glutaminase and Hepatic-Encephalopathy

There is substantial clinical and experimental evidence that ammonia is a major factor in the pathogenesis of hepatic encephalopathy. In the article is demonstrated that in hepatocellular dysfunction, ammonia detoxification to glutamine (GLN) in skeletal muscle, brain, and likely the lungs, is activated. In addition to ammonia detoxification, enhanced GLN production may exert beneficial effects on the immune system and gut barrier function. However, enhanced GLN synthesis may exert adverse effects in the brain (swelling of astrocytes or altered neurotransmission) and stimulate catabolism of branched-chain amino acids (BCAA; valine, leucine, and isoleucine) in skeletal muscle. Furthermore, the majority of GLN produced is released to the blood and catabolized in enterocytes and the kidneys to ammonia, which due to liver injury escapes detoxification to urea and appears in peripheral blood. As only one molecule of ammonia is detoxified in GLN synthesis whereas two molecules may appear in GLN breakdown, these events can be seen as a vicious cycle in which enhanced ammonia concentration activates synthesis of GLN leading to its subsequent catabolism and increase in ammonia levels in the blood. These alterations may explain why therapies targeted to intestinal bacteria have only a limited effect on ammonia levels in patients with liver failure and indicate the needs of new therapeutic strategies focused on GLN metabolism. It is demonstrated that each of the various treatment options targeting only one the of the ammonia-lowering mechanisms that affect GLN metabolism, such as enhancing GLN synthesis (BCAA), suppressing ammonia production from GLN breakdown (glutaminase inhibitors and alpha-ketoglutarate), and promoting GLN elimination (phenylbutyrate) exerts substantial adverse effects that can be avoided if their combination is tailored to the specific needs of each patient.

Topics: Amino Acids, Branched-Chain; Ammonia; Brain; Critical Illness; Drug Interactions; Enterocytes; Glutamic Acid; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Hyperammonemia; Intestines; Ketoglutaric Acids; Kidney; Liver; Microbiota; Muscle, Skeletal; Organ Specificity; Phenylbutyrates

Glutamine/glutamate homeostasis must be exquisitely regulated in mammalian brain and glutaminase (GA, E.C. 3.5.1.2) is one of the main enzymes involved. The products of GA reaction, glutamate and ammonia, are essential metabolites for energy and biosynthetic purposes but they are also hazardous compounds at concentrations beyond their normal physiological thresholds. The classical pattern of GA expression in mammals has been recently challenged by the discovery of novel transcript variants and protein isoforms. Furthermore, the interactome of brain GA is also starting to be uncovered adding a new level of regulatory complexity. GA may traffic in brain and unexpected locations, like cytosol and nucleus, have been found for GA isoforms. Finally, the expression of GA in glial cells has been reported and its potential implications in ammonia homeostasis are discussed.

Topics: Animals; Astrocytes; Brain; Brain Chemistry; Glutaminase; Hepatic Encephalopathy; Humans; Isoenzymes; Nerve Tissue Proteins

Hepatic encephalopathy is the main cognitive dysfunction in cirrhotic patients associated with impaired prognosis. Hyperammonemia plus inflammatory response do play a crucial role on hepatic encephalopathy. However, in some patients HE appeared without hyperammonemia and patients with increased levels of ammonia could not show cognitive dysfunction. This has led to investigate other factors that could act in a synergistic way. Diabetes mellitus and insulin resistance are characterized by releasing and enhancing these pro-inflammatory cytokines and, additionally, has been related to hepatic encephalopathy. Indeed, patients with diabetes showed raised risk of over hepatic encephalopathy in comparison with non-cirrhotics. Type 2 diabetes mellitus could impair hepatic encephalopathy by different mechanisms that include: a) increasing glutaminase activity; b) impairing gut motility and promoting constipation, intestinal bacterial overgrowth and bacterial translocation. Despite of insufficient clarity about the practicability of anti-diabetic therapy and the most efficacious therapy, we would have to pay a special attention to the management of type 2 diabetes mellitus and insulin resistance in cirrhotic patients.

Topics: Diabetes Complications; Diabetes Mellitus; Glutaminase; Hepatic Encephalopathy; Humans; Hyperammonemia; Systemic Inflammatory Response Syndrome

Hepatic encephalopathy complicates the course of both acute and chronic liver disease and its treatment remains an unmet clinical need. Ammonia is thought to be central in its pathogenesis and remains an important target of current and future therapeutic approaches. In liver failure, the main detoxification pathway of ammonia metabolism is compromised leading to hyperammonaemia. In this situation, the other ammonia-regulating pathways in multiple organs assume important significance. The present review focuses upon interorgan ammonia metabolism in health and disease describing the role of the key enzymes, glutamine synthase and glutaminase. Better understanding of these alternative pathways are leading to the development of new therapeutic approaches.

Topics: Adipose Tissue; Amino Acids; Ammonia; Arginine; Brain; Dipeptides; Glutamate-Ammonia Ligase; Glutaminase; Hepatic Encephalopathy; Humans; Hyperammonemia; Intestinal Mucosa; Kidney; Liver; Liver Failure; Lung; Muscles; Phenylbutyrates; Sodium Benzoate

Ammonia is a major neurotoxin implicated in hepatic encephalopathy (HE). Here we discuss evidence that many aspects of ammonia toxicity in HE-affected brain are mediated by glutamine (Gln), synthesized in excess from ammonia and glutamate by glutamine synthetase (GS), an astrocytic enzyme. The degree to which Gln is increased in brains of patients with HE was found to positively correlate with the grade of HE. In animals with HE, a GS inhibitor, methionine sulfoximine (MSO), reversed a spectrum of manifestations of ammonia toxicity, including brain edema and increased intracranial pressure, even though MSO itself increased brain ammonia levels. MSO inhibited, while incubation with Gln reproduced the oxidative stress and cell swelling observed in ammonia-exposed cultured astrocytes. Recent studies have shown that astrocytes swell subsequent to Gln transport into mitochondria and its degradation back to ammonia, which then generates reactive oxygen species and the mitochondrial permeability transition. This sequence of events led to the formulation of the "Trojan Horse" hypothesis. Further verification of the role of Gln in the pathogenesis of HE will have to account for: (1) modification of the effects of Gln by interaction of astrocytes with other CNS cells; and (2) direct effects of Gln on these cells. Recent studies have demonstrated a "Trojan Horse"-like effect of Gln in microglia, as well as an interference by Gln with the activation of the NMDA/NO/cGMP pathway by ammonia as measured in whole brain, a process that likely also involves neurons.

Topics: Ammonia; Animals; Astrocytes; Brain; Cyclic GMP; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Methionine Sulfoximine; Mitochondria; Nitric Oxide

Systemic hyperammonemia has been largely found in patients with cirrhosis and hepatic encephalopathy, and ammonia plays a major role in the pathogenesis of hepatic encephalopathy. However, controversial points remain: a) the correlation between plasma ammonia levels and neurophysiological impairment. The lack of correlation between ammonia levels and grade of hepatic encephalopathy in some cases has been considered a weakness of the ammonia hypothesis, but new methods for ammonia measurements and the implication of systemic inflammation in the modulation of ammonia neurotoxicity could explain this gap; b) the source of ammonia production. Hyperammonemia has been considered as derived from urea breakdown by intestinal bacteria and the majority of treatments were targeted against bacteria-derived ammonia from the colon. However, some data suggest an important role for small intestine ammonia production: 1) the hyperammonemia after porto-caval shunted rats has been found similar in germ-free than in non-germ-free animals. 2) In cirrhotic patients the greatest hyperammonemia was found in portal drained viscera and derived mainly from glutamine deamination. 3) The amount of time required to increase of ammonia (less than one hour) after oral glutamine challenge supports a small intestine origin of the hyperammonemia. As the main source of ammonia in cirrhotics derives from portal drained viscera owing to glutamine deamidation, increased glutaminase activity in the intestine seems to be responsible for systemic hyperammonemia. Lastly, some genetic alterations in the glutaminase gene such as the haplotype TACC could modulate intestinal ammonia production and the risk of overt hepatic encephalopathy in cirrhotics.

Topics: Ammonia; Animals; Genetic Predisposition to Disease; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Hyperammonemia; Intestine, Small; Liver Circulation; Liver Failure, Acute; Mutation

Minimal hepatic encephalopathy (MHE) is defined by the presence of neurophysiological alterations,with an important impact in the quality of life, in the risk of performing dangerous tasks as leading cars and heavy machinery and increases risk of overt hepatic encephalopathy. MHE is present in a third of cirrhotic depending on liver function. Psychometric and neurophysiologic test are used in the diagnosis of MHE, mainly PHES (Psychometric Hepatic Encephalopathy Score) battery, electroencephalogram, evoked potentials and measurement of the critical flicker frequency. Oral glutamine challenge (OGC) measures intestinal ammonia production after glutamine intake and indirectly intestinal glutaminase activity. Altered OGC in patients with MHE predicts short-time survival. In conclusion,MHE is the first stage in HE syndrome, affect to a third of cirrhotic and worsen quality of life. There are useful and easy-to-use diagnostic tests and new therapeutic options are warranted.

Topics: Glutaminase; Hepatic Encephalopathy; Humans; Hyperammonemia

Disturbed body nitrogen homeostasis due to impaired hepatic urea synthesis leads to an alteration in inter-organ ammonia trafficking, resulting in hyperammonemia. Glutamine (Gln) synthase is the alternative pathway for ammonia detoxification. Gln taken up by several organs is split by the intramitochondrial phosphate-activated enzyme glutaminase (PAG) into glutamate (Glu) and ammonia. In cirrhotic patients with portosystemic intrahepatic shunt, the main source of systemic hyperammonemia is the small intestine, and ammonia derives mainly from Gln deamidation. Recently, PAG has been found increased in cirrhotics showing minimal hepatic encephalopathy and, therefore, could be implicated in the production of systemic hyperammonemia in these patients. Intestinal PAG activity correlates with psychometric test and magnetic resonance spectroscopy findings. Moreover, nitric oxide and tumor necrosis factor seem to be the major factors regulating intestinal ammonia production in cirrhotics. In the brain, PAG localized into the astrocytes is responsible for ammonia and free-radical production. The blockade of PAG, using 6-oxo-5-norleucine, avoids the toxic effects of Gln accumulation in the brain. These data support an important role for intestinal and brain glutaminase in the pathogenesis of hepatic encephalopathy and could be a new target for future therapies.

Topics: Animals; Brain; Digestive System; Gene Expression Regulation, Enzymologic; Glutaminase; Hepatic Encephalopathy; Humans

Topics: Ammonia; Brain; Brain Edema; Dipeptides; Flumazenil; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Hyperammonemia; Inflammation; Intestine, Small; Kidney; Memantine; Models, Molecular; Nerve Tissue Proteins; Nitric Oxide

Topics: Acidosis; Animals; Aspartate Aminotransferases; Aspartic Acid; Brain; Cells, Cultured; Energy Metabolism; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamates; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Intestine, Small; Intracellular Membranes; Kidney; Liver; Mitochondria; Mitochondria, Liver; Muscles; Neoplasms; Organ Specificity; Oxidation-Reduction; Rats

Chronic hyperammonemia is a main contributor to the cognitive and motor impairment in patients with hepatic encephalopathy. Sustained hyperammonemia induces the TNFα expression in Purkinje neurons, mediated by NF-κB activation. The aims were the following: (1) to assess if enhanced TrkB activation by BDNF is responsible for enhanced NF-κB activation in Purkinje neurons in hyperammonemic rats, (2) to assess if this is associated with increased content of NF-κB modulated proteins such as TNFα, HMGB1, or glutaminase I, (3) to assess if these changes are due to enhanced activation of the TNFR1-S1PR2-CCR2-BDNF-TrkB pathway, (4) to analyze if increased activation of NF-κB is mediated by the PI3K-AKT pathway. It is shown that, in the cerebellum of hyperammonemic rats, increased BDNF levels enhance TrkB activation in Purkinje neurons leading to activation of PI3K, which enhances phosphorylation of AKT and of IκB, leading to increased nuclear translocation of NF-κB which enhances TNFα, HMGB1, and glutaminase I content. To assess if the changes are due to enhanced activation of the TNFR1-S1PR2-CCR2 pathway, we blocked TNFR1 with R7050, S1PR2 with JTE-013, and CCR2 with RS504393. These changes are reversed by blocking TrkB, PI3K, or the TNFR1-SP1PR2-CCL2-CCR2-BDNF-TrkB pathway at any step. In hyperammonemic rats, increased levels of BDNF enhance TrkB activation in Purkinje neurons, leading to activation of the PI3K-AKT-IκB-NF-κB pathway which increased the content of glutaminase I, HMGB1, and TNFα. Enhanced activation of this TrkB-PI3K-AKT-NF-κB pathway would contribute to impairing the function of Purkinje neurons and motor function in hyperammonemic rats and likely in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Topics: Animals; Brain-Derived Neurotrophic Factor; Glutaminase; Hepatic Encephalopathy; HMGB1 Protein; Hyperammonemia; Microglia; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Purkinje Cells; Rats; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha

Hyperammonemia plays a main role in the neurological impairment in cirrhotic patients with hepatic encephalopathy. Rats with chronic hyperammonemia reproduce the motor incoordination of patients with minimal hepatic encephalopathy, which is due to enhanced GABAergic neurotransmission in cerebellum as a consequence of neuroinflammation. Extracellular vesicles (EVs) could play a key role in the transmission of peripheral alterations to the brain to induce neuroinflammation and neurological impairment in hyperammonemia and hepatic encephalopathy. EVs from plasma of hyperammonemic rats (HA-EVs) injected to normal rats induce neuroinflammation and motor incoordination, but the underlying mechanisms remain unclear. The aim of this work was to advance in the understanding of these mechanisms. To do this we used an ex vivo system. Cerebellar slices from normal rats were treated ex vivo with HA-EVs. The aims were: 1) assess if HA-EVs induce microglia and astrocytes activation and neuroinflammation in cerebellar slices of normal rats, 2) assess if this is associated with activation of the TNFR1-NF-kB-glutaminase-GAT3 pathway, 3) assess if the TNFR1-CCL2-BDNF-TrkB pathway is activated by HA-EVs and 4) assess if the increased TNFα levels in HA-EVs are responsible for the above effects and if they are prevented by blocking the action of TNFα. Our results show that ex vivo treatment of cerebellar slices from control rats with extracellular vesicles from hyperammonemic rats induce glial activation, neuroinflammation and enhance GABAergic neurotransmission, reproducing the effects induced by hyperammonemia

Topics: Animals; Ataxia; Brain-Derived Neurotrophic Factor; Cerebellum; Extracellular Vesicles; Glutaminase; Hepatic Encephalopathy; Hyperammonemia; Liver Cirrhosis; Neuroinflammatory Diseases; NF-kappa B; Rats; Rats, Wistar; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha

to analyze the effect of metformin on ammonia production derived from glutamine metabolism in vitro and in vivo.. twenty male Wistar rats were studied for 28 days after a porto-caval anastomosis (n = 16) or a sham operation (n = 4). Porto-caval shunted animals were randomized into two groups (n = 8) and either received 30 mg/kg/day of metformin for two weeks or were control animals. Plasma ammonia concentration, Gls gene expression and K-type glutaminase activity were measured in the small intestine, muscle and kidney. Furthermore, Caco2 were grown in different culture media containing glucose/glutamine as the main carbon source and exposed to different concentrations of the drug. The expression of genes implicated in glutamine metabolism were analyzed.. metformin was associated with a significant inhibition of glutaminase activity levels in the small intestine of porto-caval shunted rats (0.277 ± 0.07 IU/mg vs 0.142 ± 0.04 IU/mg) and a significant decrease in plasma ammonia (204.3 ± 24.4 µg/dl vs 129.6 ± 16.1 µg/dl). Glucose withdrawal induced the expression of the glutamine transporter SLC1A5 (2.54 ± 0.33 fold change; p < 0.05). Metformin use reduced MYC levels in Caco2 and consequently, SLC1A5 and GLS expression, with a greater effect in cells dependent on glutaminolytic metabolism.. metformin regulates ammonia homeostasis by modulating glutamine metabolism in the enterocyte, exerting an indirect control of both the uptake and degradation of glutamine. This entails a reduction in the production of metabolites and energy through this pathway and indirectly causes a decrease in ammonia production that could be related to a decreased risk of HE development.

Topics: Ammonia; Animals; Caco-2 Cells; Child, Preschool; Glutaminase; Glutamine; Hepatic Encephalopathy; Humans; Hypoglycemic Agents; Male; Metformin; Rats; Rats, Wistar

In a recent study, microsatellite variations (GCA tandem repeats) in the promoter region of the (kidney-type) glutaminase gene were associated with the development of hepatic encephalopathy (HE) in Spanish patients with cirrhosis. The objective of this study was to validate the relation between microsatellite variations in the glutaminase promoter region and the development of overt HE in Korean patients with liver cirrhosis.. We performed a prospective cohort study of 154 cirrhotic patients who underwent a glutaminase microsatellite study without previous overt HE history at baseline. The primary end point was the first episode of overt HE. The microsatellite length was categorized into three groups based on its repeated number, with a cutoff value of 14; 65 (42.2%), 70 (45.5%), and 19 (12.3%) patients had the short-short, short-long, and long-long alleles, respectively.. Over a median 3.5 years of follow-up (range = 0.1-4.4), overt HE developed in 28 patients (18.2%). The 3-year cumulative incidence of overt HE was 18.4%. Multivariate Cox model indicated that past hepatocellular carcinoma history, alcoholic etiology for cirrhosis, higher Model for End-Stage Liver Disease scores and their deterioration, and serum ammonium levels were independently associated with HE development. However, microsatellite length was not associated with the development of overt HE.. In Korean patients with cirrhosis, microsatellite variations in the glutaminase promoter region were not associated with development of overt HE. Thus, additional studies are needed to identify other genetic factors related to glutaminase activity in Asians with overt HE.

Topics: Aged; Alleles; Asia, Eastern; Asian People; Female; Follow-Up Studies; Genetic Association Studies; Glutaminase; Hepatic Encephalopathy; Humans; Incidence; Kidney; Liver Cirrhosis; Male; Microsatellite Repeats; Middle Aged; Promoter Regions, Genetic; Prospective Studies; Tandem Repeat Sequences

Hepatic encephalopathy (HE) is a serious complication of liver cirrhosis. Recently, a microsatellite in the promoter region of the phosphate-activated glutaminase (GLS ) gene was associated with the risk of HE. The aim of the present study was to investigate, using the critical flicker frequency (CFF) test, whether the described GLS variant increases the risk of developing HE in patients with cirrhosis.. We recruited 158 patients (66% men; mean age 59 years, range 23-86) with liver cirrhosis. Mean model for end-stage liver disease score was 13.8 (range 5-35); 48% of patients presented with Child-Pugh score B or C. The presence and severity of HE were determined by the CFF test, with frequencies ≤39 Hz denoting cases. GLS variants were genotyped by sequencing the microsatellite in the promoter region and were classified as short, long or short-long forms (depending on the length of the macrosatellite alleles).. In total, 53% of patients had abnormal CFF results (i.e. ≤39 Hz; range for entire cohort 26-57). The GLS microsatellite distribution amongst patients was short form (20%), long form (32%) and short-long form (48%) and was consistent with Hardy-Weinberg equilibrium. CFF values differed significantly between groups (P = 0.043). Carriers of the GLS long microsatellite had a significantly higher risk of HE according to multivariate analyses (odds ratio 3.23, 95% confidence interval 1.46-7.13, P = 0.004).. CFF results were significantly lower amongst carriers of the GLS long microsatellite. Our findings support the role of the GLS long microsatellite in the development of HE; this could be important for identifying susceptible patients and for the prevention of this condition.

Topics: Adult; Aged; Aged, 80 and over; Female; Genetic Variation; Glutaminase; Hepatic Encephalopathy; Humans; Logistic Models; Male; Microsatellite Repeats; Middle Aged; Promoter Regions, Genetic

Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing Vmax and Km values of 384.62 µmol min(-1), 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.

Topics: Ammonia; Caco-2 Cells; Cell Survival; Drug Discovery; Enzyme Inhibitors; Glutaminase; Hepatic Encephalopathy; Humans; Small Molecule Libraries; Thiourea

To investigate the influence of metformin use on liver dysfunction and hepatic encephalopathy in a retrospective cohort of diabetic cirrhotic patients. To analyze the impact of metformin on glutaminase activity and ammonia production in vitro.. Eighty-two cirrhotic patients with type 2 diabetes were included. Forty-one patients were classified as insulin sensitizers experienced (metformin) and 41 as controls (cirrhotic patients with type 2 diabetes mellitus without metformin treatment). Baseline analysis included: insulin, glucose, glucagon, leptin, adiponectin, TNFr2, AST, ALT. HOMA-IR was calculated. Baseline HE risk was calculated according to minimal hepatic encephalopathy, oral glutamine challenge and mutations in glutaminase gene. We performed an experimental study in vitro including an enzymatic activity assay where glutaminase inhibition was measured according to different metformin concentrations. In Caco2 cells, glutaminase activity inhibition was evaluated by ammonia production at 24, 48 and 72 hours after metformina treatment.. Hepatic encephalopathy was diagnosed during follow-up in 23.2% (19/82): 4.9% (2/41) in patients receiving metformin and 41.5% (17/41) in patients without metformin treatment (logRank 9.81; p=0.002). In multivariate analysis, metformin use [H.R.11.4 (95% CI: 1.2-108.8); p=0.034], age at diagnosis [H.R.1.12 (95% CI: 1.04-1.2); p=0.002], female sex [H.R.10.4 (95% CI: 1.5-71.6); p=0.017] and HE risk [H.R.21.3 (95% CI: 2.8-163.4); p=0.003] were found independently associated with hepatic encephalopathy. In the enzymatic assay, glutaminase activity inhibition reached 68% with metformin 100 mM. In Caco2 cells, metformin (20 mM) decreased glutaminase activity up to 24% at 72 hours post-treatment (p<0.05).. Metformin was found independently related to overt hepatic encephalopathy in patients with type 2 diabetes mellitus and high risk of hepatic encephalopathy. Metformin inhibits glutaminase activity in vitro. Therefore, metformin use seems to be protective against hepatic encephalopathy in diabetic cirrhotic patients.

Topics: Age Factors; Ammonia; Diabetes Complications; Enzyme Inhibitors; Female; Glutaminase; Hepatic Encephalopathy; Humans; Liver Cirrhosis; Male; Metformin; Retrospective Studies; Sex Factors; Spain

Hepatic encephalopathy is a major complication of cirrhosis and is associated with a poor prognosis.. To identify mutations in the gene sequence for glutaminase in humans that could be responsible for the development of hepatic encephalopathy in patients with cirrhosis.. Cohort study.. Outpatient clinics in 6 Spanish hospitals.. 109 consecutive patients with cirrhosis in the estimation cohort, 177 patients in the validation cohort, and 107 healthy control participants.. Patients were followed every 3 or 6 months until the development of hepatic encephalopathy or liver transplantation, death, or the end of the study.. The genetic analyses showed that glutaminase TACC and CACC haplotypes were linked to the risk for overt hepatic encephalopathy. Mutation scanning of the glutaminase gene identified a section in the promoter region where base pairs were repeated (a microsatellite). Over a mean follow-up of 29.6 months, hepatic encephalopathy occurred in 28 patients (25.7%) in the estimation cohort. Multivariable Cox models were used to determine the following independent predictors: Child-Turcotte-Pugh stage (hazard ratio [HR], 1.6 [95% CI, 1.29 to 1.98]; P = 0.001), minimal hepatic encephalopathy (HR, 3.17 [CI, 1.42 to 7.09]; P = 0.006), and having 2 long alleles of the microsatellite (HR, 3.12 [CI, 1.39 to 7.02]; P = 0.006). The association between 2 long alleles of the microsatellite and overt hepatic encephalopathy was confirmed in a validation cohort (HR, 2.1 [CI, 1.17 to 3.79]; P = 0.012). Functional studies showed higher luciferase activity in cells transfected with the long form of the microsatellite, which suggests that the long microsatellite enhances glutaminase transcriptional activity.. Other genes and allelic variants might be involved in the clinical expression of hepatic encephalopathy.. This study identifies a genetic factor that is associated with development of hepatic encephalopathy in patients with cirrhosis.. Instituto de Salud Carlos III, Spanish Ministry of Health.

Topics: Aged; Female; Glutaminase; Hepatic Encephalopathy; Humans; Liver Cirrhosis; Male; Microsatellite Repeats; Middle Aged; Mutation; Promoter Regions, Genetic; Risk Factors

Topics: Glutaminase; Hepatic Encephalopathy; Humans; Liver Cirrhosis; Mutation; Promoter Regions, Genetic

To assess whether portacaval anastomosis (PCA) in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic encephalopathy (HE).. Sixteen male Wistar rats weighing 250-350 g were grouped into sham-operation control (n=8) or portacaval shunt (n=8). Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase (PAG) activity was determined by measuring ammonia production following incubation for one hour at 37 celsius degree with O-phthalaldehyde (OPA) and specific activity expressed in units per gram of protein (mukat/g of protein). Protein expression was measured by immunoblotting.. Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats (duodenum PAG activity was 976.95+/-268.87 mukat/g of protein in PCA rats vs 429.19+/-126.92mukat/g of protein in sham-operated rats; kidneys PAG activity was 1259.18+/-228.79 mukat/g protein in PCA rats vs 669.67+/-400.8 mukat/g of protein in controls, P<0.05; duodenal protein content: 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats). PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in sham-operated rats (cortex: 6646.6+/-1870.4 mukat/g of protein vs 3573.8+/-2037.4 mukat/g of protein in control rats, P<0.01; basal ganglia, PAG activity was 3657.3+/-1469.6 mukat/g of protein in PCA rats vs 2271.2+/-384 mukat/g of protein in sham operated rats, P<0.05; In the cerebellum, the PAG activity was 2471.6+/-701.4 mukat/g of protein vs 1452.9+/-567.8 mukat/g of protein in the PCA and sham rats, respectively, P<0.05; content of protein: cerebral cortex: 162%+/-40% vs 100%+/-26%, P<0.009; and basal ganglia: 140%+/-39% vs 100%+/-14%, P<0.05; but not in cerebellum: 100%+/-25% vs 100%+/-16%, P=ns).. Increased PAG activity in kidney and duodenum could contribute significantly to the hyperammonaemia in PCA rats, animal model of encephalopathy. PAG is increased in non-synaptic mitochondria from the cortex and basal ganglia and could be implicated in the pathogenesis of hepatic encephalopathy. Therefore, PAG could be a possible target for the treatment of HE or liver dysfunction.

Topics: Ammonia; Animals; Basal Ganglia; Cerebral Cortex; Disease Models, Animal; Duodenum; Glutaminase; Hepatic Encephalopathy; Humans; Kidney; Male; Portacaval Shunt, Surgical; Rats; Rats, Wistar

We performed the current study to assess the intestinal activity of enterocyte phosphate-activated glutaminase (PAG) in cirrhosis.. Forty-nine cirrhotic patients and 36 control subjects underwent endoscopic duodenal biopsies. Minimal hepatic encephalopathy (MHE) was evaluated using three psychometric tests. Oral glutamine challenge (OGC) was performed and MELD, Child-Pugh and the presence of esophageal varices were recorded. PAG was measured by enzymatic methods. Cerebral magnetic resonance spectroscopy was performed in 10 cirrhotics.. PAG was found to be higher in cirrhotics than control subjects 2.4+/-1.51 vs. 0.68+/-0.57IU/mg protein (P<0.001). PAG was also increased in patients with MHE and correlated with MELD, INR, esophageal varices and serum bile acids. A negative correlation was observed between PAG activity and intra-cerebral choline/creatine ratio (r=-0.67; P=0.035) and a positive correlation with glutamine plus glutamate/creatine ratio (r=0.78; P=0.007). In multivariate analysis using backward logistic regression, presence of MHE was the only variable independently related to altered enterocyte PAG.. Enterocyte PAG is increased in cirrhotic patients and correlates with MHE. These data support a possible role for intestinal glutaminase in the pathogenesis of hepatic encephalopathy (HE) and could be a new target for future therapies.

Topics: Adult; Female; Glutaminase; Hepatic Encephalopathy; Humans; Intestines; Liver Cirrhosis; Logistic Models; Magnetic Resonance Spectroscopy; Male; Middle Aged; Multivariate Analysis; Portal System; Psychometrics; Severity of Illness Index

The phosphate-dependent (PAG) and phosphate-independent (PIndG) glutaminase activities were measured in cerebral perikaryal mitochondria derived from rats subjected to ammonium acetate- induced "simple" hyperammonemia (SHA) or thioacetamide-induced hepatic encephalopathy (HE). These two moderately hyperammonemic conditions were previously found to be accompanied by pronounced changes in virtually all the enzyme activities coupling the tricarboxylic acid cycle to the synthesis and metabolism of the excitatory neurotransmitter glutamate. Both PAG and PIndG remained unaffected by SHA or HE, indicating that they do not contribute to the cerebral glutamine/glutamate imbalance associated with both conditions.

Topics: Acetates; Animals; Brain; Citric Acid Cycle; Glutaminase; Hepatic Encephalopathy; Male; Mitochondria; Phosphates; Rats; Rats, Wistar; Thioacetamide

To detect possible changes in the regulation of glutamate/gamma-aminobutyric acid (GABA) enzymes at the level of gene expression in a thioacetamide-induced rat model of acute hepatic encephalopathy, we have examined changes in the mRNAs of four glutamate/GABA enzymes by quantitative RNA blot hybridization analysis. Such changes could reflect cell adaptation to excess ammonia or some other associated metabolic stress. The mRNA levels of glutamate dehydrogenase (GDH) decreased similarly in three different brain regions, whereas those of glutamine synthetase (GS) and glutaminase (GA) increased. The mRNA levels of glutamate decarboxylase (GAD) were unchanged. The results indicate that some effect of liver damage, presumably hyperammonemia, affected the expression of some, but not all, genes associated with ammonia and glutamate metabolism in the brain. This adaptation of gene expression to secondary effects of ammonia on brain amino acid neurotransmitter metabolism or brain energy metabolism could play a role in the physiological changes observed in hepatic encephalopathy.

Topics: Amino Acids; Ammonia; Animals; Brain; Energy Metabolism; Glutamate Decarboxylase; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamates; Glutaminase; Hepatic Encephalopathy; Liver; Nucleic Acid Hybridization; Plasma; Rats; Rats, Inbred Strains; RNA, Messenger

Topics: Acyltransferases; Amidohydrolases; Ammonia; Animals; Asparaginase; Brain; Cats; Central Nervous System; Dogs; Energy Metabolism; Enzymes; Epilepsy; Glutamate Dehydrogenase; Glutaminase; Hepatic Encephalopathy; Humans; Hypercapnia; Hypoxia; Mice; Rats; Synapses; Transglutaminases; Urea

In vitro measurements of basal efflux and evoked release of exogenous and endogenous glutamate from rat hippocampus showed that exposition of the tissue to pathophysiological concentrations of ammonium chloride (3 mM) for 40--60 min abolished the KCl-induced release of endogenous glutamate. Basal efflux of endogenous glutamate was elevated by ammonium chloride with a delay of 30--40 min. In an attempt to link these observations to the pathogenesis of hepatic coma, rats were subjected to porta-cava anastomosis, and the hippocampal tissue was tested for ammonia sensitivity 3--4 weeks after the operation. The results showed that ammonia perfusion of the tissue was without effect effect on either basal or KCl-evoked release of endogenous glutamate. In addition, the utilization of glutamine for glutamate formation was influenced in hippocampal but not cortical tissue form porta-cava anastomized rats. The results suggest that ammonia is an important factor for the disturbed metabolism of the neurotransmitter pool of glutamate in hepatic failure.

Topics: Ammonia; Ammonium Chloride; Animals; Female; Glutamates; Glutaminase; Hepatic Encephalopathy; Hippocampus; Portacaval Shunt, Surgical; Rats