Effects of Arginine and Methionine on the Growth Depression of Rats Fed Diets High in Glycine

In order to determine if the growth retardation by dietary exceess glycine could be prevented by the addition of arginine and/or methionine, weanling rats were fed a 25% casein diet (standard) or a 10% casein diet (low protein diet) with a supplement of several combinations of glycine, arginine, or methionine.

The changes in body weight, urinary creatinine, and kidney transamidinase activity were determined. The growth depression effect by excess glycine was prevented considerably in animals receiving standard diet and completely in animals receiving low protein diet by the addition of arginine and methionine to the high glycine diets.

The total urinary creatinine was increased by the supplement of both glycine and arginine, while the growth rate was not invariably raised and kidney transamidinase activity had a tendency to decrease.     

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.     

Supplemental Effects of Arginine and Methionine on Growth,and on Formations of Urea and Creatine of Adrenalectomized Rats Fed High Glycine Diets

The effects of adrenalectomy on growth, some enzyme activities in the liver and kidney, and urinary excretion of urea, creatinine and creatine were investigated in rats fed the 10% casein diets containing 7% glycine with or without l-arginine and l-methionine (10C, 10C7G and 10C7ArgMet).

Body weight gains of the intact 10C and 10C7GArgMet groups were almost same as the corresponding adrenalectomized groups. The body weight of the adrenalectomized 10C7G group was extremely decreased though that of the intact 10C7G group was maintained almost constant; but the decrease was recovered by the administration of hydrocortisone. The activities of liver arginase and carbamylphosphate synthetase were not affected by those diets. Liver serine dehydratase and ornithine δ-aminotransferase activities were increased in the intact 10C7G and 10C7GArgMet groups, but these increases were depressed by adrenalectomy. Glutamate-pyruvate transminase activities in the liver of intact 10C7G and 10C7GArgMet groups were also enhanced, but were extremely decreased in the corresponding adrenalectomized groups. Kidney transamidinase activity was not affected by adrenalectomy. The amount of urinary excreted urea was almost unchanged by adrenalectomy, but was increased by hydrocortisone administration. The amounts of excreted creatine of the adrenalectomized groups were generally larger than the corresponding intact groups, but slightly decreased by the administration of hydrocortisone. The amount of excreted creatinine was not generally affected by adrenalectomy.     

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 Dietary Addition of Amino Acids and Proteins on the Activities of Some Urea Cycle Enzymes in Rat Liver

The activities of ornithine transcarbamylase, arginine synthetase and arginase in the liver of rats receiving basal diets containing 25% casein supplemented respectively with arginine, aspartic acid, glutamic acid, glycine, a mixture of arginine, aspartic acid and glutamic acid, egg albumin, casein, wheat gluten and gelatin have been determined.

These urea cycle enzymes in rats receiving diets supplemented with the various nitrogen sources were generally increased, but the increments were due to the increase of the ingested amount of nitrogen, and not the specific effect of the individual amino acids or proteins. The excretion of urinary urea in general was increased proportionally with the elevations of these enzyme activities, independent of the nature of the dietary nitrogen.     

Free Amino Acid Contents of Plasma and Urine,and Liver Enzyme Activities in Rats Fed High Glycine Diets

The contents of plasma free amino acids, the amounts of urinary excreted amino acids and urea, and the activities of liver serine dehydratase, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase were determined in weanling rats fed ad libitum a 10% casein diet (control), a 10% casein diet containing 7% glycine and 10% casein diets containing 7% glycine supplemented with 1.4% L-arginine and/or 0.9% L-methionine for 14 days.

The remarkable increase of glycine and the moderate increase of serine in the plasma of animals fed excess glycine diets were observed. The amount of excreted glycine in the urine of animals fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than that of animals given the excess glycine diet. Urinary excreted urea of rats fed the excess glycine diet was a little greater and that of rats fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than the control. Liver serine dehydratase activity of animals given the excess glycine diets with or without L-arginine was higher than the control and the highest activity was observed in the liver of animals fed the excess glycine diet containing L-arginine and L-methionine. The activity of liver glutamic-oxalacetic transaminase of rats fed the excess glycine diet containing L-arginine and L-methionine was a little higher than that of rats given the other diets. Liver glutamic-pyruvic transaminase activity was a little higher in animals given the excess glycine diets with or without L-arginine and further higher in animals fed the excess glycine diet containing L-arginine and L-methionine than the control.     

Repression of rat kidney L-arginine:glycine amidinotransferase synthesis by creatine at a pretranslational level

The first committed reaction in the biosynthesis of creatine is catalyzed by the enzyme L-arginine:glycine amidinotransferase, commonly called transamidinase. Creatine, the end product of the biosynthetic pathway, is known to alter the levels of kidney transamidinase activity. Rats fed a diet containing 0.3% creatine had 26% of the kidney transamidinase activity of the rats fed a creatine-free diet. This reduction in transamidinase activity was correlated with a decrease in transamidinase protein in the creatine-fed rats. The relative synthetic rates and mRNA functional activities of transmidinase were measured in control and creatine-fed rats. The relative synthetic rate of transamidinase in creatine-fed rats was 21% of that found in the control animals. The functional transamidinase mRNA in creatine-fed rats was correspondingly reduced to 37% of the amount in the control animals. Thus, creatine affects transamidinase activity by altering its rate of synthesis at a pretranslational step and represents an example of end-product repression in a higher eukaryote.     

Effects of Sulfur-containing Amino Acids and Glycine on Plasma Cholesterol Level in Rats Fed on a High Cholesterol Diet

The effects of the addition of individual amino acids on methionine-induced hypercholesterolemia (experiment 1), and the interacting effects of dietary protein level and sulfur-containing amino acids and glycine on plasma cholesterol concentration (experiment 2) were studied in growing rats fed on a high cholesterol diet. In experiment 1, rats were fed on a 25% casein-0.75% methionine (25CM) diet containing 2.5% of individual amino acids for 2 weeks. Methionine-induced hypercholesterolemia was prevented by the concurrent addition of glycine or serine, but the other amino acids tested (alanine, threonine, leucine, phenylalanine, lysine, arginine, and glutamic acid) had no effect. Histidine rather enhanced the hypercholesterolemia. In experiment 2, rats were fed on a 10%, 25%, or 50% casein diet containing 0.75% methionine, 0.60% cystine, 0.63% taurine, 2.5% glycine, or 0.75% methionine +2.5% glycine for 3 weeks. Dietary addition of 0.75% methionine increased the plasma cholesterol concentration for the 25% and 50% casein diets, but it decreased the plasma cholesterol for the 10% casein diet. When the addition level of methionine was doubled in the 10% casein diet, the plasma cholesterol concentration was significantly higher for the 1.5% methionine-added diet than for the 0.75% methionine-added diet. Cystine and taurine lowered plasma cholesterol for all dietary casein levels. Methionine-induced hypercholesterolemia with 25% and 50% casein diets was prevented by the glycine supplementation. These data suggest that sulfur-containing amino acids and glycine are important in plasma cholesterol regulation.     

Oleuropein supplementation increases urinary noradrenaline and testicular testosterone levels and decreases plasma corticosterone level in rats fed high-protein diet

The effects of oleuropein, a phenolic compound in extra virgin olive oil, on protein metabolism were investigated by measuring testicular testosterone and plasma corticosterone levels in rats fed diets with different protein levels. In Experiment 1, rats were fed experimental diets with different protein levels (40, 25 and 10 g/100 g casein) with or without 0.1 g/100 g oleuropein. After 28 days of feeding, the testosterone level in the testis was significantly higher and the plasma corticosterone level was significantly lower in rats fed the 40% casein diet with oleuropein than in those fed the same diet without oleuropein. The urinary noradrenaline level, nitrogen balance and hepatic arginase activity were significantly higher in rats fed the 40% casein diet with oleuropein supplementation than in those fed the 40% casein diet without oleuropein supplementation. In Experiment 2, the effects of oleuropein aglycone (a major phenolic compound in extra virgin olive oil and the absorbed form of oleuropein ingested in the gastrointestinal tracts) on the secretion of luteinizing hormone (LH) from the pituitary gland, which regulates testosterone production in the testis, were investigated in anesthetized rats. Plasma LH level increased dose dependently after the administration of oleuropein aglycone (P<.001, r= 0.691). These findings suggest that dietary supplementation with 0.1 g/100 g oleuropein alters the levels of hormones associated with protein anabolism by increasing urinary noradrenaline and testicular testosterone levels and decreasing plasma corticosterone level in rats fed a high-protein diet.     

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.     

Soy protein,casein, and zein regulate histidase gene expression by modulating serum glucagon

Glucagon has been postulated as an important physiological regulator of histidase (Hal) gene expression; however, it has not been demonstrated whether serum glucagon concentration is associated with the type and amount of protein ingested. The purpose of the present work was to study the association between hepatic Hal activity and mRNA concentration in rats fed 18 or 50% casein, isolated soy protein, or zein diets in a restricted schedule of 6 h for 10 days, and plasma glucagon and insulin concentrations. On day 10, five rats of each group were killed at 0900 (fasting), and then five rats were killed after being given the experimental diet for 1 h (1000). Rats fed 50% casein or soy diets showed higher Hal activity than the other groups studied. Rats fed 50% zein diets had higher Hal activity than rats fed 18% casein, soy, or zein diets, but lower activity than rats fed 50% casein or soy diets. Hal mRNA concentration followed a similar pattern. Hal activity showed a significant association with serum concentrations of glucagon. Serum glucagon concentration was significantly correlated with protein intake. Thus the type and amount of protein consumed affect Hal activity and expression through changes in serum glucagon concentrations.     

Protease and Protease Inhibitor Activity in Rat Skeletal Muscle during Growth,Protein Deficiency and Fasting

Growing rats fed a protein deficient diet showed an increase in acid and alkaline protease activity and a decrease in protease inhibitor activity. Similar results were found after two and four day fasting periods. Animals on diets of 10% and 20% casein showed parallel protease and protease inhibitor activity when compared to each other at similar body weight periods.     

Effects in chicks of arsenic,arginine, and zinc and their interaction on body weight,plasma uric acid,plasma urea,and kidney arginase activity

The interaction among arsenic, zinc, and arginine was studied in chicks using two fully crossed, three-way, two-by-two-by-two experiments. Arsenic at levels of 0 and 2 μg/g zinc at levels of 2.5 (zinc-deficient) and 25 (zinc-adequate) μg/g, and arginine at levels of 0 and 16 mg/g were supplemented to the diet. After 28 d in both experiments, growth was depressed in chicks fed diets either supplemented with arginine or deficient in zinc. Arsenic deprivation depressed growth of chicks fed diets containing the basal level of arginine and 25 μg supplemental Zn/g. Arsenic deprivation had little or no effect on growth of zinc-deprived chicks fed diets containing the basal level of arginine, or in zinc-deprived or zinc-adequate chicks fed supplemental arginine. Zinc-deficiency elevated urea in plasma and arginase activity in kidney. Those elevations, however, were more marked in arsenic-supplemented than in arsenic-deprived chicks. Also, plasma urea and kidney arginase activity were markedly elevated in chicks fed supplemental arginine; the elevations were more marked in zinc-deficient chicks. These findings support the concept that arsenic has a physiological role, associated with zinc, that can influence arginine metabolism in the chick.     

An Inverse Relationship between Liver Arginase Activity and Urea Excretion in Rats

Liver arginase activity in rats fed graded levels of diammonium citrate in high and low casein diets, was measured with simultaneous determination of urea excretion. The arginase activity changed inversely with the urea excretion.

Moreover, when the amino acid balance was quantitatively changed by varying threonine level alone, a similar relationship between total liver arginase activity and urea excretion was again observed.

From these results, the early teleological assumptions are most unlikely in that liver arginase activity increases with the decrease in dietary protein quality and that the change in activity can be used as an index of dietary protein quality.     

Analysis of regulatory factors for urea synthesis by isolated perfused rat liver. II. Comparison of urea synthesis in livers of rats subjected to different dietary conditions.

Capacities for urea synthesis and amino acid patterns in the perfused livers isolated from rats fed low and high-protein diets were compared. Urea formation with amjonium chlorode as the nitrogen source in perfused livers isolated from rats fed on a 70% casein diet was rapid and the efficiency of conversion of ammonia to urea was 97.9%. However, that in livers isolated from rats fed on a 5% casein diet was much slower and the efficiency of conversion of ammonia to urea was only 36.1%. The ratios of the rate of urea formation from ammonium chloride to activity of ornithine transcarbamylase [EC 2.1.3.3.] in the perfused livers of rats fed on 5 and 70% casein diets were calculated. The ratio of the former condition was much lower than that of the latter. The ratios reached nearly the same level by the addition of ornithine and N-acetylglutamate, the addition of which to the perfusate caused marked elevation of the ratios in both cases. In the perfused livers from rats fed on a 5% casein diet a considerable portion of the ammonia added to the perfusate was fixed into an amino ro an amide group of amino acids such as alamin, aspartate, and glutamine. On the other hand, in the perfused livers from rats fed on a 70% casein diet most of the ammonia added was converted to urea. The regulation of urea synthesis and the relation between anabolism and catabolism of amino acids in rat livers subjected to different dietary conditions were compared.     

Dietary Protein Quality and Liver Arginase Activity of Rats

The arginase activity of the liver of rats was measured after they had been maintained for 11 or 12 days on diets containing natural proteins (casein or gluten) or amino acid mixtures of various tryptophan levels.

Specific activity or total arginase activity increased with the increasing protein quality. Liver arginase activity of the casein group was significantly higher than that of the gluten group in every case for 27-, 61-, and 158-day-old rats. In the case of amino acid diets, the arginase increased with the increments of tryptophan levels up to the “tentative” minimum requirement in the diet. Moreover, these alterations in arginase activity varied inversely with the urinary urea excretion.

From the results, it was assumed that the total activity of liver arginase is not necessarily determined only by the metabolic needs for urea biosynthesis.     

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.     

Increased Plasma Homocysteine Concentration in Rats from a Low Casein Diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine β-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.     

Increased plasma homocysteine concentration in rats from a low casein diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine beta-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.     

Interrelationship of Growth and Plasma Amino Acid Concentration in the Chick Fed Casein Diets as Influenced by Dietary Protein and Amino Acid Levels

Chicks fed a 20% casein-sucrose diet showed severe growth depression, but dietary supplementation with 0.7% arginine-HCl, 0.35% glycine and 0.35% dl-methionine improved the growth rate to almost that of chicks fed a practical diet. Chicks fed high protein diets containing 64% casein showed normal growth, irrespective of the supplementation with such amino acids. Plasma arginine concentration of growth-retarded chicks was significantly lower than that of normal chicks. Plasma threonine/arginine ratio was negatively correlated with the growth rate of the chicks, the ratio of normal chicks being 3 ~ 4 whereas that of growth-retarded chicks was about 24. No lysine-arginine antagonism occurred-under high casein feeding.