concentration of 0.9 (group A) or 1.3 µmol/g (group B). As encephalopathy progressed from grade III to IV, brain ammonia concentration increased from 1.9 to 3.3 µmol/g and then decreased to 1.3 µmol/g on recovery to grade III. In contrast, brain glutamine concentration was 26, 23, and 21 µmol/g, respectively. NH4+‐infused rats pretreated with l‐methionine dl‐sulfoximine reached grade IV when brain ammonia and glutamine concentrations were 3.0 and 5.5 µmol/g, respectively; severity of encephalopathy correlates with brain ammonia, but not glutamine. In vivo GS activity, measured by NMR, was 6.8 ± 0.7 µmol/h/g for group A and 6.2 ± 0.6 µmol/h/g for group B. Hence, the in vivo activity, shown previously to increase with blood ammonia over a range of 0.4–0.64 µmol/g, approaches saturation at blood ammonia >0.9 µmol/g. This is likely to be the major cause of the observed accumulation of brain ammonia and the onset of grade IV encephalopathy.

Quantitative determination of extracellular glutamine concentration in rat brain, and its elevation in vivo by system A transport inhibitor, α-(methylamino)isobutyrate

The basal concentration of glutamine in the extracellular fluid, [GLNECF], was determined to be 385 ± 16 μm in the cortico‐striatal region of awake rats. This in vivo concentration was determined by measuring glutamine concentrations in dialysates collected at several flow rates (0.2–4 μL/min), and extrapolating to the concentration at zero flow‐rate. Dialysate glutamine concentrations in the somatosensory cortex, hippocampus and thalamus showed no statistically significant difference. In these brain regions, [GLNECF] was elevated 1.5‐ to 1.8‐fold upon perfusion of 50–250 mmα‐(methylamino)isobutyrate (MeAIB), a competitive inhibitor of glutamine uptake by system A amino acid transporter. The results show, for the first time, that MeAIB causes elevation of brain GLNECFin vivo. The MeAIB‐induced elevation of [GLNECF] provides additional support for the current view that system A GLN transporter (Gln T/SAT 1) is the major pathway for the uptake of GLNECF by neurons, while GLN release from glia is mainly mediated by a system N transporter (SN1) which is not inhibitable by MeAIB. The steady‐state GLNECF concentration and the effectiveness of MeAIB in inhibiting neuronal GLN uptake in vivo, reported in this study, will be useful, when combined with the known in vitro kinetic properties of the GLN transporters, for study of GLN transport in the intact brain.

Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and 13C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by 13C NMR and microdialysis combined with gas‐chromatography/mass‐spectrometry. Brain GLU C5 was 13C enriched by intravenous [2,5‐13C]glucose infusion, followed by [12C]glucose infusion to chase 13C from the small glial GLU pool. This leaves [5‐13C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the 13C enrichment of whole‐brain GLU C5 was significantly lower than that of extracellular GLU (GLUECF) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5‐13C]GLUECF was monitored in vivo through the formation of [5‐13C,15N]GLN during 15NH4Ac infusion. From the rate of [5‐13C,15N]GLN synthesis (1.7 ± 0.03 µmol/g/h), the mean 13C enrichment of extracellular GLU (0.304 ± 0.011) and the 15N enrichment of precursor NH3 (0.87 ± 0.014), the rate of synthesis of GLN (V′GLN), derived from neurotransmitter GLUECF, was determined to be 6.4 ± 0.44 µmol/g/h. Comparison with VGLN measured previously by an independent method showed that the neurotransmitter provides 80–90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 ± 0.44 µmol/g/h also represents a reasonable estimate for the rate of glial uptake of GLUECF, a process that is crucial for protecting the brain from GLU excitotoxicity.

Glial Alkalinization Detected In Vivo by 1H‐15N Heteronuclear Multiple‐Quantum Coherence‐Transfer NMR in Severely Hyperammonemic Rat

Abstract: Brain [5‐15N]glutamine amide protons were selectively observed in vivo by 1H‐15N heteronuclear multiple‐quantum coherence‐transfer NMR in spontaneously breathing, severely hyperammonemic rats during intravenous [15N]ammonium acetate infusion and the subsequent recovery period. The linewidth of brain [5‐15N]‐glutamine amide proton Hz increased from 36 ± 2 Hz at 3.4 h to 58 ± 6 Hz after 5.7 h of infusion, a net increase of 22 ± 6 Hz. Concomitantly, brain ammonia concentration increased from 1.7 to 3.5 ± 0.2 µmol/g and the rat progressed from grade III to grade IV encephalopathy. On recovery to grade III and decrease of brain ammonia concentration to 1.3 µmol/g, the linewidth returned to 37 ± 2 Hz. In aqueous solution, [5‐15N]glutamine amide proton Hz underwent a 17‐Hz linebroadening when pH was raised from 7.1 to 7.5 at 37°C, due to the increased rate of base‐catalyzed exchange with water proton. Hence, linebroadening is a sensitive measure of changing intracellular pH. The 22‐Hz linebroadening observed in vivo in severely hyperammonemic grade IV rats strongly suggests that the intracellular pH increases from 7.1 to about 7.4–7.5 in astrocytes where glutamine is synthesized and mainly stored. Probable mechanisms for the ammonia‐induced alkalinization and decreased intraglial buffering capacity, as well as implications of the result for pathogenesis of hepatic encephalopathy, are discussed.

Kinetics of glial glutamine efflux and the mechanism of neuronal uptake studied in vivo in mildly hyperammonemic rat brain

Kinetics of glial glutamine (GLN) transport to the extracellular fluid (ECF) and the mechanism of GLNECF transport into the neuron – crucial pathways in the glutamine–glutamate cycle – were studied in vivo in mildly hyperammonemic rat brain, by NMR and microdialysis to monitor intra‐ and extracellular GLN. The minimum rate of glial GLN efflux, determined from the rate of GLNECF increase during perfusion of α‐(methylamino)isobutyrate (MeAIB), which inhibits neuronal GLNECF uptake by sodium‐coupled amino‐acid transporter (SAT), was 2.88 ± 0.22 μmol/g/h at steady‐state brain [GLN] of 8.5 ± 0.8 μmol/g. Our previous study showed that the rate of glutamine synthesis under identical experimental conditions was 3.3 ± 0.3 μmol/g/h. At steady‐state glial [GLN], this is equal to its efflux rate to the ECF. Comparison of the two rates suggests that SAT mediates at least 87 ± 8% (= 2.88/3.3 × 100%) of neuronal GLNECF uptake. While MeAIB induced > 2‐fold elevation of GLNECF, no sustained elevation was observed during perfusion of the selective inhibitor of LAT, 2‐amino‐bicyclo[1,1,2]heptane‐2‐carboxylic acid (BCH), or of d‐threonine, a putative selective inhibitor of ASCT2‐mediated GLN uptake. The results strongly suggest that SAT is the predominant mediator of neuronal GLNECF uptake in adult rat brain in vivo.

In vivo microdialysis and gas-chromatography/mass-spectrometry for 13C-enrichment measurement of extracellular glutamate in rat brain

Extracellular glutamate (GLU(ECF)) was collected by microdialysis from the corticostriatal region of awake rats, at the basal level and after elevation by perfusion of GLU uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid. Concurrently, [2,5-(13)C]glucose was infused intravenously to 13C-enrich brain GLU predominantly at C5. The 13C enrichment of GLU(ECF) was measured, after tert-butyldimethylsilylation, by gas-chromatography/mass-spectrometry. Excellent signal-to-noise ratios of the analyte signals at three selected ion-pairs were achieved at approximately 20 pmol. The fractional 13C enrichment of basal dialysate GLU C5, collected during 0.75-1.25 h of [2,5-(13)C]glucose infusion, was 0.263+/-0.01, very close to the enrichment of whole-brain (predominantly intracellular) GLU C5 measured in parallel NMR study. The result strongly suggests that the dialysate GLU consists predominantly of neurotransmitter GLU, which was 13C-enriched in, and released from, neurons by exocytosis and had diffused to the dialysis probe; the label is undiluted by 12C-GLU(ECF) present before the enrichment. Hence, our result supports the view, proposed on the basis of Ca(2+)- and tetrodotoxin-sensitivity of dialysate GLU, that basal dialysate GLU in awake non-stimulated brain mainly represents neurotransmitter GLU. Isotope labeling provides a novel method for determining the extent to which dialysate GLU reflects synaptic GLU(ECF), and for measuring its turnover under physiological or pathological conditions.

Reversal of metabolic deficits by lipoic acid in a triple transgenic mouse model of Alzheimer's disease: a 13C NMR study.

Alzheimers disease is an age-related neurodegenerative disease characterized by deterioration of cognition and loss of memory. Several clinical studies have shown Alzheimers disease to be associated with disturbances in glucose metabolism and the subsequent tricarboxylic acid (TCA) cycle-related metabolites like glutamate (Glu), glutamine (Gln), and N-acetylaspartate (NAA). These metabolites have been viewed as biomarkers by (a) assisting early diagnosis of Alzheimers disease and (b) evaluating the efficacy of a treatment regimen. In this study, 13-month-old triple transgenic mice (a mouse model of Alzheimers disease (3xTg-AD)) were given intravenous infusion of [1-13C]glucose followed by an ex vivo13C NMR to determine the concentrations of 13C-labeled isotopomers of Glu, Gln, aspartate (Asp), GABA, myo-inositol, and NAA. Total (12C+13C) Glu, Gln, and Asp were quantified by high-performance liquid chromatography to calculate enrichment. Furthermore, we examined the effects of lipoic acid in modulating these metabolites, based on its previously established insulin mimetic effects. Total 13C labeling and percent enrichment decreased by ∼50% in the 3xTg-AD mice. This hypometabolism was partially or completely restored by lipoic acid feeding. The ability of lipoic acid to restore glucose metabolism and subsequent TCA cycle-related metabolites further substantiates its role in overcoming the hypometabolic state inherent in early stages of Alzheimers disease.

Chronic electrographic seizure reduces glutamine and elevates glutamate in the extracellular fluid of rat brain

Effects of spontaneous seizures on extracellular glutamate and glutamine were studied in the kainate-induced rat model of epilepsy in the chronic phase. Extracellular fluid from the CA1-CA3 regions of the hippocampus was collected with a 2-mm microdialysis probe every 2 min for 5h. EEG seizures with no or mild behavioral components caused 2- to 6-fold elevation of extracellular glutamate. Concomitantly, extracellular glutamine decreased at t=5h to 48% of the initial value (n=6). The changes in extracellular glutamate and glutamine correlated with the frequency and magnitude of seizure activity. In contrast, no change in either metabolite was observed in kainate-injected rats that did not undergo seizure during microdialysis (n=6). In hippocampal tissue (9.4 ± 1.1mg) that contained the region sampled by microdialysis and the site of kainate injection, intracellular glutamine concentration was significantly reduced in the seizure group, compared to that in no-seizure group. The observed elevation of extracellular glutamate strongly suggests that neurotransmitter glutamate was released at a rate faster than the rate of its uptake into glia, possibly due to down-regulation of the transporter. This reduces the availability of substrate glutamate for glutamine synthesis, as corroborated by the observed reduction of intracellular glutamine. This is likely to reduce the rate of glutamine efflux from glia and result in the observed decrease of extracellular glutamine. There remains an intriguing possibility that seizure-induced decrease of extracellular glutamine also reflects its increased uptake into neurons to replenish neurotransmitter glutamate during enhanced epileptiform activity.

Suppression of glial glutamine release to the extracellular fluid studied in vivo by NMR and microdialysis in hyperammonemic rat brain.

Release of glial glutamine (GLN) to the extracellular fluid (ECF), mainly mediated by the bidirectional system N transporter SN1, was studied in vivo in hyperammonemic rat brain, using 15N‐nuclear magnetic resonance (NMR) to monitor intracellular [5‐15N]GLN and microdialysis/gradient 1H‐15N heteronuclear single‐quantum correlation NMR to analyse extracellular [5‐15N]GLN. GLNECF was elevated to 2.4 ± 0.2 mm after 4.5 h of intravenous ammonium acetate infusion. The [GLNi]/[GLNECF] ratio (i = intracellular) was 9.6 ± 0.9, compared with 17–20 in normal brain. GLNECF then decreased substantially at t = 4.9 ± 0.1 h. Comparison of the time‐courses of intra‐ and extra‐cellular [5‐15N]GLN strongly suggested that the observed decrease reflects partial suppression of glial GLN release to ECF. Suppression also followed elevation of GLNECF to 1.9 mm, resulting in a [GLN]i/[GLNECF] ratio of 8.4, upon perfusion of α‐(methylamino)isobutyrate which inhibits neuronal uptake of GLNECF mediated by sodium‐coupled amino acid transporter (SAT). The results provide first evidence for bidirectional operation of SN1 in vivo, and clarify the effect of transmembrane GLN gradient on glial GLN release at physiological Na+ gradient. Implications of the results for SN1 as an additional regulatory site in the glutamine/glutamate cycle and utility of this approach for examining the role of GLN in an experimental model of fulminant hepatic failure are discussed.

Hypermetabolic state in the 7-month-old triple transgenic mouse model of Alzheimer's disease and the effect of lipoic acid: a 13C-NMR study

Alzheimers disease (AD) is characterized by age-dependent biochemical, metabolic, and physiologic changes. These age-dependent changes ultimately converge to impair cognitive functions. This study was carried out to examine the metabolic changes by probing glucose and tricarboxylic acid cycle metabolism in a 7-month-old triple transgenic mouse model of AD (3×Tg-AD). The effect of lipoic acid, an insulin-mimetic agent, was also investigated to examine its ability in modulating age-dependent metabolic changes. Seven-month-old 3×Tg-AD mice were given intravenous infusion of [1-13C]glucose followed by an ex vivo 13C nuclear magnetic resonance to determine the concentrations of 13C-labeled isotopomers of glutamate, glutamine, aspartate, gamma aminobutyric acid, and N-acetylaspartate. An intravenous infusion of [1-13C]glucose+[1,2-13C]acetate was given for different periods of time to distinguish neuronal and astrocytic metabolism. Enrichments of glutamate, glutamine, and aspartate were calculated after quantifying the total (12C+13C) concentrations by high-performance liquid chromatography. A hypermetabolic state was clearly evident in 7-month-old 3×Tg-AD mice in contrast to the hypometabolic state reported earlier in 13-month-old mice. Hypermetabolism was evidenced by prominent increase of 13C labeling and enrichment in the 3×Tg-AD mice. Lipoic acid feeding to the hypermetabolic 3×Tg-AD mice brought the metabolic parameters to the levels of nonTg mice.