Catabolism of arginine and ornithine in the perfused rat liver: effect of dietary protein and of glucagon

The rates of oxidation of arginine and ornithine that occurred through a reaction pathway involving the enzyme ornithine aminotransferase (EC 2.6.1.13) were determined using (14)C-labeled amino acids in the isolated nonrecirculating perfused rat liver. At physiological concentrations of these amino acids, their catabolism is subject to chronic regulation by the level of protein consumed in the diet. (14)CO(2) production from [U-(14)C]ornithine (0.1 mM) and from [U-(14)C]arginine (0.2 mM) was increased about fourfold in livers from rats fed 60% casein diets for 3-4 days. The catabolism of arginine in the perfused rat liver, but not that of ornithine, is subject to acute regulation by glucagon (10(-7) M), which stimulated arginine catabolism by approximately 40%. Dibutyryl cAMP (0.1 mM) activated arginine catabolism to a similar extent. In retrograde perfusions, glucagon caused a twofold increase in the rate of arginine catabolism, suggesting an effect of glucagon on arginase in the perivenous cells.     

The urea cycle and related pathways in the liver of Walker-256 tumor-bearing rats

The urea cycle was evaluated in perfused livers isolated from cachectic tumor-bearing rats (Walker-256 tumor). Urea production in livers of tumor-bearing rats was decreased in the presence of the following substrates: alanine, alanine + ornithine, alanine + aspartate, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine. Urea production from arginine was higher in livers of tumor-bearing rats. No difference was found with aspartate, aspartate + ammonia, citrulline, citrulline + aspartate and glutamine + aspartate. Ammonia consumption was smaller in livers from cachectic rats when the substance was infused together with lactate and pyruvate. Glucose production was smaller in the cachectic condition only when alanine was the gluconeogenic substrate. Blood urea was higher in tumor-bearing rats, suggesting higher rates of urea production. The availability of aspartate seems to be critical for urea synthesis in the liver of tumor-bearing rats, which is possibly unable to produce this amino acid in sufficient amounts from endogenous sources. The liver of tumor-bearing rats may have a different exogenous substrate supply of nitrogenous compounds. Arginine could be one of these compounds in addition to aspartate which seems to be essential for an efficient ureogenesis in tumor-bearing rats.     

Analysis of regulatory factors for urea synthesis by isolated perfused rat liver. I. Urea synthesis with ammonia and glutamine as nitrogen sources.

Urea synthesis was studied using the isolated liver perfusion with ammonium cholride and glutamine as nitrogen sources. The rate of urea formation increases with ammonium cholorde concentration up to 5mM, and the rate remained constant in the range between 5 and 20mM of ammonium chloride as the substrate. The concentration of ammonia in the medium to support the half-maximum velocity of urea formation was 0.7mM. The rate of urea formation was stimulated by the addition of 2.5mM ornithine, and the greater part of the ornithine which was taken up into the liver was accumulated as citrulline in the presence of ammonia. A considerable accelerating effect of N-acetylglutamate on the synthetic rate was observed, but a rather high concentration of N-acetylglutamate was required in order to obtain the maximum effect possibly, because its permeability into liver cells may be limited. A marked additive effect on the rate of urea formation was observed with the combined addition of ornithine and N-acetylglutamate. The metabolic conversion of glutamine nitrogen to urea in the perfused rat liver and the effect of several compounds which stimulated urea synthesis with ammonia were further examined. The process of conversion of glutamine nitrogen to urea might be composed of the following three steps. In the first lag phase, a small amount of glutamine was removed from the medium. In the second stage, the glutamine level decreased rapidly and ammonia was accumulated in the perfusate. The third stage was a period in which glutamine concentration remained at a constant low level, and the accumulated ammonia was rapidly conversed to urea. The rate of urea formation in this third stage was found to be much higher than that with ammonia as the substrate. The maximum rate of glutamine removal was obtained at pH 7.7 of the perfusate and at a concentration of 10mM glutamine. Urea formation with glutamine was also stimulated by the addition of ornithine, malate, or N-acetylglutamate, which had accelerating effects on the urea synthesis with ammonia. This stimulation was due to an effective conversion of ammonia to urea, but no change in the rate of removal glutamine was obtained.     

Effect of adding dietary L-lysine, L-threonine and L-methionine to a low gluten diet on urea synthesis in rats

Summary. We have shown that urinary urea excretion increased in rats fed a low quality protein. The purpose of present study was to determine whether an addition of dietary limiting amino acids affected urea synthesis in rats fed a low gluten diet. Experiments were done on three groups of rats given diets containing 10% gluten, 10% gluten+0.5% L-lysine or 10% gluten+0.5% L-lysine, 0.2% L-threonine and 0.2% L-methionine for 10d. The urinary excretion of urea, and the liver concentrations of serine and ornithine decreased with the addition of dietary L-lysine, L-threonine and L-methionine. The fractional and absolute rates of protein synthesis in tissues increased with the treatment of limiting amino acids. The activities of hepatic urea-cycle enzymes was not related to the urea excretion. These results suggest that the addition of limiting amino acids for the low gluten diet controls the protein synthesis in tissues and hepatic ornithine and decline urea synthesis.     

Amino acid release by isolated perfused cirrhotic livers

Livers of normal and cirrhotic rats were perfused in vitro with and without amino acid substrates (2.3 mM ornithine, 10 mM glutamine or 20 mM alanine) in order to assess urea formation and amino acid release. The rates of urea production were lower in the livers of cirrhotic rats when compared to those of controls only in perfusions with added substrates. The release of several amino acids by livers of cirrhotic rats was higher than that of controls although the pattern of amino acids in the perfusate was different from that reported in plasma during hepatic insufficiency.     

Control of urea synthesis and ammonia utilization in protein deprivation and refeeding.

Rats were fed a standard diet (20% protein) or a protein-free diet for up to 65 days. After 20 days on the protein-free diet some rats were refed the standard diet. By the 20th day the rats fed the protein-free diet showed a blood ammonia level approximately 70% higher than controls and urea excretion decreased approximately 20-fold. At this time the liver acetylglutamate decreased to approximately one-fifth of the initial and control levels, returning to normal after 3 days of refeeding the standard diet, with a concomitant increase in urea excretion. The protein-deficient diet resulted in decreased activities of liver enzymes related to ammonia metabolism. All enzyme activities assayed returned to normal values rapidly upon refeeding the standard diet, except hepatic carbamylphosphate synthetase, glutamine synthetase, and glutaminase, which took approximately 1 month to return to control values. The findings presented here are consistent with the view that urea production is controlled, at least under certain conditions, by acetylglutamate, the physiological activator of carbamylphosphate synthetase.     

The urea cycle in the liver of arthritic rats

The urea cycle in the liver of adjuvant-induced arthritic rats was investigated using the isolated perfused liver. Urea production in livers from arthritic rats was decreased during substrate-free perfusion and also in the presence of the following substrates: alanine, alanine + ornithine, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine but increased when arginine and citrulline + aspartate were the substrates. No differences were found with ammonia + aspartate, ammonia + aspartate + glutamate, aspartate, aspartate + glutamate and citrulline. Ammonia consumption was smaller in the arthritic condition when the substance was infused together with lactate or pyruvate but higher when the substance was simultaneously infused with aspartate or aspartate + glutamate. Glucose production tended to correlate with the smaller or higher rates of urea synthesis. Blood urea was higher in arthritic rats (+25.6%), but blood ammonia was lower (–32.2%). Critical for the synthesis of urea from various substrates in arthritic rats seems to be the availability of aspartate, whose production in the liver is probably limited by both the reduced gluconeogenesis and aminotransferase activities. This is indicated by urea synthesis which was never inferior in the arthritic condition when aspartate was exogenously supplied, being even higher when both aspartate and citrulline were simultaneously present. Possibly, the liver of arthritic rats has a different substrate supply of nitrogenous compounds. This could be in the form of different concentrations of aspartate or other aminoacids such as citrulline or arginine (from the kidneys) which allow higher rates of hepatic ureogenesis.     

Splanchnic sequestration of amino acids in aged rats: in vivo and ex vivo experiments using a model of isolated perfused liver

Splanchnic sequestration of amino acids (SSAA) is a process observed during aging that leads to decreased peripheral amino acid (AA) availability. The mechanisms underlying SSAA remain unknown. The aim of the present study was to determine whether a high-protein diet could increase nitrogen retention in aged rats by saturating SSAA and whether SSAA could be explained by dysregulation of hepatic nitrogen metabolism. Adult and aged male Sprague-Dawley rats were housed in individual metabolic cages and fed a normal-protein (17% protein) or high-protein diet (27%) for 2 wk. Nitrogen balance (NB) was calculated daily. On day 14, livers were isolated and perfused for 90 min to study AA and urea fluxes. NB was lower in aged rats fed a normal-protein diet than in adults, but a high-protein diet restored NB to adult levels. Isolated perfused livers from aged rats showed decreased urea production and arginine uptake, together with a release of alanine (vs. uptake in adult rats) and a hepatic accumulation of alanine. The in vivo data suggest that SSAA is a saturable process that responds to an increase in dietary protein content. The hepatic metabolism of AA in aged rats is greatly modified, and urea production decreases. This result refutes the hypothesis that SSAA is associated with an increase in AA disposal via urea production.     

Effect of ornithine and lactate on urea synthesis in isolated hepatocytes.

1. In hepatocytes isolated from 24 h-starved rats, urea production from ammonia was stimulated by addition of lactate, in both the presence and the absence of ornithine. The relationship of lactate concentration to the rate of urea synthesis was hyperbolic. 2. Other glucose precursors also stimulated urea production to varying degrees, but none more than lactate. Added oleate and butyrate did not stimulate urea synthesis. 3. Citrulline accumulation was largely dependent on ornithine concentration. As ornithine was increased from 0 to 40 mM, the rate of citrulline accumulation increased hyperbolically, and was half-maximal when ornithine was 8-12 mM. 4. The rate of citrulline accumulation was independent of the presence of lactate, but with pyruvate the rate increased. 5. The rate of urea production continued to increase as ornithine was varied from 0 to 40 mM. 6. It was concluded that intermediates provided by both ornithine and lactate are limiting for urea production from ammonia in isolated liver cells. It was suggested that the stimulatory effect of lactate lies in increased availability of cytosolic aspartate for condensation with citrulline.     

Impaired sensitivity to insulin of rat livers perfused with blood of diminished haematocrit.

1. In livers from fed rats perfused with homologous whole blood of a haematocrit value of 37%, insulin decreased the perfusate concentrations of glucose and amino acids, production of ketone bodies (3-hydroxybutyrate + acetoacetate) and increased bile flow. 2. Perfusion with blood diluted with buffer to a haematocrit value of 17% decreased hepatic O2 consumption by 40-50%. Perfusate concentrations of glucose and lactate, the rate of ketogenesis and the ratios [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] were all increased. 3. In livers perfused with blood of diminished haematocrit, effects of insulin on perfusate glucose an amino acids, ketogenesis and bile flow were abolished.     

Metabolic Effects of Arginine and Methionine Supplement on Rats Fed Excess Glycine Diets

The contents of glycogen, lipid, urea and amino acids, and some enzyme activities in plasma, liver muscle and urine were determined with rats fed 10 to 12 g of 100 g body weight per day of the 10% casein diet (control) and 10% casein diets containing 7% glycine with or without 1.4% l-arginine HC1 and l-methionine for 7 days.

Nine hours after the final feeding, the amount of liver glycogen was high in the order of rats fed 10% casein diet containing 7% glycine, 10% casein diet containing 7% glycine with l-arginine and l-methionine, and the control. The amount of muscle glycogen was decreased only in those fed the control diet. The amount of liver lipid was increased by the addition of l-arginine and l-methionine to the excess glycine diet. Plasma and urinary urea was increased in animals given the excess glycine diets with or without both amino acids. In plasma liver, and muscle of animals given either of both the excess glycine diets 3 and 9 hr after the feeding, in general, glycine and serine were increased, and threonine and alanine were decreased as compared with those of rats given the control diet. However, the increase of glycine in plasma, liver and muscle detected at 9 hr after feeding the excess glycine diet was slightly prevented by the supplementation of both amino acids to the excess glycine diet. The activities of liver glycine oxidase and ornithine δ-aminotransferase of rats given the excess glycine diet with both amino acids were higher than those of other dietary groups. Liver serine dehydratase and glutamate-oxalacetate transaminase activities were high in the order of the animals fed the control, the excess glycine diet and the excess glycine diet containing both amino acids. Glutamate-pyruvate transaminase activity in the liver of rats fed the excess glycine diets with or without both amino acids were markedly higher than that of those fed the control. The activities of phosphopyruvate carboxylase and aconitase in the liver of animals given the excess glycine diet were higher than those of other dietary groups. Liver pyruvate kinase and glutamate dehydrogenase activities were similar among those dietary groups.     

Effect of Individual Essential Amino Acid-Devoid Diets on the Activities of Liver Urea Cycle Enzymes in Rats

The activities of all urea cycle enzymes (carbamyl phosphate synthetase, ornithine trans- carbamylase, argininosuccinate synthetase, argininosuccinase and arginase) have been determined in the liver of rats forcibly fed diets lacking in individual essential amino acids from amino acid mixture simulating to a casein. In general, these enzyme activities (units/g liver and total units/body wt) in rats fed the single essential amino acid-devoid diet decreased as compared with those activities in animals fed complete diet, but their decreases were not as large as those observed in group of all amino acid-devoid diet. The degree of decrease in these enzyme activities differed somewhat from each other in individual enzymes and each essential amino acie-devoid groups. In contrast, in rats fed the arginine devoid diet, the activities (total units/body wt) of all enzymes expect the case of arginase increased more than those in the group of complete diet.     

Regulation of urea synthesis by agmatine in the perfused liver: studies with 15N

Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of l-[guanidino-15N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15N-labeled glutamine and unlabeled ammonia or 15NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15N2]agmatine is formed in the liver from perfusate l-[guanidino-15N2]arginine ( approximately 90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15N-labeled NAG and [15N]urea from 15N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15N-labeled NAG and [15N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.     

Ornithine metabolism in relation to stimulation of urea cycle,induced by high protein diet

Approximate rates of some in vivo ornithine metabolisms in rats were calculated by pulse-labeling data, on the assumption that hepatic metabolite levels are constant during a given 3-min period. The rate of ornithine catabolism was 70–110 nmol/min/g liver; that of urea output was 3–6 μmol/min/g liver; the rotary rate of the “ornithine flux” was 10–12 rpm. A change from a 25 to a 70% casein diet resulted in a 1.5-fold augmentation in the rate of ornithine catabolism and a 1.6-fold increase in the rate of urea output; however, the rate of the “ornithine flux” remained nearly constant These findings suggest that stimulation of the urea cycle is accompanied not by acceleration of the cycle rotation, but rather by increased mass of the metabolite flux.     

Regulation of urea synthesis The effect of ammonia on the N-acetylglutamate content of isolated rat liver cells

(1) Incubation of isolated rat liver cells in the presence of lactate and ammonia increases the AcGlu content. The increase is very fast in the first minutes and a steady-state concentration is reached in approx. 10 min after the addition of ammonia. (2) The amount of increase depends on the diet rats were fed before isolation of liver cells. AcGlu is increased 4-fold in hepatocytes from rats fed a carbohydrate-rich diet. If ornithine is simultaneously present with ammonia a further increase is found. (3) Urea synthesis in hepathocytes from rats fed a carbohydrate-rich diet has a marked lag period. The reason for this lag phase is the low initial AcGlu concentration. (4) Increase in AcGlu is closely associated with increase in mitochondrial glutamate content. Thus, it is concluded that the glutamate concentration is the mediator of the ammonia effect.     

The necessity of niacin in rats fed on a high protein diet

It is known that niacin itself is not necessary in rats when tryptophan is given in adequate amounts, because rats can biosynthesize niacin from tryptophan. In our experiment, young rats were fed on a 20%, 40%, 60%, or 70% casein diet with or without niacin. The rats fed on the 20%, 40%, and 60% casein diets did not require niacin for growth, but the rats fed on the 70% casein diet needed it. This phenomenon was attributed to the supposition that liver aminocarboxymuconate-semialdehyde decarboxylase activities increased according with the dietary casein levels. The conversion ratio of tryptophan-niacin in rats fed on the 70% casein diet became extremely low, and then the rats needed niacin.     

Role of anion translocation across the mitochondrial membrane in the regulation of urea synthesis from ammonia by isolated rat hepatocytes.

The regulation of urea synthesis from ammonia was investigated using isolated hepatocytes from fasted rats. Addition of ammonia alone produced only a small increase of urea formation, which was stimulated 2-fold by ornithine in conjunction with a fall of ATP levels and an accumulation of citrulline. Further addition of oleate or beta-hydroxybutyrate produced an additional 2-fold stimulation of urea formation to approximately 200 mumol/g dry weight/hour. The presence of oleate also protected against the inhibitory effect of 2,4-dinitrophenol on urea synthesis and the cellular ATP content. The data suggest that both the rate of of energy production and the rate of generation of reducing equivalents from endogensou substrates are insufficient to meet the requirements for optimal rates of urea synthesis. Urea formation from NH3 in the presence of ornithine and oleate, but iin the absence of gluconeogenic precursors, was inhibited by butylmalonate, a known inhibitor of malate-phosphate exchange across the mitochondrial membrane, and stimulated by theaddition of malate and other dicarboxylic acids and amino acids to the cell suspension...     

Control by pH of urea synthesis in isolated rat hepatocytes

Control by pH of urea synthesis has been studied in isolated rat hepatocytes incubated with a physiological mixture of amino acids. Inhibition of urea synthesis by decreasing the pH of the medium was caused by diminished production of ammonia and not, as suggested in the literature, by inhibition of entry of ammonia into the ornithine cycle. The decrease by low pH of the rate of degradation of the added amino acids, that of alanine being quantitatively the most important, was accompanied by a decrease in their intracellular concentration. It is concluded that inhibited transport of amino acids across the plasma membrane of the hepatocyte is responsible, at least in part, for the fall in urea synthesis with decreasing pH. It is proposed that inhibition by low pH of other steps in the ureogenic pathway, distal to the production of ammonia, does not affect flux through the ornithine cycle per se, but rather contributes to the buffering of the intrahepatic concentration of ammonia.     

Supplementation of orotic acid to the casein, but not to egg protein, soy protein, or wheat gluten diets, increases serum ornithine carbamoyltransferase activity

Effects of dietary supplementation of orotic acid to a diet containing the casein protein were compared with diets containing egg protein, soy protein, or wheat gluten on lipid levels in the liver and serum and activities of ornithine carbamoyltransferase (OCT) and alanine aminotransferase in the serum of rats. We found that supplementation of orotic acid to each diet increased the contents of the liver total lipids, triacylglycerol, and phospholipids compared with those not supplemented. The contents of liver total lipids, triacylglycerol, cholesterol, and phospholipids in rats fed the casein diet were significantly higher than those of rats fed the other three diets when orotic acid was supplemented. The levels of triacylglycerol, cholesterol, and phospholipids in the serum of rats fed the casein diet were markedly decreased by addition of orotic acid. The supplementation of orotic acid significantly increased the activities of both serum OCT and alanine aminotransferase in rats fed the casein diet, but not in rats fed the other diets. In conclusion, liver lipid accumulation induced by dietary orotic acid depends on the type of dietary protein. The enhancement of serum OCT activity may result from liver lipid accumulation in rats fed the casein diet supplemented with orotic acid, demonstrating hepatic damage.     

Plasma arginine and ornithine levels and selected enzyme activities in uremic rats fed various amounts of arginine

Rats weighing 100 g were made chronically uremic by partial left renal artery ligation and contralateral nephrectomy. Rats with urea clearances below 0.30 ml/min and sham-operated controls were pair-fed arginine-free diets, diets containing normal amounts of arginine or diets with high levels of arginine. After 4 to 8 weeks, rats were killed and plasma levels of arginine, ornithine and lysine were measured. In addition, activities of various urea cycle enzymes in liver and kidney and renal transamidinase were determined. Plasma amino acid levels and enzyme activities of the urea cycle remained constant in control rats fed diets differing in arginine content. However, renal transamidinase activity was elevated in control rats fed arginine-free diets. In plasma of uremic as compared with control rats, arginine levels varied with the arginine intake, and lysine levels were elevated when arginine supplements were fed. With all diets, plasma ornithine remained constant in uremic rats at slightly but not significantly increased levels. Hepatic carbamoyl phosphate synthetase activity and renal arginine synthetase activity were reduced in uremic as compared to control rats. Renal transamidinase activity, expressed per g of kidney, was elevated in uremic rats with all diets except arginine-free. When amino acid diets were fed, hepatic arginase activity was higher in uremic rats and this increase was enhanced by arginine-free diets. Other enzyme activities in uremic rats were not affected by the amount of arginine in the diet.