Inducible Arginase 1 Deficiency in Mice Leads to Hyperargininemia and Altered Amino Acid Metabolism

Arginase deficiency is a rare autosomal recessive disorder resulting from a loss of the liver arginase isoform, arginase 1 (ARG1), which is the final step in the urea cycle for detoxifying ammonia. ARG1 deficiency leads to hyperargininemia, characterized by progressive neurological impairment, persistent growth retardation and infrequent episodes of hyperammonemia. Using the Cre/loxP-directed conditional gene knockout system, we generated an inducible Arg1-deficient mouse model by crossing “floxed” Arg1 mice with CreERT2 mice. The resulting mice (Arg-Cre) die about two weeks after tamoxifen administration regardless of the starting age of inducing the knockout. These treated mice were nearly devoid of Arg1 mRNA, protein and liver arginase activity, and exhibited symptoms of hyperammonemia. Plasma amino acid analysis revealed pronounced hyperargininemia and significant alterations in amino acid and guanidino compound metabolism, including increased citrulline and guanidinoacetic acid. Despite no alteration in ornithine levels, concentrations of other amino acids such as proline and the branched-chain amino acids were reduced. In summary, we have generated and characterized an inducible Arg1-deficient mouse model exhibiting several pathologic manifestations of hyperargininemia. This model should prove useful for exploring potential treatment options of ARG1 deficiency.

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title: The human arginases and arginase deficiency
abstract: Arginase is the final enzyme in the urea cycle. Its deficiency is the least frequently described disorder of this cycle. It results primarily in elevated blood arginine, and less frequently in either persistent or acute elevations in blood ammonia. This appears to be due to a second arginase locus, expressed primarily in the kidney, which can be recruited to compensate, in part, for the deficiency of liver arginase. The liver arginase gene structure permitted study of the molecular pathology of patients with the disorder and the results of these studies and the inferences about the protein structure are presented. The conserved regions among all arginases allowed the cloning of AII, the second arginase isoform. It has been localized to the mitochondrion and is thought to be involved in ornithine biosynthesis. It shares the major conserved protein sequences, and structural features of liver arginase gene are also conserved. When AI and AII from various species are compared, it appears that the two diverged some time prior to the evolution of amphibians. The evidence for the role of AII in nitric oxide and polyamine metabolism is presented and this appears consonant with the data on the tissue distribution.

title: Human type II arginase: sequence analysis and tissue-specific expression.
abstract: A full-length cDNA encoding type II arginase was isolated from a human kidney cDNA library and its sequence compared to those of vertebrate type I arginases as well as to arginases of bacteria, fungi and plants. The predicted sequence of human type II arginase is 58% identical to the sequence of human type I arginase but is 71% identical to the sequence of Xenopus type II arginase, suggesting that duplication of the arginase gene occurred before mammals and amphibians diverged. Seven residues known to be essential for activity were found to be conserved in all arginases. Type II arginase mRNA was detected in virtually all human and mouse RNA samples tested whereas type I arginase mRNA was found only in liver. At least five mRNA species hybridizing to type II arginase cDNA were found in the human RNA samples whereas only a single type II arginase mRNA species was found in the mouse. This raises the possibility that the multiple type II arginase mRNAs in humans arise from differential RNA processing or usage of alternative promoters.

title: Cloning and characterization of the human type II arginase gene.
abstract: There are two forms of arginase in humans, both catalyzing the hydrolysis of arginine to ornithine and urea. Recent studies in animal models and in Type I arginase-deficient patients suggest that Type II arginase is inducible and may play an important role in the regulation of extra-urea cycle arginine metabolism by modulating cellular arginine concentrations. We PCR amplified and cloned the human Type II arginase gene, the first nonliver arginase gene reported in mammals. While sequence homology to Type I arginase, arginase activity data, and immunoprecipitation with an anti-AII antibody confirm the identity of this gene, Northern blot analysis demonstrates its differential expression in the brain, prostate, and kidney. Type II arginase may be an important part of the arginine regulatory system affecting nitric oxide synthase, arginine decarboxylase, kyotorphin synthase, and arginine-glycine transaminase activities and polyamine and proline biosynthesis.

title: Arginase deficiency manifesting delayed clinical sequelae and induction of a kidney arginase isozyme
abstract: Deficiency of liver arginase (AI) is characterized clinically by hyperargininemia, progressive mental impairment, growth retardation, spasticity, and periodic episodes of hyperammonemia. The rarest of the inborn errors of urea cycle enzymes, it has been considered the least life-threatening, by virtue of the typical absence of catastrophic neonatal hyperammonemia and its compatibility with a longer life span. This has been attributed to the persistence of some ureagenesis in these patients through the activity of a second isozyme of arginase (AII) located predominantly in the kidney. We have treated a number of arginase-deficient patients into young adulthood. While they are severely retarded and wheelchairbound, their general medical care has been quite tractable. Recently, however, two of the oldest (M.U., age 20, and M.O., age 22) underwent rapid deterioration, ending in hyperammonemic coma and death, precipitated by relatively minor viral respiratory illnesses inducing a catabolic state with increased endogenous nitrogen load. In both cases, postmortem examination revealed severe global cerebral edema and aspiration pneumonia. Enzyme assays confirmed the absence of AI activity in the livers of both patients. In contrast, AII activity (identified by its different cation cofactor requirements and lack of precipitation with anti-AI antibody) was markedly elevated in kidney tissues, 20-fold in M.O. and 34-fold in M.U. Terminal plasma arginine (1500μmol/1) and ammonia (1693 mmol/1) levels of M.U. were substantially higher than those of M.O. (348μmol/1 and 259μmol/1, respectively). By Northern blot analysis, AI mRNA was detected in M.O.'s liver but not in M.U.'s; similarly, anti-AI crossreacting material was observed by Western blot in M.O. only. These findings indicate that, despite their more longlived course, patients with arginase deficiency remain vulnerable to the same catastrophic events of hyperammonemia that patients with other urea cycle disorders typically suffer in infancy. Further, unlike those other disorders, an attempt is made to compensate for the primary enzyme deficiency by induction of another isozyme in a different tissue. Such substrate-stimulated induction of an enzyme may be unique in a medical genetics setting and raises novel options for eventual gene therapy of this disorder.

title: Hyperargininemia due to liver arginase deficiency.
abstract: The urea cycle is a series of six reactions necessary to rid the body of the nitrogen generated by the metabolism, primarily of amino acids, from the diet or released as the result of endogenous protein catabolism. Arginase is the sixth and final enzyme of this cycle. Arginase catalyzes the conversion of arginine to urea and ornithine, the latter recycled to continue the cycle. Hyperargininemia due to arginase deficiency is inherited in an autosomal recessive manner and gene for arginase, designated AI, has been cloned. Unlike the other urea cycle enzymes, a second gene encoding arginase, with similar structural properties and enzyme characteristics, exists and has been named Arginase II (AII). Comprehensive histories and physical examinations confirm a strikingly uniform clinical picture and one notably different from patients with other urea cycle disorders. This condition rarely presents in the neonatal period and first symptoms typically present in children between 2 and 4 years of age. First symptoms are often neurologically based. If untreated, symptoms are progressive with a gradual loss of developmental milestones. With adherence to a dietary and drug regimen, a favorable outcome can be expected, with cessation of further neurological deterioration and in some instances, of improvement. This article summarizes the clinical course of selected patients who represent the full spectrum of presentations of arginase deficiency. In addition to the clinical characterization of this disorder; the biochemical, enzymatic, and molecular evidence of disease is summarized. Treatment and prenatal diagnosis are also discussed.

title: Alternative pathway therapy for urea cycle disorders: twenty years later.
abstract: Alternative pathway therapy is currently an accepted treatment approach for inborn errors of the urea cycle. This involves the long-term use of oral sodium phenylbutyrate, arginine supplements, or both, depending on the specific enzyme deficiency, and treatment of acute hyperammonemic crises with intravenous sodium benzoate/sodium phenylacetate plus arginine. A review of 20 years of experience with this approach illustrates the strengths and limitations of this treatment. It has clearly decreased the mortality and morbidity from these disorders, but they remain unacceptably high. The medications are generally well tolerated, but severe accidental overdosage has been reported because of the infrequent use of the medication. There is also a difference in their metabolism between newborns and older children that must be addressed in determining dosage. To avoid these complications it is recommended that drug levels in blood be monitored routinely and that very specific treatment protocols and oversight be followed to avoid overdoses. Finally, it must be acknowledged that alternative pathway therapy has limited effectiveness in preventing hyperammonemia and must be combined with effective dietary management. Therefore in children with neonatal-onset disease or in those with very poor metabolic control, liver transplantation should be considered. There should also be the continued search for innovative therapies that may offer a more permanent and complete correction, such as gene therapy.

title: The gene for human liver arginase (ARG1) is assigned to chromosome band 6q23.
abstract: The human liver arginase gene, whose deficiency is responsible for argininemia (McKusick no. 20780), has been assigned to 6q23 through a combination of somatic cell hybrid analysis and in situ hybridization using a 1,550-base pair (bp) human DNA probe for this gene.

title: Human liver-type arginase gene: structure of the gene and analysis of the promoter region.
abstract: The gene for human liver-type arginase (EC 3.5.3.1), a urea cycle enzyme, was cloned and the structure was determined. This gene is 11.5 kilobases long and is split into 8 exons. The cap site was determined by nuclease S1 mapping and primer extension. A "TATA box"-like sequence is located 28 bases upstream from the cap site, and a sequence similar to the binding sites of the transcription factor CTF/NF1, a "CAAT box"-binding protein, is located 72 bases upstream. In the 5' end region, sequences resembling the glucocorticoid responsive elements, the cAMP responsive elements, and the enhancer core sequences are present. The immediately 5' flanking region of the human gene up to position -105 is 84% identical with the corresponding segment of the rat gene. In this region of the human gene, one DNase I-protected area and several hypersensitive cleavage sites were detected by footprint analysis, using nuclear extracts from the rat liver. The protected area contains the sequence similar to the binding sites of CTF/NF1 and also overlaps with the sequence resembling the glucocorticoid responsive elements.

title: Cloning of rat liver arginase cDNA and elucidation of regulation of arginase gene expression in H4 rat hepatoma cells
abstract: In order to study the regulation of expression of the two arginase genes in mammalian tissues, we undertook to clone cDNA specific for rat liver arginase. mRNA was isolated from rat liver poly somes enriched for the arginase message by immunopurification and was used to produce an 800-member cDNA library carried in pBR322. Four arginase clones were identified by hybrid selection, and one was used to find two others following colony hybridization. Clonal identity was verified by its enrichment in the cDNA made from immunopurified mRNA; by hybrid selection, immunoprecipitation, and competition by purified arginase; hybridization on Northern analysis with liver-derived RNA (high in arginase) and its absence with mRNA from tissues low in arginase; and independent identification by hybrid selection and colony hybridization. Northern analysis of mRNA from H4-II-E-C3 (H4) rat hepatoma cells in which arginase activity was induced by hydrocortisone demonstrated equal, eightfold augmentation of both arginase activity and arginase mRNA levels. Southern blot analysis of DNA from these cells indicated that no change in arrangement or copy number accompanied induction. Southern analysis also suggested that the gene for rat liver arginase is present in a single copy, without pseudogenes, and that a high degree of homology exists between it and its mouse counterpart.

title: Molecular cloning and nucleotide sequence of cDNA for human liver arginase.
abstract: Arginase (EC 3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, we isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted Mr, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

title: Mouse Model for Human Arginase Deficiency
abstract: Deficiency of liver arginase (AI) causes hyperargininemia (OMIM 207800), a disorder characterized by progressive mental impairment, growth retardation, and spasticity and punctuated by sometimes fatal episodes of hyperammonemia. We constructed a knockout mouse strain carrying a nonfunctional AI gene by homologous recombination. Arginase AI knockout mice completely lacked liver arginase (AI) activity, exhibited severe symptoms of hyperammonemia, and died between postnatal days 10 and 14. During hyperammonemic crisis, plasma ammonia levels of these mice increased >10-fold compared to those for normal animals. Livers of AI-deficient animals showed hepatocyte abnormalities, including cell swelling and inclusions. Plasma amino acid analysis showed the mean arginine level in knockouts to be approximately fourfold greater than that for the wild type and threefold greater than that for heterozygotes; the mean proline level was approximately one-third and the ornithine level was one-half of the proline and ornithine levels, respectively, for wild-type or heterozygote mice—understandable biochemical consequences of arginase deficiency. Glutamic acid, citrulline, and histidine levels were about 1.5-fold higher than those seen in the phenotypically normal animals. Concentrations of the branched-chain amino acids valine, isoleucine, and leucine were 0.4 to 0.5 times the concentrations seen in phenotypically normal animals. In summary, the AI-deficient mouse duplicates several pathobiological aspects of the human condition and should prove to be a useful model for further study of the disease mechanism(s) and to explore treatment options, such as pharmaceutical administration of sodium phenylbutyrate and/or ornithine and development of gene therapy protocols.

title: Inducible Cre mice.
abstract: The Cre/lox site-specific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. This method allows one to control gene activity in space and time in almost any tissue of the mouse, thus opening new avenues for studying gene function and for establishing sophisticated animal models of human diseases. A major technical advance in terms of in vivo inducibility was the development of ligand-dependent Cre recombinases that can be activated by administration of tamoxifen to the animal. Here we describe how tamoxifen-dependent Cre recombinases, so-called CreER recombinases, work and how they can be used to generate time- and tissue-specific mouse mutants. The focus will be on the CreER(T2) recombinase, which is currently the most successful CreER version. We will give an overview of available CreER(T2) transgenic mouse lines and present protocols that detail the generation of experimental mice for inducible gene knockout studies, the induction of recombination by tamoxifen treatment, and the analysis of the quality and quantity of recombination by reporter gene and target gene studies. Most of the protocols can also be used as general guidelines for the generation and characterization of Cre/lox-mediated genome modifications in mice.

title: Guanidino compounds in serum, urine, liver, kidney, and brain of man and some ureotelic animals.
abstract: Guanidino compound levels were quantitatively determined in serum, urine, liver, kidney, and brain of man and of some ureotelic animals. The guanidino compounds were separated over a cation exchange resin, using sodium citrate buffers, and detected with the fluorescence ninhydrin method. Species-specific differences in the levels of some guanidino compounds in the studied ureotelic animals are shown. alpha-Keto-delta-guanidinovaleric acid is a naturally occurring guanidino compound in ureotelic animals, and is not restricted to the pathobiochemistry of hyperargininemic patients. The fasting serum levels observed in beagles are the same as those found in hyperargininemic patients. In serum, liver, and kidney, the homoarginine, beta-guanidinopropionic acid, and gamma-guanidinobutyric acid levels are the highest in rats. The last two compounds have the highest levels of the studied guanidino compounds, with the exception of creatinine, in kidney. Specific high levels of gamma-guanidinobutyric acid and argininic acid are found in brain of rabbits.

title: Short-term correction of arginase deficiency in a neonatal murine model with a helper-dependent adenoviral vector.
abstract: Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 x 10(9) viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg(-/-) mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg(-/-) livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg(-/-) mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV.

title: Argininemia: A Treatable Genetic Cause of Progressive Spastic Diplegia Simulating Cerebral Palsy: Case Reports and Literature Review
abstract: Argininemia, a rare autosomal recessive urea cycle disorder, is caused by a deficiency of arginase, with resulting elevated plasma arginine and ammonia levels. Reports to date have focused little on the neurology of this disorder or the efficacy of treatments. A MEDLINE search revealed 25 previously reported cases, to which we have added two brothers who presented with late onset progressive spastic diplegia. Though their degree of enzyme deficiency was comparable, the severity of their phenotypic abnormalities differed substantially. With dietary therapy, both showed improved cognitive and motor function. Late metabolic crises occurred in both, resulting in death of the less severely affected brother. Based on analysis of our clinical database, we report on the full spectrum of neurologic abnormalities seen in argininemia with particular focus on the accompanying progressive spastic diplegia and its response to treatment; progressive decline in head growth; distinctive neuroradiologic findings; and life-threatening later complications. Current and potential future therapies and long-term outcome are summarized.

title: Tamoxifen induces rapid, reversible atrophy, and metaplasia in mouse stomach.
abstract: Tamoxifen, a selective estrogen receptor modulator, is widely used in research and clinically in patients. We find that treatment of normal mice with a single ≥3 mg/20 g body weight dose of tamoxifen leads to apoptosis of >90% of all gastric parietal cells (PCs) and metaplasia of zymogenic chief cells within 3 days. Remarkably, gastric histology returns to nearly normal by 3 weeks. Tamoxifen toxicity occurs by oral and intraperitoneal administration, in both sexes, in multiple strains, and does not depend on estrogen, though acid secretion inhibition is partially protective. Thus, substantial gastric toxicity is a heretofore unappreciated tamoxifen side effect.

title: Regulation of nitric oxide production in health and disease
abstract: Purpose of reviewThe purpose of this review is to highlight recent publications examining nitric oxide production in health and disease and its association with clinical nutrition and alterations in metabolism. Recent findingsThe role of the cofactor tetrahydrobiopterin in nitric oxide production and its relation with arginine availability is indicated as an important explanation for the arginine paradox. This offers potential for nitric oxide regulation by dietary factors such as arginine or its precursors and vitamin C. Because diets with a high saturated fat content induce high plasma fatty acid levels, endothelial nitric oxide production is often impaired due to a reduction in nitric oxide synthase 3 phosphorylation. Increasing the arginine availability by arginine therapy or arginase inhibition was, therefore, proposed as a potential therapy to treat hypertension. Recent studies in septic patients and transgenic mice models found that inadequate de-novo arginine production from citrulline reduces nitric oxide production. Citrulline supplementation may, therefore, be a novel therapeutic approach in conditions of arginine deficiency. SummaryBoth lack and excess of nitric oxide production in diseases can have various important implications in which dietary factors can play a modulating role. Future research is needed to expand our understanding of the regulation and adequate measurement of nitric oxide production at the organ level and by the different nitric oxide synthase isoforms, also in relation to clinical nutrition.

title: Guanidino compounds in hyperargininemia.
abstract: Plasma and cerebrospinal fluid (CSF) concentrations and urinary excretion of guanidino compounds were investigated in a patient with hyperargininemia during the treatment with low-protein diet, oral administration of an essential amino acid mixture and sodium benzoate, or enzyme replacement therapy such as exchange transfusion or erythrocyte transfusion. In the patient, alpha-keto-delta-guanidinovaleric acid (GVA), N-alpha-acetylarginine (NAA), argininic acid (ArgA) and homoarginine concentrations in plasma were elevated as well as arginine. Urinary excretion of GVA, ArgA, NAA and gamma-guanidinobutyric acid (GBA) were also increased. CSF concentrations of ArgA, homoarginine and arginine were also elevated. On the other hand, guanidinosuccinic acid (GSA), which is usually detected in all samples, was not detected in plasma, CSF and urine of the patient. The present results suggest that in patients with hyperargininemia other factors such as arginine and its metabolites including GVA, GAA, ArgA and homoarginine may cause the neurological symptoms. Furthermore, it is suggested that in patients with hyperargininemia, arginine may be catabolized via other pathways and nitrogen may be excreted partially in urine in the form of some of guanidino compounds.

title: Guanidino compound levels in blood, cerebrospinal fluid, and post-mortem brain material of patients with argininemia.
abstract: The paucity of hyperammonemic crises together with spasticity, only seen in human arginase I deficient patients and not in patients with other urea cycle disorders, forces a search for candidates other than ammonia to associate with the pathophysiology and symptomatology. Therefore, we determined arginine together with some catabolites of arginine in blood and cerebrospinal fluid of these patients as well as in extremely rare post-mortem brain material of two patients with argininemia. The levels of alpha-keto-delta-guanidinovaleric acid, argininic acid and alpha-N-acetylarginine correlate with the arginine levels in blood and cerebrospinal fluid of patients with imposed or spontaneous protein restriction. The levels in blood are higher than the upper limit of normal in all studied patients. In addition to the highly increased levels of these same compounds in blood of a child with argininemia, the increase of guanidinoacetic acid, 24h before death, is remarkable. However, the manifest increases of these studied catabolites of arginine are not seen in post-mortem brain material of the same pediatric patient. Otherwise a clear increase of guanidinoacetic acid in post-mortem brain material of an adult patient was shown. A similar, comparable increase of homoarginine in both studied post-mortem brain materials is observed. Therefore the study of the pathobiochemistry of arginine in argininemia must be completed in the future by the determination of the end catabolites of the nitric oxide and agmatine biosynthesis pathways in the knockouts as well as in the patients to evaluate their role, together with the here studied catabolites, as candidates for association with pathophysiology and symptomatology.

title: Increased plasma and tissue guanidino compounds in a mouse model of hyperargininemia.
abstract: In humans, arginase I (AI)-deficiency results in hyperargininemia, a metabolic disorder with symptoms of progressive neurological and intellectual impairment, spasticity, persistent growth retardation, and episodic hyperammonemia. A deficiency of arginase II (AII) has never been detected and the clinical disorder, if any, associated with its deficiency has not been defined. Since the spasticity and paucity of hyperammonemic crises seen in human AI-deficient patients are not features of the other urea cycle disorders, the likelihood of ammonia as the main neuropathogenic agent becomes extremely low, and the modest elevations of arginine seen in the brains of our mouse model of hyperargininemia make it an unlikely candidate as well. Specific guanidino compounds, direct or indirect metabolites of arginine, are elevated in the blood of patients with uremia. Other guanidino compounds are also increased in plasma and cerebrospinal fluid of hyperargininemic patients making them plausible as neurotoxins in these disorders. We analyzed several guanidino compounds in our arginase single and double knockout animals and found that alpha-keto-delta-guanidinovaleric acid, alpha-N-acetylarginine, and argininic acid were increased in the brain tissue from the AI knockout and double knockout animals. Several compounds were also increased in the plasma, liver, and kidneys. This is the first time that several of the guanidino compounds have been shown to be elevated in the brain tissue of an arginase-deficient mammal, and it further supports their possible role as the neuropathogenic agents responsible for the complications seen in arginase deficiency.

title: Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress.
abstract: The potential of proline to suppress reactive oxygen species (ROS) and apoptosis in mammalian cells was tested by manipulating intracellular proline levels exogenously and endogenously by overexpression of proline metabolic enzymes. Proline was observed to protect cells against H(2)O(2), tert-butyl hydroperoxide, and a carcinogenic oxidative stress inducer but was not effective against superoxide generators such as menadione. Oxidative stress protection by proline requires the secondary amine of the pyrrolidine ring and involves preservation of the glutathione redox environment. Overexpression of proline dehydrogenase (PRODH), a mitochondrial flavoenzyme that oxidizes proline, resulted in 6-fold lower intracellular proline content and decreased cell survival relative to control cells. Cells overexpressing PRODH were rescued by pipecolate, an analog that mimics the antioxidant properties of proline, and by tetrahydro-2-furoic acid, a specific inhibitor of PRODH. In contrast, overexpression of the proline biosynthetic enzymes Delta(1)-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in 2-fold higher proline content, significantly lower ROS levels, and increased cell survival relative to control cells. In different mammalian cell lines exposed to physiological H(2)O(2) levels, increased endogenous P5CS and P5CR expression was observed, indicating that upregulation of proline biosynthesis is an oxidative stress response.

title: Plasma concentrations of arginine and related amino acids following traumatic brain injury: Proline as a promising biomarker of brain damage severity.
abstract: The aim of this study was to find a plasma biomarker, in relation with nitric oxide (NO), as a sign of brain damage severity following traumatic brain injury (TBI). We first investigated the post-traumatic evolution of the plasma concentrations of NO via the level of NO end-products metabolites (nitrite plus nitrate, NO(x)), that of l-arginine (Arg) and amino acids involved in its metabolism as well as the time course of neurological score in a rat model of lateral fluid percussion brain injury. First, the level of NO(x) was increased in plasma at 24 and 48 h post-TBI with a marked increase at 72 h. In contrast, this elevation was neither accompanied by a modification of plasma concentrations of Arg, nor of amino acids involved in NO and Arg metabolism, l-ornithine (Orn), l-glutamate (Glu), and l-glutamine (Gln). Second, TBI induced a fall of plasma l-proline (Pro) concentrations. The time course of post-TBI neurological deficit showed also a decrease of neurological score at 24, 48, and 72 h. Furthermore, there is a weak negative correlation between the neurological score and the plasma level of NO(x) (r=-0.305; P<0.05), while a marked positive correlation has been found between the neurological score and the plasma level of Pro (r=0.563; P<0.001). In conclusion, the plasma concentrations of Pro could be a promising marker of post-traumatic neurological deficit.

title: Dietary L-arginine supplementation reduces white fat gain and enhances skeletal muscle and brown fat masses in diet-induced obese rats.
abstract: Previous studies showed that dietary L-arginine supplementation decreased white fat mass in genetically obese rats. This study tested the effectiveness of L-arginine in diet-induced obesity. Male Sprague-Dawley rats were fed for 15 wk a high-fat (HF) (40% energy) or low-fat (LF) (10% energy) diet beginning at 4 wk of age, resulting in 18% higher body weight gains and 74% higher weights of major white fat pads (retroperitoneal, epididymal, subcutaneous, and mesenteric adipose tissues) in HF than in LF fed rats. Starting at 19 wk of age, rats in each dietary group were supplemented for 12 wk with 1.51% L-arginine-HCl or 2.55% L-alanine (isonitrogenous control) (n = 8 per treatment) in drinking water and arginine groups were individually pair-fed to alanine controls. Despite similar energy intake, absolute weights of white fat pads increased by 98% in control rats over a 12-wk period but only by 35% in arginine-supplemented rats. The arginine treatment reduced the relative weights of white fat pads by 30% and enhanced those of soleus muscle by 13%, extensor digitorum longus muscle by 11%, and brown fat by 34% compared with control rats. Serum concentrations of insulin, adiponectin, growth hormone, corticosterone, triiodothyronine, and thyroxine did not differ between control and arginine-supplemented rats. However, arginine treatment resulted in lower serum concentrations of leptin, glucose, triglycerides, urea, glutamine, and branched-chain amino acids, higher serum concentrations of nitric-oxide metabolites, and improvement in glucose tolerance. Thus, dietary arginine supplementation shifts nutrient partitioning to promote muscle over fat gain and may provide a useful treatment for improving the metabolic profile and reducing body white fat in diet-induced obese rats.

title: Acute hyperammonemia activates branched-chain amino acid catabolism and decreases their extracellular concentrations: different sensitivity of red and white muscle
abstract: Hyperammonemia is considered to be the main cause of decreased levels of the branched-chain amino acids (BCAA), valine, leucine, and isoleucine, in liver cirrhosis. In this study we investigated whether the decrease in BCAA is caused by the direct effect of ammonia on BCAA metabolism and the effect of ammonia on BCAA and protein metabolism in different types of skeletal muscle. M. soleus (SOL, slow-twitch, red muscle) and m. extensor digitorum longus (EDL, fast-twitch, white muscle) of white rat were isolated and incubated in a medium with or without 500 μM ammonia. We measured the exchange of amino acids between the muscle and the medium, amino acid concentrations in the muscle, release of branched-chain keto acids (BCKA), leucine oxidation, total and myofibrillar proteolysis, and protein synthesis. Hyperammonemia inhibited the BCAA release (81% in SOL and 60% in EDL vs. controls), increased the release of BCKA (133% in SOL and 161% in EDL vs. controls) and glutamine (138% in SOL and 145% in EDL vs. controls), and increased the leucine oxidation in EDL (174% of controls). Ammonia also induced a significant increase in glutamine concentration in skeletal muscle. The effect of ammonia on intracellular BCAA concentration, protein synthesis and on total and myofibrillar proteolysis was insignificant. The data indicates that hyperammonemia directly affects the BCAA metabolism in skeletal muscle which results in decreased levels of BCAA in the extracellular fluid. The effect is associated with activated synthesis of glutamine, increased BCAA oxidation, decreased release of BCAA, and enhanced release of BCKA. These metabolic changes are not directly associated with marked changes in protein turnover. The effect of ammonia is more pronounced in muscles with high content of white fibres.

title: Protein intake, brain amino acid and serotonin concentrations and protein self-selection.
abstract: Analysis of evidence of associations among dietary protein content, brain amino acid and serotonin concentrations, and protein self-selection by rats suggests that 1) protein intake is not regulated precisely, although rats will select between low and high protein diets to obtain an adequate, but not excessive, amount of protein; 2) associations between brain serotonin concentration and protein intake are weak, although consumption of single meals of protein-deficient diets will elevate brain serotonin concentration; 3) the nature of signals that drive rats to avoid diets containing inadequate or excessive amounts of protein remains obscure; (4) whole brain amino acid and serotonin concentrations are quite stable over the usual range of protein intakes, owing to competition among amino acids for uptake across the blood-brain barrier and effective metabolic regulation of blood amino acid concentrations; 5) protein intake and preference are not in themselves regulated, but what appears to be regulation of intake and preference is a reflection of the responses of systems for control of plasma amino acid concentrations; and (6) the relative stability of the average protein intake of groups of self-selecting rats (which gives the appearance of regulation) results from averaging the variable behavioral responses--learned aversions and preferences--of rats to the variety of sensory cues arising from diets that differ in protein content.

title: Multiple neural systems controlling food intake and body weight
abstract: Discovery of the leptin receptor and its downstream peptidergic pathways has reconfirmed the crucial role of the hypothalamus in the regulation of food intake and energy balance. Strategically located in the midst of the mammalian neuraxis, the hypothalamus receives at least three distinct types of relevant information via direct or indirect neural connections as well as hormone receptors and substrate sensors bestowed on hypothalamic neurons. First, the medial and to a lesser extent the lateral hypothalamus receive a rich mix of information pertaining to the internal state of relative energy repletion/depletion. Second, specific hypothalamic nuclei receive information about the behavioral state, such as diurnal clock, physical activity-level, reproductive cycle, developmental stage, as well as imminent (e.g. fight and flight) and chronic (e.g. infection) stressors, that can potentially impact on short-term availability of fuels and long-term energy balance. Third, the hypothalamus, particularly its lateral aspects, receives information from areas in the forebrain involved in the acquisition, storage, and retrieval of sensory representations of the external food space and internal food experience, as well as from the executive forebrain involved in behavior selection and initiation. In addition, rich intrahypothalamic connections facilitate further distribution of incoming information to various hypothalamic nuclei. On the other hand, the hypothalamus has widespread neural projections to the same cortical areas it receives inputs, and many hypothalamic neurons are one synapse away from most endocrine systems and from both sympathetic and parasympathetic effector organs involved in the flux, storage, mobilization, and utilization of fuels. It is argued that processing within cortico-limbic areas and communication with hypothalamic areas are particularly important in human food intake control that is more and more guided by cognitive rather than metabolic aspects in the obesigenic environment of affluent societies. A distributed neural network for the control of food intake and energy balance consisting of a central processor and several parallel processing loops is hypothesized. Detailed neurochemical, anatomical, and functional analysis of reciprocal connections of the numerous peptidergic neuron populations in the hypothalamus with extrahypothalamic brain areas will be necessary to better understand what hypothalamus, forebrain, and brainstem tell each other and who is in charge under specific conditions of internal and external nutrient availability.

title: Lethal phenotype in conditional late-onset arginase 1 deficiency in the mouse.
abstract: Human arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia, which lead to neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation; uncommonly, patients suffer early death from this disorder. In a murine targeted knockout model, onset of the phenotypic abnormality is heralded by weight loss at around day 15, and death occurs typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. This discrepancy between the more attenuated juvenile-onset human disease and the lethal neonatal murine model has remained suboptimal for studying and developing therapy for the more common presentation of arginase deficiency. These investigations aimed to address this issue by creating an adult conditional knockout mouse to determine whether later onset of arginase deficiency also resulted in lethality. Animal survival and ammonia levels, body weight, circulating amino acids, and tissue arginase levels were examined as outcome parameters after widespread Cre-recombinase activation in a conditional knockout model of arginase 1 deficiency. One hundred percent of adult female and 70% of adult male mice died an average of 21.0 and 21.6 days, respectively, after the initiation of tamoxifen administration. Animals demonstrated elevated circulating ammonia and arginine at the onset of phenotypic abnormalities. In addition, brain and liver amino acids demonstrated abnormalities. These studies demonstrate that (a) the absence of arginase in adult animals results in a disease profile (leading to death) similar to that of the targeted knockout and (b) the phenotypic abnormalities seen in the juvenile-onset model are not exclusive to the age of the animal but instead to the biochemistry of the disorder. This adult model will be useful for developing gene- and cell-based therapies for this disorder that will not be limited by the small animal size of neonatal therapy and for developing a better understanding of the characteristics of hyperargininemia.

title: Clinical biochemistry parameters in C57BL/6J mice after blood collection from the submandibular vein and retroorbital plexus.
abstract: Collection of blood from the submandibular vein allows simple and rapid processing of many animals without anesthesia and facilitates rapid recovery with no signs of pain and discomfort in the mice. Here we compared the submandibular vein and retroorbital plexus blood collection methods, to determine the potential effect of the sampling technique on several clinical biochemistry parameters in C57BL/6J mice. We found statistically significant differences for 8 of the 9 biochemical parameters studied between the 2 blood sampling techniques. Compared with samples collected from the retroorbital plexus, blood obtained from the submandibular vein had higher levels of AST, ALT, protein, albumin, triglycerides, total cholesterol, and creatinine. Glucose values of retroorbital blood were higher than those from the submandibular vein. Urea levels were similar for both sampling techniques. Our results demonstrate that the technique used to obtain blood samples affects parameters commonly used to assess animal health. We recommend caution when comparing results of biochemical analysis of blood obtained from the submandibular vein in mice with reference values obtained by other blood sampling techniques.

title: Long-term survival of the juvenile lethal arginase-deficient mouse with AAV gene therapy.
abstract: Arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia. Human patients suffer from neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation. In a murine model, onset of the phenotypic abnormality is heralded by weight loss beginning around day 15 with death occurring typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. The goal of this study was to address the development of a gene therapy approach for arginase deficiency beginning in the neonatal period. Lifespan extension, body weight, circulating amino acids and ammonia levels were examined as outcome parameters after gene therapy with an adeno-associated viral vector expressing arginase was administered to mice on the second day of life (DOL). One-hundred percent of untreated arginase-deficient mice died by DOL 24, whereas 89% of the adeno-associated virus (AAV)-treated arginase deficient mice have survived for >8 months. While animals at 8 months demonstrate elevated glutamine levels, ammonia is less than three times that of controls and arginine levels are normal. These studies are the first to demonstrate that AAV-based therapy for arginase deficiency is effective and supports the development of gene therapy for this and the other urea cycle disorders.

title: AAV-based Gene Therapy Prevents Neuropathology and Results in Normal Cognitive Development in the Hyperargininemic Mouse
abstract: Complete arginase I deficiency is the least severe urea cycle disorder, characterized by hyperargininemia and infrequent episodes of hyperammonemia. Patients suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation and seizures, and is associated with intellectual disability. In mice, onset is heralded by weight loss beginning around day 15; gait instability follows progressing to inability to stand and development of tail tremor with seizure-like activity and death. Here we report that hyperargininemic mice treated neonatally with an adeno-associated virus (AAV)-expressing arginase and followed long-term lack any presentation consistent with brain dysfunction. Behavioral and histopathological evaluation demonstrated that treated mice are indistinguishable from littermates, and that putative compounds associated with neurotoxicity are diminished. In addition, treatment results in near complete resolution of metabolic abnormalities early in life; however, there is the development of some derangement later with decline in transgene expression. Ammonium challenging revealed that treated mice are affected by exogenous loading much greater than littermates. These results demonstrate that AAV-based therapy for hyperargininemia is effective and prevents development of neurological abnormalities and cognitive dysfunction in a mouse model of hyperargininemia; however, nitrogen challenging reveals that these mice remain impaired in the handling of waste nitrogen.