Dietary supplements of mixtures of indispensable amino acids lacking threonine, phenylalanine or histidine increase the activity of hepatic threonine dehydrogenase, phenylalanine hydroxylase or histidase, respectively, and prevent growth depressions in chicks caused by dietary excesses of threonine, phenylalanine, or histidine*

Experiments were carried out to determine whether the addition of a mixture of indispensable amino acids (IAA) lacking in threonine, phenylalanine or histidine, respectively, to a nutritionally complete diet would increase the hepatic activities of the rate-limiting enzymes for catabolism of threonine, phenylalanine or histidine and prevent the adverse effects of the amino acid on growth when the dietary level of the amino acid is excessive. Week old Leghorn chicks were fed semi-purified diets containing 19% crude protein to which were added no IAA supplement or 10% crude protein from an IAA mix and 5 graded levels of either L-threonine, L-phenylalanine or L-histidine in a 2 x 5 factorial arrangement of treatments. Each amino acid was investigated in a separate experiment involving four replicate pens (seven chicks each) per diet. Weight gains and feed consumptions were determined on the fourteenth day of each experiment. The groups receiving no excess, and 1.0% or 2.0% excesses of amino acids were sampled on the fifteenth day for enzyme activities and plasma amino acid concentrations. Weight gain and/or feed consumption were lower, and plasma concentrations of threonine, phenylalanine and histidine were higher, in chicks receiving 1.5 to 2.0% dietary additions of threonine, phenylalanine, and histidine, respectively, than in chicks that did not receive these amino acids. Chicks that received the amino acids in diets that also contained the IAA supplement had better growth and feed consumption, lower plasma concentrations of threonine, phenylalanine or histidine, higher plasma concentrations of other indispensable amino acids, and higher activities of threonine dehydrogenase, phenylalanine hydroxylase, and histidase than chicks receiving excess amino acids in the absence of IAA supplements. We conclude that the dietary level of protein, not the dietary level of individual amino acids, is the primary determinant of the activity of amino acid degrading enzymes in liver. The increased activity of these enzymes may be the mechanism by which dietary protein alleviates the adverse effects of excessive levels of individual amino acids.     

Histidine degradation enzymes in rat liver: Induction by high protein intake

Summary High protein dietary content stimulates urea formation in ureotelic animals but does not exert almost any effect on ammonia production from L-amino acids in vitro. L-histidine and L-threonine are the only amino acids which are most actively deaminated by ureotelic animals fed on a high protein diet.All the steps of L-histidine metabolism have been studied: it has been found that both the histidine transaminase pathway and the histidase pathway are stimulated. Glutamic acid is also a product of histidine catabolism through the histidase pathway, but its catabolism is unaffected by the dietary protein content. These data suggest the existence of independent mechanism controlling the catabolism of the two amino acids.     

Cloning and expression of rat histidase. Homology to two bacterial histidases and four phenylalanine ammonia-lyases

Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.     

Dietary arginine influences Rous sarcoma growth in a major histocompatibility B complex progressor genotype.

L-Arginine (L-Arg) can serve as a substrate for the production of reactive nitrogen intermediates. One of these metabolites, nitric oxide, has been shown to possess significant antitumor properties in vitro. To investigate the importance of this system in vivo, we have examined the dietary L-Arg host tumor interaction in the chicken. Since chickens are incapable of de novo L-Arg synthesis, concentration of this amino acid is readily controlled by diet. Line UNH 105 New Hampshire chickens having the major histocompatibility complex genotype, B24/B24, were used to study in vivo effects of dietary L-Arg on Rous sarcoma growth. After 5 weeks on a standard diet, 119 chicks were fed either a basal (0.92% L-Arg) diet or a high arginine (2.40% L-Arg) diet. One week later, chicks were wing-web inoculated with subgroup A Rous sarcoma virus. Tumor growth was monitored weekly for 12 weeks after inoculation. Plasma L-Arg levels and body weights from birds on each dietary treatment were analyzed. Neither body weight gains nor latent period for tumor development was affected by diet. However, plasma L-Arg levels were significantly different between dietary treatments (basal, 0.245 +/- 0.01 mumol/ml; high, 0.738 +/- 0.03 mumol/ml). In addition, mean tumor size scores were significantly (P less than 0.05) lower over time in chickens fed the high L-Arg diet. The results suggest that dietary L-Arg in excess of the amount required for growth reduces tumor load.     

Characterization and expression of chicken selenoprotein W

As an essential trace element, selenium (Se) deficiency results in White Muscle Disease in livestock and Keshan disease in humans. The main objectives of this study were to clone and characterize the chicken selenoprotein W (SeW) gene and investigate SeW mRNA expression in chicken tissues. The deduced amino acid (AA) sequence of chicken SeW contains 85 AAs with UAG as the stop codon. Like all SeW genes identified in different species, chicken SeW contains one well-conserved selenocysteine (Sec) at the 13th position encoded by the UGA codon. The proposed glutathione (GSH)-binding site at the Cys₃₇ of SeW is not conserved in the chicken, but Cys₉ and Sec₁₃, with possible GSH binding, are conserved in SeWs identified from all species. There are 23–59% and 50–61% homology in cDNA and deduced AA sequences of SeW, respectively, between the chicken and other species. The predicted secondary structure of chicken SeW mRNA indicates that the selenocysteine insertion sequence element is type II with invariant adenosines within the apical bulge. The SeW mRNA expression is high in skeletal muscle followed by brain, but extremely low in other tissues from chickens fed a commercial maize-based diet. The SeW gene is ubiquitously expressed in heart, skeletal muscle, brain, testis, spleen, kidney, lung, liver, stomach and pancreas in chickens fed a commercial diet supplemented with sodium selenite. These results indicate that dietary selenium supplementation regulates SeW gene expression in the chicken and skeletal muscle is the most responsive tissue when dietary Se content is low.     

Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida.

The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli. The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approximate molecular weight 53,600, in the location previously identified for the histidase gene by Tn1000 mutagenesis. Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence. This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits. Comparison of the P. putida histidase gene sequence with that of a Bacillus subtilis region encoding histidase revealed 42% identity at the protein level. Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.     

Purification of histidase from Streptomyces griseus and nucleotide sequence of the hutH structural gene.

Histidine ammonia-lyase (histidase) was purified to homogeneity from vegetative mycelia of Streptomyces griseus. The enzyme was specific for L-histidine and showed no activity against the substrate analog, D-histidine. Histidinol phosphate was a potent competitive inhibitor. Histidase displayed saturation kinetics with no detectable sigmoidal response. Neither thiol reagents nor a variety of divalent cations had any effect on the activity of the purified enzyme. High concentrations of potassium cyanide inactivated histidase in the absence of its substrate or histidinol phosphate, suggesting that, as in other histidases, dehydroalanine plays an important role in catalysis. The N-terminal amino acid sequence of histidase was used to construct a mixed oligonucleotide probe to identify and clone the histidase structural gene, hutH, from genomic DNA of the wild-type strain of S. griseus. The cloned DNA restored the ability of a histidase structural gene mutant to grow on L-histidine as the sole nitrogen source. The deduced amino acid sequence of hutH shows significant relatedness with histidase from bacteria and a mammal as well as phenylalanine ammonia-lyase from plants and fungi.     

Suppressive effect of excess dietary histidine on the expression of hepatic metallothionein-1 in rats

The gene expression of liver metallothionein-1 in excess dietary histidine was investigated by feeding rats ad libitum on either a basal or histidine-excess (50 g of L-histidine per kg of diet) diet for 5 d. The copper content of the liver and zinc level in the serum of the rats fed on the histidine-excess diet were lower by 21% and 61%, respectively of the figures for the rats fed on the basal diet, but the zinc content of the liver and copper level in the serum were not affected. Excess dietary histidine caused an increase in the urinary output of both copper and zinc. The level of liver metallothionine-1 mRNA was markedly lower at 19% in the rats fed on the a histidine-excess diet compared to the level with the basal diet. It thus appears that such a response by the level of liver metallothionein-1 mRNA might have been be due to the lower content of liver copper.     

Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro

The activity of histidase (l-histidine ammonia-lyase EC 4. 3. 1. 3) of rat liver was inhibited by cystine competitively with the substrate histidine. Histidase was inactivated by preincubation with cystine, but the inactivated enzyme was reactivated by the addition of glutathione to the reaction mixture. Cysteine, at a lower level, partly reactivated the enzyme inactivated by cystine, but, at a higher level, did not reactivate it. Cysteine itself acted inhibitory on the enzyme. Glutathione did not reactivate histidase activity inhibited by cysteine. No other amino acid affected histidase activity at the level of 2 mm. Apparent Km of rat liver histidase was 1.8 × l0^?m.

Neither inhibitory nor activating effect on histidase activity was observed by the addition of liver homogenate obtained from rats fed a histidine imbalanced diet to that from those fed a basal diet and vice versa.     

Studies on Hormonal Effects on Hepatic Histidase and Alanine Transaminase of Rats Fed on Either Histidine Controlled or Imbalanced Diet

Effects of several hormones on liver histidase activity were investigated to study the mechanism of elevation of the activity of histidase when rats were fed on a histidine imbalanced diet.

Continuous injection of thyroxine (5~10 µg/rat/day) diminished histidase activity, and hypophysectomy increased histidase activity as compared with that of non treated animals. No change was observed on alanine transaminase (GPT) by above treatments.

Neither histamine, insulin, triamcinolone, adrenaline, nor adrenalectomy affected on the activity of histidase. Injection of histidine to rats fed on a histidine imbalanced diet made the activities of histidase and GPT tend to decrease.     

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.     

Effects of breeds and dietary protein levels on the growth performance, energy expenditure and expression of avUCP mRNA in chickens

The physiological mechanisms of thermogenesis, energy balance and energy expenditure are poorly understood in poultry. The aim of this study was designed to investigate the physiological roles of avian uncoupling protein (avUCP) regulating in energy balance and thermogenesis by using three chicken breeds of existence striking genetic difference and feeding with different dietary protein levels. Three chicken breeds including broilers, hybrid chickens, and non-selection Wuding chickens were used in this study. Total 150 chicks of 1 day of age, with 50 from each breed were reared under standard conditions on starter diets to 30 days. At 30 days of age, forty chicks from each breed chicks were divided into two groups. One group was fed low protein diet (LP, 17.0 %), and the other group was fed high protein diet (HP, 19.5 %) for 60 days. Wuding chickens showed the lowest feed conversion efficiency (FCE) and the highest expressions of avUCP mRNA association with high plasma T3 and insulin concentrations. Hybrid chickens showed the lowest expressions of avUCP mRNA association with high FCE and energy efficiency. Expressions of avUCP mRNA association with diet-induced thermogenesis (DIT) were only observed in broiler and hybrid chickens. The expressions of avUCP mRNA were positive association with plasma insulin, T3 and NEFA concentrations. Age influence on the expression of avUCP mRNA were observed only for hybrid and broiler chickens. It seems that both roles of avUCP regulation thermogenesis and lipid utilisation as fuel were observed in the present study response to variation in dietary protein and breeds.     

Studies on Dietary Effects on Hepatic Histidase and Alanine Transaminase of Rats Fed on either Histidine Controlled or Imbalanced Diet

Liver glutamic pyruvic transaminase (GPT) activity of rats fed on a histidine imbalanced diet was higher than those of animals fed on a histidine controlled or corrected diet. Histidase activity was not changed by varying the amount of histidine in diets from zero to 0.8% when all other amino acids were presented in a balanced composition, and the excessive amount of histidine in a low protein diet increased the activity of histidase slightly. Histidase activity increased linearly with each increase of the amount of phenylalanine in the diets from zero to 0.4%, but the further increase of phenylalanine (2%) did not increase the enzyme activity any more. On the other hand, GPT decreased by increaseing the amount of both histidine and phenylalanine in the diet along with the increase of biological value of the diet. Replacing the carbohydrate with hydrogenated coconuts oil did not alter the activity of histidase, and increased the activity of GPT.     

Hepatic histidase and muscle branched chain aminotransferase gene expression in experimental nephrosis

Protein and amino acid metabolism is altered during nephrotic syndrome. However, the expression of the amino acid degrading enzymes has not been well studied. The objective of this work was to assess the expression of hepatic histidase (Hal) and skeletal muscle mitochondrial branched chain amino transferase (BCATm) in rats with experimental nephrotic syndrome induced by a single injection of puromycin aminonucleoside (150 mg/kg). Six days after the injection rats were killed and hepatic Hal and skeletal muscle BCATm activities were measured. Also, total mRNA from both tissues was isolated and Hal and BCATm mRNA expression were analyzed by Northern blot. Rats with NS showed a reduction in food intake with respect to the control group. Hepatic Hal activity increased significantly in nephrotic and pair fed rats by 59% compared to control group. This change in activity was associated with a corresponding increase in Hal mRNA abundance. On the other hand, skeletal muscle BCATm activity and mRNA abundance were similar in the three groups studied. These results suggest that the increase in Hal expression was associated with the reduced food intake and not to the NS. However, BCAT expression did not change indicating the importance of BCAA in body nitrogen conservation.     

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.     

Effects of dietary grape seed extract on growth performance,amino acid digestibility and plasma lipids and mineral content in broiler chicks

Polyphenols are chemically and biologically active compounds. Grape seed extracts (GSEs) have been widely used as a human food supplement for health promotion and disease prevention. However, there is little information regarding its application in animal feeds. An experiment was conducted to investigate the effect of inclusion of GSE at 0.025, 0.25, 2.5 and 5.0 g/kg in a wheat soya bean control diet on growth performance, protein and amino acid (AA) digestibility and plasma lipid and mineral concentrations in broiler chickens at 21 days of age. Performance was not affected by dietary treatment except in the case of birds fed the diet with the highest GSE concentration, which showed a worsening of weight gain and feed conversion. Apparent ileal digestibility (AID) of protein was significantly reduced in the birds fed the highest concentration of GSE, which also had a reduction on the AID of arginine, histidine, phenylalanine, cystine, glutamic acid and proline compared with those fed control diet. The inclusion of graded concentration of GSE in the chicken diets caused a significant linear decrease in the concentrations of plasma copper, iron and zinc. Plasma cholesterol, triglycerides and lipoproteins (high-density lipoprotein, low-density lipoprotein and very-low-density lipoprotein) concentrations were not affected by dietary GSE. In conclusion, this study demonstrated that incorporation of GSE in chicken diets up to 2.5 g/kg had no adverse effect on growth performance or protein and AA digestibility. Feed conversion was reduced and growth rate was retarded, when chickens were fed 5 g/kg of GSE. This study also indicated that grape polyphenols reduce the free plasma minerals.     

Molecular cloning of chicken metallothionein. Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA.

A cDNA library was constructed using RNA isolated from the livers of chickens which had been treated with zinc. This library was screened with a RNA probe complementary to mouse metallothionein-I (MT), and eight chicken MT cDNA clones were obtained. All of the cDNA clones contained nucleotide sequences homologous to regions of the longest (376 bp) cDNA clone. The latter contained an open reading frame of 189 bp, and the deduced amino acid sequence indicates a protein of 63 amino acids of which 20 are cysteine residues. Amino acid composition and partial amino acid sequence analyses of purified chicken MT protein agreed with the amino acid composition and sequence deduced from the cloned cDNA. Amino acid sequence comparisons establish that chicken MT shares extensive homology with mammalian MTs, but is more closely related to the MT-II than to the MT-I isoforms from various mammals. The nucleotide sequence of the coding region of chicken MT shares approximately 70% homology with the consensus sequence for the mammalian MTs. Southern blot analysis of chicken DNA indicates that the chicken MT gene is not a part of a large family of related sequences, but rather is likely to be a unique gene sequence. In the chicken liver, levels of chicken MT mRNA were rapidly induced by metals (Cd2+, Zn2+, Cu2+), glucocorticoids and lipopolysaccharide. MT mRNA was present in low levels in embryonic liver and increased to high levels during the first week after hatching before decreasing again to the basal levels found in adult liver. The results of this study establish that MT is highly conserved between birds and mammals and is regulated in the chicken by agents which also regulate expression of mammalian MT genes. However, in contrast to the mammals, the results suggest the existence of a single isoform of MT in the chicken.     

Activities of Threonine Dehydratase and Histidase of Rats Fed on either Threonine or Histidine Imbalanced Diet

Liver threonine dehydratase and histidase activities of rats fed on a threonine and a histidine imbalanced diets respectively, were measured, each of which was the degradation enzyme of the most limiting amino acid for rats on each imbalanced diet. Rats of the imbalanced groups initially lost their body weight, and began to grow again after a few days. Threonine dehydratase activity decreased by changing from stock diet to the experimental diets, and no difference was observed among the control and the imbalanced groups. Histidase activity decreased gradually on the control diet, but, the enzyme activity of the histidine imbalanced group was maintained at the higher level. The inconsistency among the enzyme activities and growth showed that neither the increase of threonine dehydratase activity in threonine imbalance nor that of histidase activity in histidine imbalance would be the main cause of the imbalance.     

Variation of glutathione level and synthesis activity in chick liver due to selenium and vitamin E deficiencies

Chicks were fed an amino acid mixture-based diet (basal diet) or one supplemented with selenium (Se, 0.2 micrograms/g as Na2SeO3) and/or vitamin E (100 micrograms/g as alpha-tocopherol). The group receiving the basal diet devoid of Se and vitamin E showed a tendency to grow slowly, but not significantly so, compared to the non-deficient control and manifested a symptom of exudative diathesis after the feeding period of 4 weeks. Supplementation of the basal diet with Se or vitamin E prevented the deficiency symptoms in the chicks. The hepatic GSH level and GSH synthesis activity were about three times as much in the Se- and vitamin E-deficient group as in the control. This was also the case for in vivo sulfur incorporation into hepatic GSH for 10 h post-injection with [35S]methionine. The increased level of GSH may partly compensate the hepatocytes for peroxidative damage.     

Glycine-serine interrelations in the chick.

Glycine and serine are involved in numerous important functions in the body in addition to protein synthesis. Glycine is synthesized by higher animals; however, the rate is not adequate to support maximal growth of the chick. Studies indicate that chicks fed a glycine-serine free crystalline amino acid diet grow at approximately 80% the rate of chicks fed the same diet supplemented with glycine. An equimolar quantity of L-serine has been shown to support equal chick performance as glycine, thus indicating that serine conversion is adequate to meet the dietary need for glycine. The serine-glycine interconversion is catalyzed by the folic acid containing enzyme sering hydroxymethyltransferase, and a deficiency of this vitamin decreases the effectiveness of serine in meeting the chick's dietary need for glycine. Studies with chicks fed normal and high levels of a crystalline amino acid mixture devoid of glycine and serine indicate that the chick has the metabolic potential to synthesize more of these two amino acids than should be required for normal growth. The observed dietary response to glycine or serine or both indicates, however, that this synthetic potential is not being utilized.