Cerebral Cortex Ammonia and Glutamine Metabolism During Liver Insufficiency-Induced Hyperammonemia in the Rat

Hyperammonemia has been suggested to induce enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis and accumulation, and finally net glutamine release into the blood stream, but this has never been confirmed in liver insufficiency models. Therefore, cerebral cortex ammonia- and glutamine-related metabolism was studied during liver insufficiency-induced hyperammonemia by measuring plasma flow and venous-arterial concentration differences of ammonia and amino acids across the cerebral cortex (enabling estimation of net metabolite exchange), 1 day after portacaval shunting and 2, 4, and 6 h after hepatic artery ligation (or in controls). The intra-organ effects were investigated by measuring cerebral cortex tissue ammonia and amino acids 6 h after liver ischemia induction or in controls. Arterial ammonia and glutamine increased in portacaval-shunted rats versus controls, and further increased during liver ischemia. Cerebral cortex net ammonia uptake, observed in portacaval-shunted rats, increased progressively during liver ischemia, but net glutamine release was only observed after 6 h of liver ischemia. Cerebral cortex tissue glutamine, gamma-aminobutyric acid, most other amino acids, and ammonia levels were increased during liver ischemia. Glutamate was equally decreased in portacaval-shunted and liver-ischemia rats. The observed net cerebral cortex ammonia uptake, cerebral cortex tissue ammonia and glutamine accumulation, and finally glutamine release into the blood suggest that the rat cerebral cortex initially contributes to net ammonia removal from the blood during liver insufficiency-induced hyperammonemia by augmenting tissue glutamine and ammonia pools, and later by net glutamine release into the blood. The changes in cerebral cortex glutamate and gamma-aminobutyric acid could be related to altered ammonia metabolism.     

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.     

Cerebral Cortex Ammonia and Glutamine Metabolism in Two Rat Models of Chronic Liver Insufficiency-Induced Hyperammonemia: Influence of Pair-Feeding

Abstract: Enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis, and glutamine release into the bloodstream have been hypothesized to deplete cerebral cortex glutamate pools. We investigated this hypothesis in rats with chronic liver insufficiency-induced hyperammonemia and in pair-fed controls to rule out effects of differences in food intake. Cerebral cortex plasma flow and venous-arterial concentration differences of ammonia and amino acids, as well as cerebral cortex tissue concentrations, were studied 7 and 14 days after surgery in portacaval-shunted/bile duct-ligated, portacaval-shunted, and sham-operated rats, while the latter two were pair-fed to the first group, and in normal unoperated ad libitum-fed control rats. At both time points, arterial ammonia was elevated in the chronic liver insufficiency groups and arterial glutamine was elevated in portacaval shunt/biliary obstruction rats compared to the other groups. In the chronic liver insufficiency groups net cerebral cortex ammonia uptake was observed at both time points and was accompanied by net glutamine release. Also in these groups, cerebral cortex tissue glutamine, many other amino acid, and ammonia levels were elevated. Tissue glutamate levels were decreased to a similar level in all operated groups compared with normal unoperated rats, irrespective of plasma and tissue ammonia and glutamine levels. These results demonstrate that during chronic liver insufficiency-induced hyperammonemia, the rat cerebral cortex enhances net ammonia uptake and glutamine release. However, the decrease in tissue glutamate concentrations in these chronic liver insufficiency models seems to be related primarily to nutritional status and/or surgical trauma.     

Ammonia Assimilation in Rumen Bacteria: A Review

In the rumen bacteria, ammonia as the end product of nitrogen is incorporated into carbon skeleton (α-ketoglutarate) to yield glutamine and glutamate which are important nitrogen donors in nitrogenous compounds metabolism in cells. The enzymes glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase are involved in these processes. Some experimental results have proven that the global nitrogen regulation system may participate in the regulation of assimilation of ammonia in rumen bacteria. This review offers a current perspective on the pathways and key enzymes of ammonia assimilation in rumen bacteria with the possible molecular regulation strategy, while points out the further research direction.     

Brain Quinolinic Acid in Chronic Experimental Hepatic Encephalopathy: Effects of an Exogenous Ammonium Acetate Challenge

Abstract: Elevated brain concentrations of the neurotoxin and NMDA receptor agonist quinolinic acid (QUIN) have been demonstrated in portacaval-shunted (PCS) rats, a chronic hepatic encephalopathy (HE) model. Increased brain QUIN levels have also been shown in acute hyperammonemic rats. In the present study, the plasma and brain (neocortical) QUIN levels in chronic PCS rats were investigated. The study also included a single exogenous ammonium acetate (NH₄Ac; 5.2 mmol/kg, i.p.) challenge to precipitate a reversible hepatic coma. Compared with sham-operated controls, chronic PCS rats exhibited decreased rather than increased plasma and brain QUIN levels. The plasma-to-brain QUIN ratio was not found to be altered. The NH₄Ac administration induced coma in all of the PCS rats 20–25 min after the challenge, and this coma was resolved within 60–75 min. No relevant temporal relationship between changes in brain QUIN levels and the neurological status in the PCS rats was observed. Therefore, our results do not support the contention that increased brain QUIN levels per se are involved in the pathogenesis of HE.     

重组CHO-GS细胞降低氨毒副作用的代谢研究

在重组CHOGS细胞无血清批培养过程中,由于GS系统的引入,使氨对细胞的毒副作用显著降低,从而引起细胞生长和代谢途径发生变化。当起始氨浓度为1.42mmolL时,细胞最高密度可达到15.6×105cellsmL,随着氨浓度的增加,尽管细胞生长受到一定的抑制,但在氨浓度为12.65mmolL时,细胞密度仍可达到8.9×105cellsmL。当起始氨浓度从0.36mmolL增加到12.65mmolL时,细胞对葡萄糖的得率系数和乳酸对葡萄糖的得率系数降低,己糖激酶(HK)、丙酮酸激酶(PK)和乳酸脱氢酶(LDH)酶活分别提高了43%、140%和25%,表明细胞对葡萄糖的利用增加,糖代谢更倾向于高能量生成途径。在谷氨酰胺代谢途径中,氨促进了谷丙转氨酶(GPT)酶活,谷氨酸到α酮戊二酸的转化逐渐倾向于谷丙转氨途径,谷氨酸脱氢酶(GDH)酶活降低,脱氨途径相应受到抑制。此外,氨浓度的增加使细胞群体处于G0G1期的比例逐渐升高,当氨浓度为12.65mmolL时,重组蛋白比生产速率比氨浓度为0.36mmolL时提高了2.1倍。     

Altered Cerebral Protein Turnover in Rats Following Prolonged In Vivo Treatment with Nicotine

Turnover rates of cerebral proteins were examined in control adult rats and in those subjected to prolonged in vivo treatment with "low" (0.02 mg/ml) or "high" (0.04 mg/ml) doses of nicotine (added to drinking water), using [14C]bicarbonate as the label. It was found that the turnover of proteins in various subcellular fractions consisted of two distinct components turning over at a "fast" or a "slow" rate and having relatively short or long half-lives, respectively. Thus in control animals the half-lives of the protein components turning over at a fast rate ranged from 1.31 to 3.61 days whereas for those turning over at a slow rate the half-lives ranged from 8.56 to 24.28 days. Treatment with low doses of nicotine resulted in a more rapid turnover of nuclear fast turning over component with a concomitant decreased turnover of homogenate, cytosol, mitochondrial, and microsomal proteins; in the synaptosomal membranes this component disappeared altogether. The half-lives of the slow turning over components decreased in general from 14.3 to 33.3% with the exception of the nuclear proteins, where the half-live increased by 71.1%. Turnover of microsomal proteins was not affected. When the animals were given a high dose of nicotine, the turnover of fast components became even more rapid for nuclear, myelin, and microsomal proteins with a decrease in half-life from 26.6 to 32.3%. By contrast, half-lives of synaptosomal and mitochondrial proteins increased by 16.1-89.3%. These changes were not reflected in the turnover rate of whole homogenate proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction Between the Glutamine Cycle and the Uptake of Large Neutral Amino Acids in Astrocytes

Abstract: When astrocyte cultures are incubated with glutamate and ammonium, the clearance of these substrates followed by the formation and export of glutamine simulates the action of the "glutamine cycle" that is believed to function in the CNS. In the present study this process was found to increase the uptake of large neutral amino acids (LNAAs), namely, histidine, kynurenine, leucine, phenylalanine, and tryptophan, by two-to threefold in mouse cerebral astrocytes. The enhancement of kynurenine uptake was shown to depend on the formation of glutamine and to saturate at low levels of glutamine. LNAAs transiently lowered the glutamine content of astrocytes that were incubated with glutamate and ammonium, but they did not affect net export of glutamine to the solution at normal physiological pH. However, on adjustment of the pH of the solution to 7.8, which causes a large increase in glutamine content without affecting export, kynurenine now significantly increased net glutamine export. These findings relate to proposed mechanisms of cerebral dysfunction in hyperammonemia.     

Regional Cerebral Glucose Utilization During Insulin-Induced Hypoglycemia in Unanesthetized Rats

Regional cerebral glucose utilization (rCMRgl) was studied during insulin-induced hypoglycemia in unanesthetized rats. Rats were surgically prepared using halothane and nitrous oxide anesthesia and allowed 5 h to recover from the anesthesia before rCMRgl was measured. The rCMRgl was measured using [6-14C]glucose in a normoglycemic control group and two hypoglycemic groups, A (30 min after insulin injection) and B (2 h after insulin injection). The mean plasma glucose level was 7.03 mumol/ml in the normoglycemic group, 1.96 mumol/ml in hypoglycemic group A, and 1.40 mumol/ml in hypoglycemic group B. The rCMRgl in hypoglycemic group A decreased 8-18% in 17 brain regions measured; five changes were statistically significant. The rCMRgl in hypoglycemic group B decreased significantly in all but one of the brain regions measured; the decrease ranged from 15% in the pyramidal tract to 36% in the motor and auditory cortices. The rCMRgl in every brain region decreased when the plasma glucose level fell below 1.5-2.5 mumol/ml. No brain region could maintain rCMRgl at plasma glucose concentrations lower than predicted by regional glucose influx described in previous studies. Glucose utilization in all brain regions appears to be limited by the influx of glucose.     

Regional Differences in the Capacity for Ammonia Removal by Brain Following Portocaval Anastomosis

Portocaval anastomosis (PCA) in the rat leads, within 4 weeks, to severe liver atrophy, sustained hyperammonemia, and increased brain ammonia. Because brain is not equipped with an effective urea cycle, removal of ammonia involves glutamine synthesis and PCA results in significantly increased brain glutamine. Glutamine synthetase activities, however, are decreased by 15% in cerebral cortex and are unchanged in brainstem of shunted rats. Administration of ammonium acetate to rats following PCA results in severe encephalopathy (loss of righting reflex and, ultimately, coma). Glutamine concentrations in brainstem of comatose rats are increased a further two-fold, whereas those of cerebral cortex are unchanged. Consequently, ammonia levels in cerebral cortex reach disproportionately high levels (of the order of 5 mM). These findings suggest a limitation in the capacity of cerebral cortex to remove additional blood-borne ammonia by glutamine formation following PCA. Such mechanisms may explain the hypersensitivity of rats with PCA and of patients with portal-systemic shunting to small increases of blood ammonia. Disproportionately high levels of brain ammonia in certain regions, such as cerebral cortex, may then result in alterations of inhibitory neurotransmission and, ultimately, loss of cellular (astrocytic) integrity.     

Superoxide Production and Antioxidant Enzymes in Ammonia Intoxication in Rats

Injection of large doses of ammonium salts lead to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of injecting 7 mmol/kg of ammonium acetate on the production of superoxide and on the activities of some antioxidant enzymes in rat liver, brain, erythrocytes and plasma. Glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver and brain (both in cytoso-lic and mitochondrial fractions) and also in blood red cells, while glutathione reductase activity remained unchanged. Superoxide production in submitochon-drial particles from liver and brain was increased by more than 100% in both tissues. Both diminished activity of antioxidant enzymes and increased superoxide radical production could lead to oxidative stress and cell damage, which could be involved in the mechanism of acute ammonia toxicity.     

Physiological Levels of Ammonia Regulate Glutamine Synthesis from Extracellular Glutamate in Astrocyte Cultures

The effect of ammonia on glutamate accumulation and metabolism was examined in astrocyte cultures prepared from neonatal rat cortices. Intact astrocytes were incubated with 70 microM L-[14C(U)]glutamate and varying amounts of ammonium chloride. The media and cells were analyzed separately by HPLC for amino acids and labelled metabolites. Extracellular glutamate was reduced to 8 microM by 60 min. Removal of glutamate from the extracellular space was not altered by addition of ammonia. The rate of glutamine synthesis was increased from 3.6 to 9.3 nmol/mg of protein/min by addition of 100 microM ammonia, and intracellular glutamate was reduced from 262 to 86 nmol/mg of protein after 30 min. The metabolism of accumulated glutamate was matched nearly perfectly by the synthesis of glutamine, and both processes were proportional to the amount of added ammonia. The transamination and deamination products of glutamate were minor metabolites that either decreased or remained unchanged with increasing ammonia. Thus, ammonia addition stimulates the conversion of glutamate to glutamine in intact astrocyte cultures. At physiological concentrations of ammonia, glutamine synthesis appears to be limited by the rate of glutamate accumulation and the activity of competing reactions and not by the activity of glutamine synthetase.     

Ammonia assimilation and metabolism byBeggiatoa alba

Beggiatoa alba B18LD was investigated for its pathways of ammonia assimilation. The increase in growth yields ofB. alba with excess acetate was linear from 0.1 to 2.0 mM ammonia.B. alba had strong glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, irrespective of the ammonia concentration in the medium. Glutamate dehydrogenase activity was not found, and alanine dehydrogenase (aminating) was observed only whenB. alba was grown at high (2.0 mM) ammonia. Methionine sulfoximine, an inhibitor of GS, inhibited growth ofB. alba irrespective of the ammonia concentration in the medium. Thus it appears the primary pathway for ammonia assimilation inB. alba is via the GS-GOGAT pathway at both low and high ammonia concentrations. Preliminary experiments were unable to discern if theB. alba GS is modified by covalent modification.Non-standard abbreviations GS Glutamine synthetase - GOGAT glutamate-oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - MSX methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate aminotransferase     

Metabolism of Acetate to Amino Acids in Brains of Rats After Complete Hepatectomy

Abstract: The concentration of glutamine increases in the brain after hepatectomy. In the present studies the conversion of intravenously given [¹⁴C]acetate to [¹⁴C]glutamate and [¹⁴C]glutamine was studied in control rats and in rats at 6 h after complete hepatectomy. The incorporation of label into glutamate was only slightly inhibited, but the further incorporation into glutamine was greatly inhibited, after hepatectomy. These data, and previous data using [¹⁴C]glucose as precursor, indicate that synthesis of glutamine in brain is inhibited after hepatectomy, and suggest that its concentration must increase because degradation is inhibited to an even greater extent.     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.     

Acute and Chronic Hyperammonemia Modulate Antioxidant Enzymes Differently in Cerebral Cortex and Cerebellum

Studies on acute hyperammonemic models suggest a role of oxidative stress in neuropathology of ammonia toxicity. Mostly, a low grade chronic type hyperammonemia (HA) prevails in patients with liver diseases and causes derangements mainly in cerebellum associated functions. To understand whether cerebellum responds differently than other brain regions to chronic type HA with respect to oxidative stress, this article compares active levels of all the antioxidant enzymes vis a vis extent of oxidative damage in cerebral cortex and cerebellum of rats with acute and chronic HA induced by intra-peritoneal injection of ammonium acetate (successive doses of 10 × 10³ & 8 × 10³ μmol/kg b.w. at 30 min interval for acute and 8 × 10³ μmol/kg b.w. daily up to 3 days for chronic HA). As compared to the respective control sets, cerebral cortex of acute HA rats showed significant decline (P < 0.01–0.001) in the levels of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) but with no change in glutathione reductase (GR). In cerebellum of acute HA rats, SOD, catalase and GR though declined significantly, GPx level was found to be stable. Contrary to this, during chronic HA, levels of SOD, catalase and GPx increased significantly in cerebral cortex, however, with a significant decline in the levels of SOD and GPx in cerebellum. The results suggest that most of the antioxidant enzymes decline during acute HA in both the brain regions. However, chronic HA induces adaptive changes, with respect to the critical antioxidant enzymes, in cerebral cortex and renders cerebellum susceptible to the oxidative stress. This is supported by ∼ 2- and 3-times increases in the level of lipid peroxidation in cerebellum during chronic and acute HA respectively, however, with no change in the cortex due to chronic HA.     

Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes

Abstract: The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 m M [U-¹³C]glutamate 85% of the ¹³C metabolized was converted to glutamine. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 m M glutamate, ¹³C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [¹³C]lactate was essentially unchanged within the range of 0.2–0.5 m M glutamate, whereas the amount of [¹³C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 m M , suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 m M and astrocytes from both rat and mouse brain are consistent with these findings.     

Influence of Pathological Concentrations of Ammonia on Metabolic Fate of 14C-Labeled Glutamate in Astrocytes in Primary Cultures

Rates of glutamine formation and of carbon dioxide production (as an indication of oxidative deamination of glutamate) were determined in primary cultures of astrocytes exposed to 50 microM labeled glutamate in the absence or presence of added ammonia (0.1-3 mM). Glutamine formation (1.7 nmol/min/mg protein) was unaffected by all concentrations of added ammonia. This probably reflects the presence of a low content of ammonia (0.1-0.2 mM), originating from degradation of glutamine, in the cells even in the absence of added ammonia, and it shows that pathophysiological concentrations of ammonia do not increase the formation of glutamine from exogenous glutamate. The carbon dioxide production rate was 5.9 nmol/min/mg protein, i.e., three to four times higher than the rate of glutamine formation. It was significantly reduced (to 3.5 nmol/min/mg protein) in the presence of 1 mM or more of ammonia. This is in keeping with suggestions by others that toxic levels of ammonia affect oxidative metabolism.