Ornithine Cycle Enzymes Research Articles

Gene expression profiling indicates a shift in ammonia assimilation capacity along the hepatic acinus induced by different forms of selenium in vitamin–mineral mixes fed to beef steers grazing on toxic endophyte-infected tall fescue

Recently, we demonstrated that supplementing (with 3 mg Se per day) the diets of growing beef steers grazing on Se-deficient toxic endophyte-infected tall fescue-based forage with either organic Se (OSe, SEL-PLEX) or a 1:1 blend (MIX) of OSe and inorganic Se (ISe, sodium selenite) in vitamin–mineral mixes, rather than inorganic forms of Se, ameliorated several classic serum symptoms of fescue toxicosis. Importantly, higher levels of hepatic glutamine synthetase activity were observed in MIX and OSe steers. Accordingly, transcriptome level and targeted mRNA expression analyses were conducted on the same liver tissue to determine if Se treatments affected other hepatic metabolic pathways, especially those that are responsible for supplying substrates of glutamine synthetase. The effect of ISe, OSe, and MIX treatments (n = 8/treatment) on the relative abundances of mRNA [determined using microarray and real-time reverse-transcription PCR (RT-PCR)] and protein (determined using Western blotting) in liver tissue was assessed by ANOVA. Fisher’s protected LSD procedure was used to separate treatment means, with significance being declared at p ≤ 0.05. Microarray analysis identified (p< 0.01, false discovery rate of< 33%) 573 annotated differentially expressed gene (DEG) transcripts. Canonical pathway analysis identified the DEGs that are central to glutamine and glutamate biosynthesis/degradation and proline biosynthesis. Targeted RT-PCR analyses found that MIX and OSe steers had lower periportal ammonia-assimilation and urea-synthesizing capacities (lower glutaminase 2, key ornithine cycle enzymes, and mitochondrial ornithine/citrulline exchanger mRNA) than ISe steers. In addition, MIX and OSe steers had a higher capacity for pericentral ammonia assimilation (higher glutamine synthetase activity) and a higher capacity for the production of glutamate in pericentral hepatocytes from α-ketoglutarate (higher levels of glutamine dehydrogenase, and decreased levels of arginase 2 and ornithine aminotransferase and mRNA). The form of supplemental Se also affected steers’ capacity for hepatic proline metabolism, with OSe steers having a higher capacity for proline synthesis and MIX steers having a higher capacity for pyroline-5-carboxylate synthesis. In conclusion, supplementing the diets of growing beef steers grazing on toxic endophyte-infected tall fescue with MIX and OSe in vitamin–mineral mixes, rather than with inorganic forms of Se, shifts hepatic ammonia assimilation from periportal urea production to pericentral glutamine production, thus potentially increasing whole-animal N efficiency by increasing the supply of hepatic-synthesized glutamine.

C/EBPα knock-in hepatocytes exhibit increased albumin secretion and urea production

The CCAAT/enhancer-binding protein alpha (C/EBP alpha) is essential for maintaining the differentiated state of hepatocytes in vivo. C/EBP alpha activates albumin transcription and coordinates the expression of multiple ornithine cycle enzymes involved in urea production. We have examined the effects of the C/EBP alpha knock-in gene on the mRNA and protein expression of genes involved in liver-specific functions, such as albumin and urea production. Albumin mRNA and protein levels are higher in knock-in hepatocytes compared with wild-type hepatocytes. mRNA levels for the ornithine cycle enzymes, namely arginase, carbamylphosphate synthetase 1, and ornithine transcarbamylase, also increase. Albumin secretion and urea production are sustained at higher levels in culture in knock-in hepatocytes. We have previously established that C/EBP alpha induces early liver glycogen storage in C/EBP alpha knock-in mice. Our present observations underline the importance of C/EBP alpha in liver metabolism and suggest that the induction of C/EBP alpha expression enhances hepatocyte function.

Argininosuccinate synthetase and argininosuccinate lyase: two ornithine cycle enzymes from Agaricus bisporus

Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. In this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase ( ass) and argininosuccinate lyase ( asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47 266 Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61–63 % and 51–55 % identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52 337 Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59–60 % identity. Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3 h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology.

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.

CCAAT/enhancer-binding protein β is required for activation of genes for ornithine cycle enzymes by glucocorticoids and glucagon in primary-cultured hepatocytes

Transcription of genes for enzymes of the ornithine cycle is activated by hormones such as glucocorticoids and glucagon. Promoters and enhancers of several genes for the enzymes interact with the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, and C/EBPβ has been suggested to mediate glucocorticoid response of the gene for arginase, the last enzyme of the cycle. To determine the contribution of C/EBPβ to hormonal regulation of genes for ornithine cycle enzymes, we examined mice with targeted disruption of the C/EBPβ gene. Induction of genes for the enzymes by intraperitoneal injection of dexamethasone and glucagon was almost intact in the liver of C/EBPβ-deficient mice. On the other hand, in primary-cultured hepatocytes derived from C/EBPβ-deficient mice, induction of genes for the first enzyme carbamylphosphate synthetase, as well as for arginase, in response to dexamethasone and/or glucagon was severely impaired. Therefore, C/EBPβ is required for hormonal induction of the genes for ornithine cycle enzymes in primary-cultured hepatocytes, while the deficiency of C/EBPβ is compensated for in vivo.

Hypoglycemia-associated Hyperammonemia Caused by Impaired Expression of Ornithine Cycle Enzyme Genes in C/EBPα Knockout Mice

Ammonia produced by amino acid metabolism is detoxified through conversion into urea by the ornithine cycle in the liver, whereas carbon skeletons of amino acids are converted to glucose by gluconeogenic enzymes. Promoter and enhancer sequences of several genes for ornithine cycle enzymes interact with members of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family. Disruption of the C/EBPalpha gene in mice causes hypoglycemia associated with the impaired expression of gluconeogenic enzymes. Here we examined the expression of ornithine cycle enzyme genes in the livers of C/EBPalpha-deficient mice. mRNA levels for the first, third, fourth, and fifth enzymes of five enzymes in the cycle were decreased in C/EBPalpha-deficient mice. Protein levels for the first, second, fourth, and fifth enzymes were also decreased. In situ hybridization analysis revealed that the enzyme mRNAs were distributed normally in the periportal region but were disordered in C/EBPalpha-deficient mice with relatively higher mRNA levels in the midlobular region. Blood ammonia concentrations in the mutant mice were severalfold higher than in wild-type mice. Thus, C/EBPalpha is crucial for ammonia detoxification by ornithine cycle enzymes and for coordination of gluconeogenesis and urea synthesis.

Arginine-metabolizing enzymes in the developing rat small intestine.

Before weaning, arginine biosynthesis from citrulline most likely takes place in the small intestine rather than in the kidney. We studied the expression of ornithine cycle enzymes in the rat small intestine during perinatal development. The spatiotemporal patterns of expression of ornithine aminotransferase, carbamoylphosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase mRNAs were studied by Northern blot analysis and in situ hybridization. In addition, the expression of carbamoylphosphate synthetase and argininosuccinate synthetase protein was studied by immunohistochemistry. Before birth, the developmentally more mature proximal loops of the intestine expressed the mRNAs at higher concentrations than the more distal loops. After birth, this difference was no longer obvious. The mRNAs of argininosuccinate synthetase and argininosuccinate lyase, the enzymes that metabolize citrulline to arginine, were detectable only in the upper part of the villi, whereas the other mRNAs were concentrated in the crypts. The distribution of argininosuccinate synthetase protein corresponded with that of the mRNA, whereas carbamoylphosphate synthetase protein was present in all enterocytes of the crypts and villi. Hepatic arginase mRNA could not be detected in the enterocytes. The spatial distribution of the respective mRNAs and proteins along the villus axis of the suckling small intestine indicates that the basal enterocytes synthesize citrulline, whereas the enterocytes in the upper half of the villus synthesize arginine.

The Glucocorticoid-responsive Gene Cascade: ACTIVATION OF THE RAT ARGINASE GENE THROUGH INDUCTION OF C/EBPβ

The gene for liver-type arginase, an ornithine cycle enzyme, is induced by glucocorticoids in a delayed secondary manner. An enhancer element located around intron 7 of the rat arginase gene shows delayed glucocorticoid responsiveness, and it harbors two sites binding with members of the CCAAT/enhancer binding protein (C/EBP) family. Here, we investigate the role of these C/EBP binding sites in glucocorticoid response of the arginase gene. When inserted in front of the herpes simplex virus thymidine kinase promoter, these C/EBP sites exhibited glucocorticoid responsiveness in reporter transfection assay using rat hepatoma H4IIE cells. In footprint analysis using nuclear extracts of H4IIE cells, profiles of the protected areas of the two C/EBP sites changed when cells were treated with dexamethasone. In gel shift analysis, the complex formation for the two C/EBP sites was augmented in response to dexamethasone. Antibody supershift/inhibition analysis demonstrated that a major portion of the binding proteins induced by dexamethasone is C/EBPbeta. Induction of arginase mRNA by dexamethasone was preceded by augmentation of the C/EBP site-binding activities, which followed increase in C/EBPbeta mRNA. These results were consistent with the notion that the glucocorticoid response of the arginase gene is mediated by C/EBPbeta.

Development of the ornithine cycle in rat liver: Zonation of a metabolic pathway

Ammonia-fixation in mammalian livers is, via the ornithine cycle and glutamine synthetase, strictly compartmentalized, occurring in a wide upstream periportal compartment and in the very last downstream pericentral hepatocytes, respectively. This conclusion is based on the well-known distribution patterns of carbamoyl phosphate synthetase I (CPS) and glutamine synthetase in the developing and adult liver. To determine to what extent the expression patterns of the ornithine cycle enzymes are coordinated with that of CPS, we investigated the patterns of expression of the individual messenger RNAs (mRNAs) of the ornithine cycle pathway in developing and adult rat liver. Our results show that, within the liver, all mRNAs of the ornithine cycle pathway are zonated. The patterns of expression of the different mRNAs match almost perfectly, with variations only in the steepness of the gradients of the mRNAs, suggesting that, in the rat, common regulatory factors are involved in the establishment of the zonation pattern. (Hepatology 1996 Aug;24(2):407-11)

Development of the ornithine cycle in rat liver: zonation of a metabolic pathway.

Ammonia-fixation in mammalian livers is, via the ornithine cycle and glutamine synthetase, strictly compartmentalized, occurring in a wide upstream periportal compartment and in the very last downstream pericentral hepatocytes, respectively. This conclusion is based on the well-known distribution patterns of carbamoyl phosphate synthetase I (CPS) and glutamine synthetase in the developing and adult liver. To determine to what extent the expression patterns of the ornithine cycle enzymes are coordinated with that of CPS, we investigated the patterns of expression of the individual messenger RNAs (mRNAs) of the ornithine cycle pathway in developing and adult rat liver. Our results show that, within the liver, all mRNAs of the ornithine cycle pathway are zonated. The patterns of expression of the different mRNAs match almost perfectly, with variations only in the steepness of the gradients of the mRNAs, suggesting that, in the rat, common regulatory factors are involved in the establishment of the zonation pattern.

Transcriptional regulation of genes for ornithine cycle enzymes.

Transcriptional regulation of genes for ornithine cycle enzymes.

The delayed glucocorticoid-responsive and hepatoma cell-selective enhancer of the rat arginase gene is located around intron 7.

Liver-selective transcription of the gene for rat arginase, an ornithine cycle (urea cycle) enzyme, is induced by glucocorticoids in a delayed secondary manner; the mRNA induction by the hormones requires de novo protein synthesis, and is preceded by a time lag of several hours. We searched for a DNA element mediating the glucocorticoid induction of the arginase gene with a transient transfection system using hepatoma cell lines. Within the 233-base pair region that is located 11 kilobases downstream from the transcription start site and that spans the junction of intron 7 and exon 8, we detected an enhancer element that is glucocorticoid-responsive and hepatoma cell-selective. The time course of the glucocorticoid induction through this enhancer element was delayed compared to that through the primary glucocorticoid-responsive mouse mammary tumor virus promoter. Footprint analysis revealed four protein-binding sites in this enhancer region. In gel retardation analysis, each site exhibited a complicated profile characterized by a number of shifted bands, some of which were tissue-selective and others ubiquitous. Gel shift competition and antibody supershift/inhibition analysis demonstrated that two of the four sites are recognized by members of the CCAAT/enhancer binding protein (C/EBP) family, some of which are liver-enriched.

Expression patterns of ammonia-metabolizing enzymes in the liver, mesonephros, and gut of human embryos and their possible implications.

Human and ungulate embryos can catabolize amino acids for energy production, whereas rodent embryos cannot, raising the question whether studies of rodent model systems are suitable for extrapolation to the human situation. Therefore, we investigated the expression of the amino acid- and ammonia-metabolizing enzymes glutaminase, glutamate dehydrogenase, glutamine synthase, carbamoylphosphate synthase, and arginase immunohistochemically in a graded series of human embryos and fetuses. During human development the expression of these enzymes is first seen in the liver, then in the mesonephric kidney, and finally in the small intestine. Such a simultaneous expression of nitrogen-metabolizing enzymes was not seen in any other organ. The early appearance of the enzymes involved in amino acid and ammonia metabolism in the human liver, compared to, for example, the rat liver, suggests that catabolism of amino acids may provide an important supply of metabolic energy for the human embryo. The coexpression of glutaminase, glutamate dehydrogenase, and carbamoylphosphate synthase, but not of arginase, in the mesonephros and the small intestine suggests that these organs are involved in the biosynthesis of intermediates of the ornithine cycle, e.g., arginine or citrulline. From a comparison of the developmental appearance of ornithine cycle enzymes in different mammalian species we postulate that an early appearance of these enzymes is generally associated with a relatively slow prenatal growth rate and the use of amino acids as metabolic fuel.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses transcription from the promoter of the gene for ornithine transcarbamylase in a manner antagonistic to hepatocyte nuclear factor-4 (HNF-4)

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and hepatocyte nuclear factor-4 (HNF-4) are orphan members of the steroid/thyroid receptor superfamily and exhibit ubiquitous and liver-enriched tissue distribution, respectively. The gene for rat ornithine transcarbamylase (OTC), an ornithine cycle enzyme, is mainly expressed in the liver and is under the control of the promoter and the 11-kilobase upstream enhancer, both of which are liver-selective. Two sites of the promoter region and two sites of the enhancer region of the OTC gene, as well as the ovalbumin promoter site, were recognized by both HNF-4 and COUP-TF, showing that these two factors have closely related binding specificities. Since HNF-4 activated expression from the OTC promoter in cotransfection analysis, this factor appears to participate in liver-selective activation of the OTC gene. On the other hand, COUP-TF repressed the expression from the OTC promoter, whereas it activated expression from several other promoters. Therefore, COUP-TF plays a dual regulatory role depending on the promoter context. Repression of a tissue-specific promoter by a ubiquitous transactivator and derepression by a related tissue-enriched transactivator is potentially an important mechanism for tissue-specific activation of a gene.

Fenitrothion‐induced alterations in ammonia metabolism in the crab, Oziotelphusa senex senex fabricius

AbstractFresh water crabs, (Oziotelphusa senex senex) were subjected to short‐term (1 day) and long‐term (30 days) exposure to a sublethal concentration of fenitrothion (0.04 mg litre‐1), and the hepatopancreatic nitrogen metabolism was investigated. Differential patterns of nitrogen metabolism were observed in the hepatopancreas in response to both short‐term and long‐term exposure. Levels of ammonia in hemolymph of crab after short‐term exposure were markedly increased, with an increase in excretion of ammonia. The activity levels of aspartate aminotransaminase (AAT), alanine aminotransaminase (AIAT) and glutamate dehydrogenase (GDH) were found to increase in the hepatopancreas during short‐term exposure, which suggests increased transamination and deamination of amino acids. The AMP‐deaminase activity increased in the hepatopancreas during short‐term exposure indicating increased deamination of nucleic acids. The activity levels of ornithine cycle enzymes such as arginino succinate synthetase (ArSS) and arginase indicated diminished activities resulting in a low level of operation of the urea cycle and consequent accumulation of ammonia during short‐term exposure. The whole view of ammonia production and utilization mechanisms suggests the probable synergistic manifestation of ammonia toxicity during short‐term fenitrothion poisoning.However, during long‐term exposure, the haemolymph had a low ammonia level. The excretion of ammonia was also found to decrease. The activity levels of AAT, AIAT and GDH in the hepatopancreas were found to increase. In contrast to these, the AMP‐deaminase activity decreased in the hepatopancreas suggesting decreased deamination of nucleic acids. ArSS and arginase activities increased indicating active operation of the urea cycle and consequent accumulation of urea. Glutamine synthetase activity rose with the accumulation of glutamine which may help in averting toxic effects of ammonia. The possible effects of fenitrothion toxicity on energy requirements, transdeamination patterns, ureogenesis and glutamine production have been discussed.

Distribution of arginase and other ornithine cycle enzymes in the gut of the earthworm Lumbricus terrestris L., and some physiological and comparative implications

Abstract 1. 1. Guts of fresh and dehydrated-starved L. terrestris were compared as to tissue and anterior-posterior distribution of arginase, OCT (ornithine carbamoyl phosphate transferase) and AS (arginosuccinase). 2. 2. In fresh worms the anterior two thirds of the midgut was practically devoid of arginase activity, while in starved worms this region exhibited a high arginase activity. Both fresh and starved worms had high arginase activity in the posterior part of the midgut and in the hindgut, and this arginase activity did not increase during starvation. The distribution of AS and OCT was relatively uniform along the gut regardless of state of the worm. 3. 3. The main site of the constant posterior arginase activity was the intestinal epithelium, so part of the posterior midgut epithelium may possibly carry out both ammonia (Prento P. (1989) Comp. Biochem. Physiol. B., in press) and urea production. OCT and AS were always present in both chloragog tissue and epithelium. 4. 4. Starved worms had about four times higher arginase, AS and OCT activities than in fresh worms. Most of the arginase activity (about 70%) was in the chloragog tissue, while the remaining activity was in the intestinal epithelium. This distribution supports Tillinghast (Tillinghast E. K. (1987) J. exp. Zool166, 295–300; and (1986) Comp. Biochem. Physiol.24B 621–623.) as to the excretory route for urea. About 66% of the gut OCT and AS activities were found in the chloragog tissue. 5. 5. The distribution of arginase suggests that both for the intestinal epithelium and for the chloragog tissue high external ammonia levels induce arginase synthesis. The distribution of OCT and AS suggest that arginine synthesis is a property of both the chloragog tissue and the intestinal epithelium.

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.

The influence of ornithine cycle enzyme activity on the plasma urea concentration in sheep

The influence of plane of nutrition and protein content of the diet on the in vitro hepatic activity of the rate-limiting enzyme of the ornithine cycle, arginine synthetase, and the relationship between arginine synthetase activity and plasma urea concentration were studied in lambs and mature sheep. Arginine synthetase activity was positively related to the protein content of the diet, and was reduced when food and thus protein intake was restricted. These findings indicated that in sheep arginine synthetase activity was influenced by the daily protein intake. The plasma urea concentration was positively related to arginine synthetase activity irrespective of the base used to express enzyme activity. There were differences between experiments in the regression equations describing the relationships between plasma urea concentration and arginine synthetase activity, which suggest that the relationships may not have broad application. The implications of these findings are discussed in relation to the control of plasma urea concentration in sheep.

Urea as a selective inhibitor of argininosuccinate lyase.

The effect of urea on various ornithine cycle enzymes has been investigated. It was demonstrated that argininosuccinate lyase was the only ornithine cycle enzyme inhibited by urea in a competitive manner. Based on the data presented the possible role of urea in maintaining a physiological range of intracellular and extracellular urea concentration by controlling hepatic ureogenesis was discussed.

Ornithine cycle and uricolytic enzymes in four bivalve molluscs

1. 1. The digestive diverticula, kidney and mantle tissue of Anodonta kennerlyi (Lea), Mytilus californianus (Conrad), Saxidomus giganteus and Compsomyax subdiaphana (Carpenter) were investigated. 2. 2. Ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase and arginase were detected in the digestive diverticula of all species examined. 3. 3. Argininosuccinate synthetase was found to be rate limiting in urea biosynthesis. 4. 4. The full complement of uricolytic enzymes—uricase, allantoinase, allantoicase and urease—was found in the digestive diverticula of all four species. 5. 5. Uricase and allantoicase were found to be equally rate limiting. Carbam ylphosphate synthetase activity could not be detected. 6. 6. None of the enzymes investigated were found in the kidney or the mantle.