Effect of glucagon on phenylalanine metabolism and phenylalanine-degrading enzymes in the rat

Glucagon administered subcutaneously to rats for 10 days had no significant effect on liver phenylalanine hydroxylase activity, but induced liver dihydropteridine reductase more than twofold. In rats administered a phenylalanine load orally, glucagon treatment stimulated oxidation and depressed urinary phenylalanine excretion. These responses could not be related to an effect of glucagon on hepatic tyrosine-alpha-oxoglutarate aminotransferase activity. Even in rats with phenylalanine hydroxylase activity depressed to 50% of control values by p-chlorophenylalanine administration, glucagon treatment increased the phenylalanine-oxidation rate substantially. Although hepatic phenylalanine-pyruvate aminotransferase was increased tenfold in glucagon-treated rats, glucagon treatment did not increase urinary excretion of phenylalanine transamination products by rats given a phenylalanine load. Glucagon treatment did not affect phenylalanine uptake by the gut or liver, or the liver content of phenylalanine hydroxylase cofactor. It is suggested that dihydropteridine reductase is the rate-limiting enzyme in phenylalanine degradation in the rat, and that glucagon may regulate the rate of oxidative phenylalanine metabolism in vivo by promoting indirectly the maintenance of the phenylalanine hydroxylase cofactor in its active, reduced state.     

Pteridine cofactor of phenylalanine hydroxylase in fetal rat liver

1. Pteridine cofactor of phenylalanine hydroxylase (EC 1.14.16.1) and dihydropteridine reductase (EC 1.6.99.7) in the phenylalanine hydroxylating system have been studied in the fetal rat liver. 2. Activities of pteridine cofactor and dihydropteridine reductase were measured as about 6 and 50%, respectively, of the levels of adult liver in the liver from fetuses on 20 days of gestation, at this stage the activity of phenylalanine hydroxylase was almost negligible in the liver. 3. Development of the activity of sepiapterin reductase (EC 1.1.1.153), an enzyme involved in the biosynthesis of pteridine cofactor, was studied in rat liver during fetal (20-22 days of gestation), neonatal and adult stages comparing with the activity of dihydrofolate reductase (EC 1.5.1.3). Activities of the enzymes were about 80 and 50%, respectively, of the adult levels at 20 days of gestation. 4. Some characteristics of sepiapterin reductase and dihydropteridine reductase of fetal liver were reported.     

Liver phenylalanine hydroxylase activity in relation to blood concentrations of tyrosine and phenylalanine in the rat

The plasma concentration of phenylalanine and tyrosine decreases in normal rats during the first few postnatal days; subsequently, the concentration of phenylalanine remains more or less constant, whereas that of tyrosine exhibits a high peak on day 13. The basal concentrations of the two amino acids were not altered by injections of thyroxine or cortisol, except in 13-day-old rats, when an injection of cortisol decreased the concentration of tyrosine. In young rats (13-15 days old), treatment with cortisol increased the activity of phenylalanine hydroxylase in the liver (measured in vitro) and accelerated the metabolism of administered phenylalanine: the rate constant of the disappearance of phenylalanine from plasma and the initial increase in tyrosine in plasma correlated quantitatively with the activity of phenylalanine hydroxylase in the liver. In adult rats, the inhibition of this enzyme (attested by assay in vitro) by p-chlorophenylalanine resulted in a proportionate decrease in tyrosine formation from an injection of phenylalanine. However, the quantitative relationship between liver phenylalanine hydroxylase activity and phenylalanine metabolism within the group of young rats was different from that observed among adult rats.     

The adaptive behaviour of isoenzyme forms of rat liver alanine amino transferases during development

1. The activities of the mitochondrial and cytosol isoenzyme forms of l-alanine–glyoxylate and l-alanine–2-oxoglutarate aminotransferases were determined in rat liver during foetal and neonatal development. 2. The mitochondrial glyoxylate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. The cytosol glyoxylate aminotransferase and the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities first appear prenatally, increase further after birth and then rise to the adult values during weaning. 4. In foetal liver the mitochondrial glyoxylate aminotransferase and the cytosol 2-oxoglutarate aminotransferase activities are increased after injection in utero of glucagon, dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) or thyroxine. The cytosol glyoxylate aminotransferase and the mitochondrial 2-oxoglutarate aminotransferase activities are increased after injection in utero of cortisol or thyroxine. 5. After birth the further normal increases in the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities can be hastened by cortisol injection, whereas the increase in cytosol glyoxylate aminotransferase activity requires cortisol treatment together with the intragastric administration of casein. 6. The results are discussed with reference to the metabolic patterns and the changes in regulatory stimuli (hormonal and dietary) that occur during the period of development.     

The quantitative determination of phenylalanine hydroxylase in rat tissues. Its developmental formation in liver

A sensitive method was developed for determining the phenylalanine hydroxylase activity of crude tissue preparations in the presence of optimum concentrations of the 6,7-dimethyl-5,6,7,8-tetrahydropterin cofactor (with ascorbate or dithiothreitol to maintain its reduced state) and substrate. Tissue distribution studies showed that, in addition to the liver, the kidney also contains significant phenylalanine hydroxylase activity, one-sixth (in rats) or half (in mice) as much per g as does the liver. The liver and the kidney enzyme have similar kinetic properties; both were located in the soluble phase and were inhibited by the nucleo-mitochondrial fraction. Phenylalanine hydroxylase, like most rat liver enzymes concerned with amino acid catabolism, develops late. On the 20th day of gestation, the liver (and the kidney) is devoid of phenylalanine hydroxylase and at birth contains 20% of the adult activity. During the second postnatal week of development, when the phenylalanine hydroxylase activity was about 40% of the adult value, an injection of cortisol doubled this value. Cortisol had no significant effect on phenylalanine hydroxylase in adult liver or on phenylalanine hydroxylase in kidney at any age.     

A comparative analysis of the ontogenic development of rat liver sequential enzymes-tyrosine -ketoglutarate aminotransferase, p-hydroxyphenylpyruvate hydroxylase, and homogenitsate oxygenase

The specific activities (per milligram of DNA) of the three rat liver sequential enzymes—tyrosine aminotransferase (TAT), p-hydroxyphenyl pyruvate hydroxylase (PHPP hydroxylase), and homogentisate oxygenase develop coordinately in a stepwise pattern from birth to adulthood.Just after birth about 80% of TAT but only 20–30% of PHPP hydroxylase appears to be active in vivo. The active fraction of TAT remains essentially constant during development of the rat but that of PHPP hydroxylase increases with age to reach about 90% in adulthood.The differences in the enzymes of the infant and adult rat (ontogenic changes) are, in general, similar to those observed in the enzymes, respectively, of reptile and mammal (phylogenic changes).The biochemical mechanisms involved in the in vivo activation of PHPP hydroxylase and the in vitro reactivation of the adult enzyme inactivated by various reagents are analyzed.     

The regulation of phenylalanine hydroxylase in rat tissues in vivo. Substrate- and cortisol-induced elevations in phenylalanine hydroxylase activity.

Injections of phenylalanine increased a 2.5-fold in 9 h the hepatic phenylalanine hydroxylase activity of 6-day-old or adult rats that had been pretreated (24h earlier) with p-chlorophenylalanine; without such pretreatment, phenylalanine did not raise the enzyme concentration. This difference is paralleled by the much greater extent to which the injected phenylalanine accumulated in livers of the pretreated compared with the normal animals. The hormonal induction of hepatic phenylalanine hydroxylase activity obeyed different rules: an injection of cortisol was without effect on adult livers but caused a threefold rise in phenylalanine hydroxylase activity of immature ones, both without and after pretreatment with p-chlorophenylalanine. In the latter instance, the effects of cortisol, and of phenylalanine were additive. Actinomycin inhibited the cortisol- but not the substrate-induced increase of phenylalanine hydroxylase, whereas puromycin inhibited both. The results indicate that substrate and hormone, two potential positive regulators of the amount of the hepatic (but not the renal) phenylalanine hydroxylase, act independently by two different mechanisms. The negative effector, p-chlorophenylalanine, also appears to interact with the synthetic (or degradative) machinery rather than with the existing phenylalanine hydroxylase molecules: 24h were required in vivo for an 85% decrease to ensue, and no inhibition occurred in vitro when incubating the enzyme with p-chlorophenylalanine or with liver extracts from p-chlorophenylalanine-treated rats.     

The isolation and properties of phenylalanine hydroxylase from human liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.     

The metabolism of L-phenylalanine and L-tyrosine by liver cells isolated from adrenalectomized rats and from streptozotocin-diabetic rats.

Flux through, and maximal activities of, key enzymes of phenylalanine and tyrosine degradation were measured in liver cells prepared from adrenalectomized rats and from streptozotocin-diabetic rats. Adrenalectomy decreased the phenylalanine hydroxylase flux/activity ratio; this was restored by steroid treatment in vivo. Changes in the phosphorylation state of the hydroxylase may mediate these effects; there was no significant change in the maximal activity of the hydroxylase. Tyrosine metabolism was enhanced by adrenalectomy; this was not related to any change in maximal activity of the aminotransferase. Steroid treatment increased the maximal activity of the aminotransferase. Both acute (3 days) and chronic (10 days) diabetes were associated with increased metabolism of phenylalanine; insulin treatment in vivo did not reverse these changes. Although elevated hydroxylase protein concentration was a major factor, changes in the enzyme phosphorylation state may contribute to differences in phenylalanine degradation in the acute and chronic diabetic states. Tyrosine metabolism, increased by diabetes, was partially restored to normal by insulin treatment in vivo. These changes can, to a large extent, be interpreted in terms of changes in the maximal activity of the aminotransferase.     

Tyrosine aminotransferase activity in human fetal liver

There are at least two enzymes in adult human liver that transaminate tyrosine: cytoplasmic tyrosine aminotransferase (EC 2.6.1.5) and mitochondrial aspartate aminotransferase (EC 2.6.1.1). Total tyrosine aminotransferase activity in the supernatant fraction of adult human liver was 19.8 nmol of p-hydroxyphenylpyruvate formed per min/mg of protein as compared to 0.53 in fetuses of 12--22 weeks of gestational age and 2.0 in the newborn. The presence of specific tyrosine aminotransferase (EC 2.6.1.5) could be demonstrated by isoelectric focusing techniques in fetal human liver during the first trimester. No specific tyrosine aminotransferase could be detected in the placenta. Total tyrosine aminotransferase activity was elevated by dexamethasone and tyrosine administration to organ cultures of fetal liver.     

The influence of starvation and tryptophan administration on the metabolism of phenylalanine, tyrosine and tryptophan in isolated rat liver cells.

Liver cells from fed Sprague-Dawley rats metabolized phenylalanine, tyrosine and tryptophan at rates consistent with the known kinetic properties of the first enzymes of each pathway. Starvation of rats for 48 h did not increase the maximal activities of phenylalanine hydroxylase, tryptophan 2,3-dioxygenase and tyrosine aminotransferase in liver cell extracts, when results were expressed in terms of cellular DNA. Catabolic flux through the first two enzymes was unchanged; that through the aminotransferase was elevated relatively to enzyme activity. This is interpreted in terms of changes in the concentrations of 2-oxoglutarate and glutamate. Cells from tryptophan-treated animals exhibited significant increases in the catabolism of tyrosine and tryptophan, but not of phenylalanine. The activities of tyrosine aminotransferase and tryptophan 2,3-dioxygenase were also increased, although the changes in flux and enzyme activity did not correspond exactly. These results are discussed with reference to the control of aromatic amino acid catabolism in liver; the role of substrate concentration is emphasized.     

Properties of phenylalanine hydroxylase of cultured hepatoma cells.

The kinetic and immunologic properties of phenylalanine hydroxylase of adult rat liver were compared to the properties of the similar enzyme present in cultured H4-II-E-C3 hepatoma cells. The enzymes from the two sources could not be distinguished by the Km values for either phenylalanine or 6,7-dimethyltetrahydropterin. Analysis by double immunodiffusion showed that phenylalanine hydroxylase from the two sources had identical immunologic determinants, but immunotitrations revealed a small but significant difference between the enzyme of the normal adult rat liver and the enzyme of cultured hepatoma cells. The results of double immunodiffusion and immunotitration experiments indicated also that the increased levels of phenylalanine hydroxylase seen in the hepatoma cells grown in the presence of hydrocortisone resulted from the accumulation of enzyme protein, but it could not be decided whether this accumulation resulted from an increased rate of synthesis or decreased rate of degradation.     

Comparison of alpha-methylphenylalanine and p-chlorophenylalanine as inducers of chronic hyperphenylalaninaemia in developing rats.

alpha-Methylphenylalanine is a very weak competitive inhibitor of rat liver phenylalanine hydroxylase in vitro but a potent suppressor in vivo. The loss of the hepatic activity (the renal one is unaffected) becomes maximal (70-75% decrease; cf. control) 18h after the administration (per 10g body wt.) of 24 mumol of alpha-methylphenylalanine with or without 52 mumol of phenylalanine. Chronic suppression of hepatic phenylalanine hydroxylase was obtained by injections of alpha-methylphenylalanine plus phenylalanine to suckling rats, and by their addition to the diet after weaning. A series of comparisons of the effects of this treatment, and one with p-chlorophenylalanine, was then carried out. In both cases there was a rise (1.3-2-fold) in phenylalanine-pyruvate amino-transferase activity (but no change in four other enzyme activities) in the liver; in brain there was a rise in phosphoserine phosphatase activity, but the total activity and subcellular distribution of nine enzymes revealed no other abnormalities in cerebral development. Striking increases in the concentration of plasma phenylalanine during 26 of the 31 experimental days (with a transient fall at 18-22 days) were maintained by treatment with both analogues plus phenylalanine. However, p-chlorophenylalanine-treated animals had a 30-60% mortality rate and 27-52% decrease in body weight. Developing rats treated with alpha-methylphenylalanine, showing no growth deficit or signs of toxicity (e.g. cataracts), appear to be a more suitable model for the human disease of phenylketonuria. Their phenylalanine concentrations exhibited at least 20-40-fold increase during 50% of each of the first 18 days of life, and 30-fold after weaning.     

Hydrocortisone induction of phenylalanine hydroxylase isozymes in cultured hepatoma cells.

The hydrocortisone stimulation of phenylalanine hydroxylase activity in Reuber H4 hepatoma cells is shown to be associated with an alteration in phenylalanine hydroxylase isozyme composition. Three forms of phenylalanine hydroxylase were identified in H4 cells which have been treated with hydrocortisone; however, only one of these forms appears to be present prior to glucocorticoid treatment. The relative amounts, as well as the total amount, of the three forms and their chromatographic behavior on hydroxylapatite are nearly identical to the three phenylalanine hydroxylase isozymes found in adult rat liver. The hydroxylase isozyme composition in 2 day old rats is similar to that found in adult rats and in H4 cells treated with hydrocortisone.     

Modification of the multiple forms of rat hepatic phenylalanine hydroxylase by in vitro phosphorylation

The two major forms of rat liver phenylalanine hydroxylase have been isolated and partially purified. The tetrahydrobiopterin-dependent activity of these forms can be differentially stimulated by exposure to enzymatic phosphorylating conditions. This $\text{in vitro}$ treatment is associated with incorporation of 32p into the enzymes and generates a further, chromatographically distinct, species. These results suggest that the multiple forms of rat liver phenylalanine hydroxylase are due to different degrees of phosphorylation.     

Effects on induction of tyrosine aminotransferase in fetal mouse liver in vitro of prednisolone,insulin and thyroxine

The hormonal requirements for formation of tyrosine aminotransferase (EC 2.6.1.5) in fetal mouse liver were investigated in organ culture using chemically defined medium. The hormones tested were insulin, thyroxine and prednisolone. Prednisolone alone resulted in a two-fold increase in tyrosine amino-transferase activity in explanted liver in hormone-free medium on day 6, and its effect was dose dependent, but neither insulin nor thyroxine alone induced the enzyme. Addition of prednisolone plus thyroxine and prednisolone plus insulin increased the enzyme activity 1.4- and 1.3-fold, respectively, over that of explants with prednisolone alone. These three hormones together had the greatest effect, causing induction of 1.5-fold more activity than that with prednisolone plus insulin or plus thyroxine. The three hormones were not all needed continuously during the culture period: prednisolone and insulin were required during the early part of cultivation and thyroxine during the later part. The effects of these hormones were blocked by actinomycin D or puromycin, suggesting that these hormones increase de novo synthesis of tyrosine aminotransferase. Phase-contrast microscopy showed that prednisolone stimulated liver epithelial cell outgrowth, probably acting with insulin.     

Enzymes in intracellular organelles of adult and developing rat brain

Eighty percent of the hexokinase and about a half of the lactate dehydrogenase, pyruvate kinase, and aldolase activities of adult rat cerebral homogenates is particulate, associated to a large extent, with the sediment (P₂) obtained by centrifugation at 17,000g. Centrifugation of P₂ into sucrose gradients shows that all four enzymes are associated with synaptosomes: their peak concentration coincides with that of glutamate decarboxylase rather than with those of mitochondrial enzymes, glutamate dehydrogenase, and aspartate aminotransferase. After hypoosmotic shock and high-speed centrifugation considerable portions of synaptosomal enzymes are recovered in the supernatant phase; the composition of this fluid, as indicated by the higher specific activity of several enzymes, is different from that of the soluble fraction of whole homogenates.The concentration of the seven enzymes studied is considerably lower in fetal than in adult brain and, in general, a larger fraction of the total is soluble. Preferential accumulation with age in the particulate fraction is especially striking in the case of hexokinase. Between fetal and adult life there are changes in the enzymic composition as well as increases in the amount of the total protein attributable to the synaptosomal fraction. Glutamate decarboxylase and lactate dehydrogenase are the synaptosomal enzymes to rise first (before or at birth), followed by hexokinase and, in the third postnatal week, by aldolase and pyruvate kinase. The upsurge of mitochondrial enzymes (that of glutamate dehydrogenase at term and of aspartate aminotransferase 10 days later) is accompanied by insignificant or small increases in the total protein content of the same fraction. The results indicate that the maturation of subcellular organelles involves a stepwise enrichment with various enzymes; some signs of biochemical differentiation precede and others coincide with the development of cerebral functions known to occur in 2- to 4-wk-old rats.     

Mechanism of inactivation of phenylalanine hydroxylase by p-chlorophenylalanine in hepatome cells in culture. Two possible models.

The mechanism by which p-chlorophenylalanine specifically reduces phenylalanine hydroxylase activity in rat liver in vivo and in Reuber H4 hepatoma cells in culture has been investigated. Chromatography on hydroxylapatite of liver extract from rats injected with p-chlorophenylalanine showed that the compound differentially affected the three normal phenylalanine hydroxylase isoenzymes (I, II, and III); isoenzymes II and III were completely absent after the treatment, but isoenzyme I was only reduced in quantity compared with normal adult rats. Normal Reuber H4 cells only possess isoenzyme I; treatment with p-chlorophenylalanine yielded a reduced level of enzyme activity which appeared to be noraml isoenzyme I by both chromatographic and kinetic criteria. There is evidence, based on immunochemical techniques, that cultures grown in the presence of p-chlorophenylalanine have significantly reduced levels of phenylalanine hydroxylase antigen, and that p-chlorophenylalanine inactivates phenylalanine hydroxylase at or near the time of enzyme synthesis. The bulk of enzyme synthesized prior to the addition of the compound appears unaffected by it. There is no indication that protein synthesis itself is affected by p-chlorophenylalanine. In addition, p-chlorophenylacetate was found to inactivate phenylalanine hydroxylase in an apparently identical manner with p-chlorophenylalanine, which almost certainly eliminates from consideration any mechanism of inactivation specifically requiring an amino acid. Finally, effects of cycloheximide and chlorophenylalanine were compared. Taken together, the data lead to two possible models for the inactivation of the enzyme. The model most consistent with all data requires (predicts) the existence of a proenzyme form of phenylalanine hydroxylase which can be specifically inactivated by p-chlorophenylalanine.     

Role of thyroxine in the postnatal development of rat hepatic tryptophan oxygenase and ornithine aminotransferase

The activities of tryptophan oxygenase and ornithine aminotransferase are known to increase markedly in rat liver during the postnatal period. The aim of this study was to determine whether thyroxine regulates the development of these two enzymes. It was found that hyperthyroidism had no effect on the activity of tryptophan oxygenase, but caused a modest increase of ornithine aminotransferase activity at 10 days of age. The latter effect persisted in adrenalectomized animals, indicating that it was not secondary to elevation of plasma corticosterone. When thyroxine was administered together with cortisone acetate, elevation of ornithine aminotransferase activity was substantially greater than that observed with cortisone acetate alone. It is concluded that the postnatal development of hepatic ornithine aminotransferase is primarily controlled by glucocorticoids, but that the effect of these hormones may be potentiated by thyroxine.     

Immunoquantification of epoxide hydrolase and cytochrome P-450 isozymes in fetal and adult human liver microsomes

Epoxide hydrolase and three cytochrome P-450 isozymes were immunochemically determined in microsomes from adult and fetal human liver and tentatively correlated with some enzyme activities. The P-450 isozymes 5, 8 and 9 present in adult liver could not be positively correlated with the total cytochrome P-450 concentration spectrophotometrically determined. In fetal liver microsomes, isozyme 8 could not be detected by either electrophoretic or immunochemical procedures. Isozyme 5 was the major isozyme present in the fetal liver and its concentration increased in close relation with the total P-450 level. As shown previously, arylhydrocarbon hydroxylase activity was related to the concentration of isozyme 8 in adult liver. In fetal preparations, the absence of isozyme 8 was associated with a very low arylhydrocarbon hydroxylase activity. Aldrin epoxidase and benzphetamine-N-demethylase activities were correlated with isozyme 5 concentration, but with different slopes for adult and fetal microsomes: adult preparations catalyzed these two reactions more efficiently. Conversely, the dehydroepiandrosterone 16 beta-hydroxylase, also associated with isozyme 5 concentration, was more active in fetal than in adult microsomes. Moreover, if acetanilide hydroxylase increased with isozyme 5 concentration in adult samples, no correlation occurred between activity and P-450 isozyme level in fetal microsomes. Hydroxylations of lauric acid in positions 11 and 12 and of dehydroepiandrosterone in position 16 alpha increased with total P-450 concentration but not with isozyme concentrations whatever the age considered. Lastly, epoxide hydrolase activity towards benzopyrene 4,5-oxide was closely associated with its immunochemically determined level. These results clearly suggest that multiple mechanisms are involved in the regulation of different drug-metabolizing enzymes in the human fetus.