High protein diet induces pericentral glutamate dehydrogenase and ornithine aminotransferase to provide sufficient glutamate for pericentral detoxification of ammonia in rat liver lobules

 The liver plays a central role in nitrogen metabolism. Nitrogen enters the liver as free ammonia and as amino acids of which glutamine and alanine are the most important precursors. Detoxification of ammonia to urea involves deamination and transamination. By applying quantitative in situ hybridization, we found that mRNA levels of the enzymes involved are mainly expressed in periportal zones of liver lobules. Free ammonia, that is not converted periportally, is efficiently detoxified in the small rim of hepatocytes around the central veins by glutamine synthetase preventing it from entering the systemic circulation. Detoxification of ammonia by glutamine synthetase may be limited due to a shortage of glutamate when the nitrogen load is high. Adaptations in metabolism that prevent release of toxic ammonia from the liver were studied in rats that were fed diets with different amounts of protein, thereby varying the nitrogen load of the liver. We observed that mRNA levels of periportal deaminating and transaminating enzymes increased with the protein content in the diet. Similarly, mRNA levels of pericentral glutamate dehydrogenase and ornithine aminotransferase, the main producers of glutamate in this zone, and pericentral glutamine synthetase all increased with increasing protein levels in the diet. On the basis of these changes in mRNA levels, we conclude that: (a) glutamate is produced pericentrally in sufficient amounts to allow ammonia detoxification by glutamine synthetase and (b) in addition to the catalytic role of ornithine in the periportally localized ornithine cycle, pericentral ornithine degradation provides glutamate for ammonia detoxification. Accepted: 16 March 1999     

Urea formation in the green vegetable bug Nezara viridula

Urea comprises 7·7 per cent of the total nitrogen excretion of Nezara viridula. The bug is capable of oxidizing uric acid to allantoin, which is also excreted, but the uricolytic pathway is not active beyond this point. Of the enzymes of the ornithine cycle, arginase and ornithine transcarbamalase are active, but there is no evidence for the arginine synthetase system. Carbamyl phosphate synthetase has a low activity detectable only by the use of radioactive substrates. Confirmation of the operation of only part of the ornithine cycle is seen in the incorporation of bicarbonate carbon into citrulline, but not into arginine or urea, by homogenates of bug tissue. It is concluded that urea in the excreta is derived from excess arginine in the diet by the action of the enzyme arginase. Free arginine is present in the cell sap of the bean pods on which the bugs feed in amounts sufficient to account for the urea excreted.     

Nitrogen assimilation in the facultative methylotroph Hyphomicrobium X

A survey of the possible nitrogen assimilation pathways in Hyphomicrobium X showed that when the nitrogen source was satisfied by ammonium sulphate or methylamine and the supply was in excess, NADPH-dependent glutamate dehydrogenase was used to assimilate nitrogen. When the nitrogen supply was limited the cells expressed high levels of glutamine synthetase and NADH-dependent glutamine:2-oxoglutamate aminotransferase activity whilst the activity of the glutamate dehydrogenase was lower. When nitrate was the N-source, the glutamine synthetase/glutamine oxoglutamate aminotransferase pathway was utilised irrespective of the nitrogen concentration in the medium. Evidence was obtained to suggest that the glutamine synthetase activity was regulated by adenylylation/deadenylylation. Carbon-limited chemostat cultures showed low glutamine synthetase activity levels but the synthesis of the enzyme was derepressed when the cultures became N-limited.     

Metabolic alterations in the red and white muscles of rat to acute ethanol treatment

Lactate (LDH) and succinate (SDH) dehydrogenases activities decreased in red and white muscles of rat under acute ethanol loading indicating the inhibition of energy metabolism and stepped up lactic acid formation under stress conditions. Aspartate aminotransferase (AAT) and glutamate dehydrogenase (GDH) were found to increase. In contrast to these, the AMP deaminase activity decreased in white muscle suggestive of decreased deamination of nucleic acids. The ornithine cycle enzymes such as argininosuccinate synthetase (ArSS) and arginase indicated diminished activities showing low level of operation of urea cycle and consequent accumulation of ammonia was observed in red muscle with low production of glutamine, whereas in the case of white muscle this trend is reversed. The possible alterations of ethanol toxicity on energy requirements, transdeamination patterns, ureogenesis and glutamine production have been discussed.     

Effect of arginine,ornithine and citrulline supplementation upon performance and metabolism of trained rats

During intense exercise there is an augmented production of ammonia and IMP in the exercised muscle that could be related to the establishment of peripheral fatigue. In order to prevent this accumulation, the urea cycle in the liver eliminates ammonia in the form of urea and the skeletal muscle buffers the increase of ammonia via transamination reactions. In the present study we evaluated the effect of arginine, citrulline and ornithine supplementation, intermediates of the urea cycle, on the performance of sedentary and swimming-trained rats submitted to a single bout of exhaustive exercise. We also measured the glycogen content of the soleus and gastrocnemius muscles and of the liver, as well as the plasma concentrations of ammonia, urea, glutamine, glucose and lactate. The results indicate that arginine, citrulline and ornithine supplementation increased the flux of substrate through the reaction catalysed by glutamine synthetase, leading to increased glutamine production after an exhaustive bout of exercise, and of the mechanism involved in ammonia buffering.     

Perturbations in nitrogen metabolism of brain and liver of rat following repeated benthiocarb administration

Effects of repeated administration of benthiocarb on the nitrogen metabolism of hepatic and neuronal systems have been studied. Repeated benthiocarb treatment was associated with significant decrease in proteins with a concomitant increase in free amino acids (FAA) and specific activity levels of proteases suggesting impaired protein synthesis or elevated proteolysis. The glycogenic aminotransferases showed a significant elevation in both the tissues indicating high feeding of ketoacids into oxidative pathway for efficient operation of TCA cycle to combat energy crisis during induced benthiocarb stress. However, the activity levels of branched-chain aminotransferases decreased suggesting their reduced contribution of intermediates to TCA cycle. A comparative evaluation of the activity levels of ammonogenic enzymes, AMP deaminase, adenosine deaminase and glutamate dehydrogenase (GDH) indicated that ammonia was mostly contributed by nucleotide deamination rather than by oxidative deamination. GDH exhibited reduced activity due to low availability of glutamate. In accordance with increased levels of urea, the activity levels of arginase, a terminal enzyme of urea cycle was increased suggesting increased urea cycle operation in order to combat the increased ammonia content. As the presence of urea cycle in the brain is rather doubtful, the conversion of ammonia to glutamine for the synthesis of GABA is envisaged in brain whereas in liver, excess ammonia was converted to urea through ornithine-arginine reacting system. The increased glutaminase activity observed during benthiocarb intoxication is accounted for counteracting acidosis or maintenance of metabolic homeostasis. Arginase, a terminal enzyme of ornithine cycle showed increased activity denoting the efficient potentiality of tissues to avert ammonia toxicity. The changes observed in tissues of rat administered with benthiocarb reflects a shift in nitrogen metabolism for efficient mobilization of end products of protein catabolism.     

The ornithine cycle enzyme arginase from Agaricus bisporus and its role in urea accumulation in fruit bodies

An extensive survey of higher fungi revealed that members of the family Agaricaceae, including Agaricus bisporus, accumulate substantial amounts of urea in their fruit bodies. An important role of the ornithine cycle enzymes in urea accumulation has been proposed. In this work, we present the cloning and sequencing of the arginase gene and its promoter region from A. bisporus. A PCR-probe based on fungal arginase was used to identify the A. bisporus arginase gene from a cDNA library. The arginase cDNA encodes a 311-aa protein which is most likely expressed in the cytosol. Expression of the cDNA in Escherichia coli was established as a His-tagged fusion protein. The arginase gene was used as a molecular marker to study expression and regulation during sporophore formation and postharvest development. The expression of the arginase gene was significantly up-regulated from developmental stage 3 onwards for all the tissues studied. A maximum of expression was reached at stage 6 for both stipe and cap tissue. In postharvest stages 5, 6 and 7 the level of expression observed was similar to normal growth stages 5, 6 and 7. A good correlation was found between arginase expression and urea content of stipe, velum, gills, cap and peel tissue. For all tissues the urea content decreased over the first four stages of development. From stage 4 onwards urea accumulated again except for stipe tissue where no significant changes were observed. The same trend was also observed for postharvest development, but the observed increase of urea in postharvest tissues was much higher.     

STUDIES ON FUNCTIONAL AND METABOLIC ROLE OF UREA CYCLE INTERMEDIATES IN BRAIN

Abstract— The distribution of argininosuccinate synthetase, argininosuccinase and arginase, and the synthesis of urea in cerebullum. cerebral cortex and brain stem have been studied. Cerebral cortex had high levels of argininosuccinate synthetase and argininosuccinase. and a high ability to synthesize urea from aspartic acid and citrulline. Of the three regions, cerebullum had the highest arginase activity. The activities of the enzymes transamidinase and ornithine aminotransferase in the metabolism of arginine and ornithine in pathways other than urea formation have been studied in the three regions of the rat brain. The activity of creatine phosphokinase in all regions was the same: carbamylphosphatase activity was highest in cerebullum. Cerebral cortex had a high activity of aspartic acid transcarba-mylase. The brain stem, among the three regions, had the lowest activities of glutamine synthetase and glutaminase. The activities of these enzymes in the different regions are discussed in relation to urea production and the utilization of the urea cycle intermediates.
Intraperitoneal injection of high amounts of citrulline brought about a rise in the glutamine synthetase activity of cerebellum and brain stem and a rise in ornithine aminotransferase in cerebral cortex and liver. These results are discussed in relation to the mechanism of action of citrulline in alleviating the toxicity in hyperammonaemic states.     

Evidence for urea cycle activity in Sporosarcina ureae

Sporosarcina ureae BS 860, a motile, sporeforming coccus, possesses the enzymes required for a functioning urea (ornithine) cycle. This is only the second known example of urea cycle activity in a prokaryote. Specific activities are reported for ornithine carbamoyltransferase, argininosuccinase, arginase, and urease. Although argininosuccinate synthetase activity could not be detected directly in crude cell extracts, indirect evidence from radiocarbon tracing data for arginine synthesis from the substrate, l-[1-¹⁴C]-ornithine, strongly suggest the presence of this or other similar enzyme activity. Furthermore, good growth in defined media containing either 1.0% glutamine, ornithine, or citrulline as sole carbon sources suggests argininosuccinate synthetase activity is necessary for arginine synthesis. The effect of varying pH on arginase and urease activities indicate that these two enzymes may function within the context of the urea cycle to generate ammonia for amino acid synthesis, as well as for raising the pH of the growth micro-environment.     

Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias)

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).     

Glutamine and ammonium handling by anaesthetized rats

The infusion of ether anesthaetized rats with 0.2 M (1 mmols in total) ammonium acetate or glutamine were compared with the infusion of 0.2 M NaCl. The levels of circulating glucose, amino acids, lactate, urea and ammonium were measured as well as liver glycogen and tissue amino acids and the liver and muscle activities of carbamoyl phosphate synthetases I and II, glutamate dehydrogenase, glutamine synthetase and adenylate deaminase. Neither treatment altered the glucose and glycogen homeostasis. The infusion of ammonium did not result in increases in circulating ammonium, but resulted in increased circulating urea after a short delay; the infusion of glutamine resulted also in urea production but much later on. Glutamine infusion also resulted in increased tissue free amino-acid levels. There was little alteration in enzyme activities, except for decreased glutamine synthetase and adenylate deaminase activity in muscle of glutamine-infused rats and higher tissue carbamoyl phosphate synthetase II. The results agree with a fast removal of infused ammonium, and maintenance of glutamine, with their channeling towards urea production at a rate comparable with that of infusion, that did not alter significantly the homeostasis of the experimental animals.     

Cell growth in arginine- and ornithine-deprived medium and conversion of glutamate to ornithine and arginine in rat hepatoma cells

Summary A subline growing in medium without arginine and ornithine was established from a rat Reuber hepatoma cell line (R-Y121B·cho). The subline designated R-Y117B·cho was able to grow in glutamine, arginine and ornithine-free, glutamate-supplemented medium. Arginine synthesis from glutamate requires four urea cycle enzymes and another two enzymes, glutamate semialdehyde dehydrogenase and ornithine aminotransferase. Since R-Y121B·cho cells have all the urea cycle enzymes, two other enzyme activities were determined. The activities of ornithine aminotransferase and glutamate semialdehyde dehydrogenase were similar in R-Y117B·cho and its parental R-Y121B·cho cells, but R-Y117B·cho cells had higher conversion of glutamate to arginine than parental cells.     

Distribution of amino acids and amino-acid enzymes in whole kidney and renal cortex. Effect of 24-h starvation

The levels of activity of amino-acid enzymes (alanine-, aspartate- and tyrosine transaminases, serine dehydratase, glutamate dehydrogenase, glutamine synthetase, arginase and adenylate deaminase) in the kidney cortex and whole kidney of control and 24-h starved adult rats have been determined from crude homogenates. The individual amino-acid content of the two kidney fractions indicated has also been studied. Serine dehydratase was found mainly in the cortex, whilst glutamine synthetase presence was mainly limited to the medulla. The distribution of adenylate deaminase and glutamate dehydrogenase is remarkably uniform along the kidney. Starvation induced decreases in arginase and adenylate deaminase activities as well as a decrease in cortical serine dehydratase. Analysis of the variance of the enzyme data showed no significant overall changes with respect to distribution and starvation. The same analysis applied to aminograms indicated only significant changes when the distribution was studied on starved samples as a whole, with changes mainly in the urea cycle and some essential amino acids. The results suggested a remarkable degree of uniformity in kidney composition both with respect to amino acid and enzyme distribution. This uniformity is not markedly affected by starvation despite the important r?le of kidney in the overall amino-acid economy of the Mammal.     

Changes in glutamate dehydrogenase activity of Chlamydomonas reinhardtii under different trophic and stress conditions

Abstract. Under stress conditions (darkness, nitrogen starvation, high ammonium concentrations, glutamine synthetase and glutamate synthase inhibition) glutamate dehydrogenase animating activity levels of Chlamydomonas cells varied inversely to those of glutamine synthetase. Nitrogen and carbon sources also influenced glutamate dehydrogenase levels in Chlamydomonas , the highest values being found in cells cultured mixotrophically with ammonium, under which conditions glutamate dehydrogenase and glutamine synthetase levels were likewise inversely related. These facts, together with the analysis of internal fluctuations of ammonium, 2-oxoglutarate, and the amino acid pool as well as the variations of certain enzymes involved in carbon metabolism indicate that glutamate dehydrogenase animating activity is adaptative, being involved in the maintenance of intracellular levels of L-glutamate when they cannot be maintained by the GS-GOGAT cycle, and probably more connected with carbon than nitrogen metabolism.     

Purification and properties of ornithine carbamoyl transferase from liver of Squalus acanthias

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.     

The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low-salinity challenge

Urea synthesis via the hepatic ornithine urea cycle (OUC) has been well described in elasmobranchs, but it is unknown whether OUC enzymes are also present in extrahepatic tissues. Muscle and liver urea, trimethylamine oxide (TMAO), and other organic osmolytes, as well as selected OUC enzymes (carbamoyl phosphate synthetase III, ornithine transcarbamoylase, arginase, and the accessory enzyme glutamine synthetase), were measured in adult little skates (Raja erinacea) exposed to 100% or 75% seawater for 5 d. Activities of all four OUC enzymes were detected in the muscle. There were no changes in muscle OUC activities in skates exposed to 75% seawater; however, arginase activity was significantly lower in the liver, compared to controls. Urea, TMAO, and several other osmolytes were significantly lower in the muscle of little skates exposed to 75% seawater, whereas only glycerophosphorylcholine was significantly lower in the liver. Urea excretion rates were twofold higher in skates exposed to 75% seawater. Taken together, these data suggest that a functional OUC may be present in the skeletal muscle tissues of R. erinacea. As well, enhanced urea excretion rates and the downregulation of the anchor OUC enzyme, arginase, in the liver may be critical in regulating tissue urea content under dilute-seawater stress.     

Changes in urea cycle-related metabolites in the mouse after combined administration of valproic acid and an amino acid load

Increased blood ammonia was induced in fasting mice by ip administration of 200 mg/kg Na-valproate followed 1 h later by 13 and 4 mmol/kg alanine and ornithine, respectively. When valproate was not used blood or liver ammonia was not increased, but increases were observed in liver glutamate (5-fold), glutamine (2-fold), aspartate (5-fold), acetylglutamate (15-fold), citrulline (35-fold), argininosuccinate (11-fold), arginine (11-fold), and urea (3-fold). The level of carbamoyl phosphate (less than 2 nmol/g) was, by far, the lowest of all urea cycle intermediates. The large increase in citrulline indicates that argininosuccinate synthesis was limiting, and that the increase in acetylglutamate induced a considerable activation of carbamoyl phosphate synthetase, which agrees with theoretical expectations, irrespective of the actual KD value for acetylglutamate. Pretreatment with valproate resulted in lower hepatic levels of glutamate, glutamine, aspartate, acetyl-CoA, and acetylglutamate. At the level found of acetylglutamate the activation of carbamoyl phosphate synthetase would be expected to be similar to that without valproate. Indeed, the levels of citrulline were similar with or without valproate. Argininosuccinate, arginine, and urea levels exhibited little if any change. Although the model used may not replicate exactly the situation in patients, from our results it appears that changes in citrullinogenesis or in other steps of the urea cycle do not account for the increase in blood ammonia induced by valproate, and it is proposed that valproate may alter glutamine metabolism.     

Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH₃ assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH₃ is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.     

Reversible reaction of cyanate with a reactive sulfhydryl group at the glutamine binding site of carbamyl phosphate synthetase.

Carbamyl phosphate synthetase from Escherichia coli reacts stoichiometrically (one to one) with [14C]cyanate to give a 14C-labeled complex which can be isolated by gel filtration. The formation of the complex is prevented if L-glutamine is present or if the enzyme is first reacted with 2-amino-4-oxo-5-chloropentanoic acid, a chloro ketone analog of glutamine which has been shown to react with a specific SH group in the glutamine binding site. The rate of complex formation is increased by ADP and decreased by ATP and HCO3-. The isolated complex is inactive with respect to glutamine-dependent synthetase activity. However, the reaction of cyanate with the enzyme is reversible. The rate of dissociation of the isolated complex is not affected by pH (over the pH range 6-10), is greatly increased by ATP and HCO3-, and is decreased by ADP. The allosteric effectors ornithine and UMP have no effect on either the rate of formation or the rate of dissociation of the complex; however, the apparent affinity of the enzyme for ATP is decreased by UMP and increased by ornithine. The site of reaction of cyanate with carbamyl phosphate synthetase, which is composed of a light and a heavy subunit, is with an SH group in the light subunit to give an S-carbamylcysteine residue. The binding of L-[14C]glutamine to the enzyme and the inhibition of glutamine-dependent synthetase activity by the chloroketone analog are both prevented by the presence of cyanate. The reaction with cyanate is considered to be with the same essential SH group which is located in the glutamine binding site and is alkylated by 2-amino-4-oxo-5-chloropentanoic acid. The bicarbonate-dependent effects of ATP suggest that formation of the activated carbon dioxide intermediate is accompanied by changes in the heavy subunit which functionally alter the properties of the glutamine binding site on the light subunit. The allosteric effects of ornithine and UMP are probably not related to this intersubunit interaction.     

Insulin regulation of RNA synthesis

During periods of nitrogen exportation from the cell, mitochondrial carbamoyl phosphate is synthesized, thus initiating the urea cycle. During times of nitrogen conservation by the liver cell, carbamoyl phosphate is synthesized in the cytosol of the cell, whereupon the de novo pyrimidine synthesis pathway is initiated. The de novo pathway provides pyrimidines for increased ribonucleic acid synthesis. Formerly, it was believed that these two pathways functioned irrespective of one another. However, recent experimental evidence indicates that, when excess ammonia is present, mitochondrial carbamoyl phosphate passes from the mitochondria into the cell cytosol, where it is metabolized by the de novo pyrimidine synthesis pathway. When ornithine and excess ammonia are both present, mitochondrial carbamoyl phosphate no longer passes from the mitochondria into the cytosol to be metabolized by the de nova pathway. Thus the metabolic fate of mitochondrial carbamoyl phosphate, and that of excess nitrogen, is determined by the presence or absence of ornithine. In turn, this key molecule is the substrate for the cytoplasmic enzyme ornithine decarboxylase. When ornithine decarboxylase is stimulated by insulin, ornithine is metabolized to putrescine. The activated ornithine decarboxylase combines with ribonucleic acid polymerase, activating the later enzyme. When ornithine is acted upon by ornithine decarboxylase, it is no longer available for the perpetuation of the urea cycle and mitochondrial carbamoyl phosphate levels rise until the carbamoyl phosphate passes into the cytosol to be metabolized by the de novo pathway. Increased amounts of pyrimidines are available for the activated ribonucleic acid polymerase. Therefore insulin, through its stimulation of ornithine decarboxylase, achieves cellular nitrogen retention by regulating nitrogen incorporation into newly synthesized ribonucleic acid.