L-tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo

The administration of l-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo. When l-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy-dl-tryptophan and indole administration in amounts equimolar to l-tryptophan also result in similar increases in activity whereas α-methyl-dl-tryptophan produces little or no increase.Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.     

Influence of azacytidine on the induction of tyrosine aminotransferase and the NAD metabolism in rat liver

5-Azacytidine was found to inhibit the induction of tyrosine aminotransferase caused by L-tyrosine and hydrocortisone. The inhibitory effect can be overcome by L-methionine. There was only a slight inhibition of the tryptophan induction by 5-azacytidine in normal animals 4 hr after the administration of tryptophan. At 8 and 12 hr, a superinduction could be observed. The NAD content in adrenalectomized animals increased after application of tryptophan and 5-azacytidine. There was a slight inhibition of ADPR transferase in the rat liver.     

Immunological study of carbon tetrachloride-mediated induction of tyrosine aminotransferase in rat liver

Administration of CCl₄ (1.0 ml/kg) to rats resulted in a rise of liver tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) activity to a maximum of about 3.6 times the normal level 6 hr later. An immunological titration study proved that the phenomenon was due to increased enzyme content. Using an isotopic-immunochemical procedure the half-life of liver tyrosine aminotransferase at 3.5 hr after CCl₄ administration was shown to be 11.9 hr in contrast to 2.1 hr in the normal liver. Immunochemical analysis revealed that enzyme synthesis was decreased by CCl₄. Thus, in the early stage of CCl₄ poisoning, enzyme synthesis proceeded at a moderate rate while degradation was markedly impaired, resulting in the rise of tyrosine aminotransferase in the liver tissue.Several hours after administration of hydrocortisone to adrenalectomized rats, induced tyrosine aminotransferase reached its peak activity and then subsided to the basal level. At any time following hydrocortisone administration, administration of CCl₄ consistently caused an elevation of the enzyme activity above the level in controls not treated with CCl₄. Actinomycin D (5 mg/kg) also increased the enzyme at an early period of induction cycle but failed to do so at a later period.The CCl₄-mediated “superinduction” of hormonally preinduced tyrosine aminotransferase, like the induction of this enzyme by CCl₄ at a basal level, was found to be caused by the differential inhibitory effect of CCl₄ on the synthesis and degradation of this enzyme.     

Activation of tyrosine aminotransferase expression in fetal liver by 5-azacytidine

Rat fetuses of 20 days gestational age were treated in utero with the inhibitor of DNA methylation, 5-azacytidine. The liver enzyme tyrosine aminotransferase, normally expressed at very low levels until several hours after birth, was increased by the drug in the fetal livers after a lag period of about 9 hours, reaching a level 70-fold above control levels 18 hours after treatment. The high levels attained after 5-azacytidine treatment are comparable to those of glucocorticoid-treated adult livers, and were not further increased by administration of hydrocortisone to dams carrying treated fetuses. Cytidine and two other analogs, cytosine arabinoside and 6-azacytidine, were essentially without effect.     

Influence of Carbon Tetrachloride on Inducible Liver Enzymes and Response to Endotoxin in Mice

Injection of mice with a sublethal dose of endotoxin 2 hr after administration of 0.1 ml of carbon tetrachloride (CCl(4)) killed 75% of the animals. CCl(4) alone killed no controls. Significant protection against this effect was afforded by 5 mg of cortisone and by 5 mg of nicotinamide adenine dinucleotide (NAD). With a larger dose of endotoxin, cortisone did not give protection. Liver tryptophan pyrrolase activity was lowered 2 hr after CCl(4) injection and reached a minimum after 17 hr. Induction of tryptophan pyrrolase by cortisone, followed by administration of CCl(4), resulted in rapid loss of activity. A significant induction of tyrosine-alpha-ketoglutarate transaminase was observed in CCl(4)-treated mice. The induction of this enzyme by cortisone was somewhat impaired when CCl(4) was administered concurrently with the hormone. CCl(4) did not lower tryptophan pyrrolase in endotoxin-tolerant mice after 4 hr, but at 17 hr the activity was decreased as much as in control mice. Oxidized pyridine nucleotides were decreased 17 hr after administration of CCl(4). This loss was prevented by administration of NAD (5 mg) or by cortisone (5 mg). Carbon clearance from blood was reduced after treatment with CCl(4). These results indicate a degree of similarity between the metabolic effects of endotoxin and CCl(4) in that both depress tryptophan pyrrolase and prevent its induction by cortisone, and both induce tyrosine transaminase in intact mice. Both substances appear to exert these effects through some type of mediated reaction.     

Acute effects of aspartame on large neutral amino acids and monoamines in rat brain

The dipeptide aspartame (APM; aspartylphenylalanine methylester), an artificial sweetener, was studied $\text{in vivo}$ for its ability to influence brain levels of the large neutral amino acids and the rates of hydroxylation of the aromatic amino acids. The administration by gavage of APM (200 mg/kg) caused large increments in blood and brain levels of phenylalanine and tyrosine by 60 minutes. Brain tryptophan level was occasionally reduced significantly, but the brain levels of the branched-chain amino acids were always unaffected. Smaller doses (50, 100 mg/kg) also raised blood and brain tyrosine and phenylalanine, but did not reduce brain tryptophan levels. At the highest dose (200 mg/kg), APM gavage caused an insignificant increase in dopa accumulation (after NSD-1015), and a modest reduction in 5-hydroxytryptophan accumulation. No changes in the brain levels of serotonin, 5-hydroxyindoleacetic acid, dopamine, dihydroxyphenylacetic acid, homovanillic acid, or norepinephrine were produced by APM administration (200 mg/kg). These results thus indicate that APM, even when administered in amounts that cause large increments in brain tyrosine and phenylalanine, produce minimal effects on the rates of formation of monoamine transmitters.     

On the induction of the hepatic tyrosine aminotransferase by quinolinic acid.

The induction of tyrosine aminotransferase by quinolinic acid is inhibited completely by cycloheximide and by alpha-amanitin, but only partially during the first 3 hours by 5-azacytidine and 8-azaguanine; longer treatment with 8-azaguanine, however, also prevents the major increase in enzyme activity. The hepatic concentration of cyclic AMP does not change after administration of quinolinic acid. Insulin, like hydrocortisone, acts additively to qlinolinic acid. The isoenzyme pattern of tyrosine aminotransferase is not changed cosniderably during induction of quinolinic acid. Most likely, quinolinic acid acts through its own mechanism of induction.     

Effects of age and sex on the induction by triiodothyronine of cortisone delta4-5alpha-reductase and glucose 6-phosphate dehydrogenase of rat liver and adrenal

The effects of age and sex on the induction by 3,5,3′-triiodothyronine (T-3) of cortisone Δ⁴-5α-reductase and glucose 6-phosphate dehydrogenase (G 6-P D) in liver and the latter in adrenal have been investigated. Levels of cortisone Δ⁴-(5α, 5β)-reductase and G 6-P D were measured in homogenates of tissue from normal and T-3 injected male and female rats, $\text{1 1}\text{4}$ to 21 months of age. Increases in the levels of the reductase seen under T-3 stimulation were ascribed to induction of the 5α-reductase alone. T-3 caused induction of cortisone Δ⁴-5α-reductase only in the livers of male rats $\text{1}\text{3}\text{4}$ months of age. There was induction of total G 6-P D at most ages except in the livers of old male and young female rats and adrenals of young and old male rats. At all ages in normal animals of both sexes the maximum activity of glucose 6-phosphate dehydrogenase was much greater than that of cortisone Δ⁴-(5α, 5β)-reductase. It is concluded that the amount of G 6-P D in normal liver may be sufficient to handle an increase in cortisone reduction, and factors other than cortisone Δ⁴-reductase or G 6-P D levels alone must regulate increased reduction of the steroid.     

Effect of substrate and small doses of cortisone on the induction of Tryptophan 2,3-dioxygenase (author's transl)]

A number of enzymes are induced by steroid hormones. In this paper the reaction of tryptophan 2,3-dioxygenase is further analyzed. In particular we show in which way the substrate and low doses of cortisone cause an induction. 1) For the induction of tryptophan 2,3-dioxygenase in adrenalectomized rats by 2.5 mg cortisone/kg, the presence of the substrate is necessary as well. Under these conditions an induction of the enzyme can already be registered in the presence of 12.5 mg L-tryptophan/kg. 2) In animals treated before with cortisone, the enzyme maximum appears 30 min after L-tryptophan injection, The enhancement of enzyme activity in animals which are treated with 2.5 mg cortisone/kg before is blocked by actidione only until 30 min after L-tryptophan injection. 3) Experiments with antibodies in animals treated with a low dosis of cortisone show that L-tryptophan acts mainly via enzyme degradation or the saturation with the coenzyme hematin, respectively.     

Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol l-serine Pyruvate aminotransferase by dibutyryl adenosine 3′,5′-monophosphate

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine: pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have no effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serine aminotransferase diminishes with the age of animals. Increase in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyrl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol L-serrine. Pyruvate aminotransferase by dibutyryl adenosine 3,5-monophosphate.

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutyryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine:pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have not effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serin aminotransferase diminishes with the age of animall. Increases in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyryl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

Induction of tyrosine aminotransferase under the influence of D-galactosamine

The induction of the tyrosine aminotransferase by tyrosine and by tryptophan + methionine is completely inhibited by 375 mg/kg D-galactosamine-HCl. The hydrocortisone induction is reduced in dependence on the amount of D-galactosamine. Tryptophan protects to some extent the influence of low doses of D-galactosamine on the hydrocortisone induction of tyrosine aminotransferase.     

Effects of pyrazole on rat liver tryptophan oxygenase

The effects of pyrazole administration on rat liver tryptophan oxygenase have been studied both under basal conditions and after induction by cortisol or activation by tryptophan.Pyrazole administration is followed by a decrease of the basal holoenzyme and total enzyme activities. It induces furthermore a considerable inhibition of the cortisol mediated tryptophan oxygenase induction. These effects are not mediated by a modification of a tryptophan oxygenase effector, as shown by mixed homogenate experiments. The tryptophan enhancement of total tryptophan oxygenase activity is not affected by pyrazole administration contrary to the holoenzyme activity. Pyrazole added $\text{in vitro}$ inhibits liver tryptophan oxygenase activity only when used at concentrations which are considerably higher that those occuring $\text{in vivo}$ after pyrazole administration.     

Stimulation of tyrosine aminotransferase activity by dl-alpha-methyltryptophan.

DL-alpha-Methyltryptophan (alphaMeTrp), a synthetic analogue of tryptophan, has been found to be a potent inducer of hepatic tyrosine aminotransferase activity in the adrenalectomized rat. alphaMeTrp is inactive in vitro. Unlike the action of other known inducers (tryptophan, hydrocortisone, adenosine cyclic 3:5-monophosphate, and glucagon), maximal stimulation of enzyme activity occurs only 16 to 30 hours after alphaMeTrp administration and the activity is still elevated at 96 hours. Only the L isomer of alphaMeTrp is active, and addition of a hydroxyl group to position 5 of the indole ring renders an inactive compound. The induction can be prevented by actinomycin D or cycloheximide but not galactosamine. Administration of alphaMeTrp together with hydrocortisone produced an additive stimulation of enzyme activity. alphaMeTrp given along with glucagon or adenosine cyclic 3:5-monophosphate caused a further but not additive increase in enzyme activity. Tryptophan given along with alphaMeTrp promoted no extra stimulation whatsoever. These data indicate that alphaMeTrp and tryptophan may act via a common pathway which in part requires RNA synthesis. Other enzymes, namely alanine and aspartate aminotransferase, ornithine aminotransferase, ornithine carbamoyltransferase, serine dehydratase, and histidine ammonialyase, were not affected by treatment of rats with alphaMeTrp.     

Caffeine injection raises brain tryptophan level,but does not stimulate the rate of serotonin synthesis in rat brain

Acute caffeine injection (100 mg/kg) elevates brain levels of tryptophan (TRP), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA). Experiments were performed to determine if the increases in 5HT and 5HIAA result from a stimulation of the rate of 5HT synthesis. Both the rate of 5-hydroxytryptophan (5HTP) accumulation following NSD-1015 injection, and the rate of ³H-5-hydroxyindole synthesis from ³H-tryptophan were measured $\text{in vivo}$ following caffeine administration and found to be normal. Tryptophan hydroxylase activity, as measured $\text{in vitro}$ in brain homogenates, was also unaffected by caffeine. The results suggest that the elevations in brain 5HT and 5HIAA levels produced by caffeine do $\text{not}$ reflect enhanced 5HT synthesis, despite significant elevations in brain TRP level. Some other mechanism(s) must therefore be responsible for these elevations in brain 5-hydroxyindole levels.     

Phenotypic conversion of cultured mouse embryo cells by aza pyrimidine nucleosides.

Cells of the $\text{C3H}\text{10}\text{T}\text{1}\text{2}\text{CL8}$ line, which are nonmyoblastic in nature, form functional myotubes when treated with low concentrations of 5-azacytidine. Further characterization of the myotubes revealed that they arise from the fusion of mononucleated precursors and not as a result of endoreplication. They accumulate histochemically detectable myosin ATPase activity as well as acetylcholine receptors capable of binding radioactively labeled α-bungarotoxin. The deoxy analog, 5-aza-2′-deoxycytidine, induced myogenic conversion at one-tenth of the maximally effective concentration of 5-azacytidine. The ability of both analogs to induce myotube formation and to cause cytotoxicity was strongly influenced by cotreatment with certain pyrimidine nucleosides. These effects were consistent with a requirement for metabolism of both aza compounds to phosphorylated derivatives and with a mechanism of action based on their incorporation into DNA. Concentrations of the analogs causing myogenic conversion did not substantially alter rates of DNA, RNA, or protein synthesis as measured by precursor incorporation into intact cells. The induction of myotubes by 5-azacytidine in cells synchronized by two different methods required that treatment with the analog was carried out at a critical phase early in S phase. Thus the mechanism of drug action appears to be linked to specific DNA synthesis.     

Changes in the intracellular levels of pyridoxal 5'-phosphate affect the induction of tyrosine aminotransferase by glucocorticoids

In the present study a cell culture system was used to correlate the intracellular levels of pyridoxal 5′-phosphate with the induction of the hepatic enzyme, tyrosine aminotransferase, by glucocorticoids. Increased intracellular levels of pyridoxal 5′-phosphate produced antiglucocorticoid effects whereas a reduction in pyridoxal 5′-phosphate content increased the sensitivity of cells to glucocorticoids. The data strongly implicate pyridoxal 5′-phosphate as an $\text{in}\text{vivo}$ modulator of the glucocorticoid receptor. The mechanism by which pyridoxal 5′-phosphate modulates the receptor is presumably through its binding to the DNA-binding site of the “activated” form of the receptor complex.     

Inhibition of hepatic enzyme induction as a sensitive assay for endotoxin

Bacterial endotoxins in mice reduced the induction by cortisone of two hepatic enzymes, tryptophan oxygenase, and phosphoenolpyruvate carboxykinase, they prevented the glyconeogenesis in liver induced by the same hormone, and they induced in intact animals the liver enzyme tyrosine-alpha-ketoglutarate transaminase, all in proportion to their ld(50). When cortisone was given in the least amount (100 mug), it resulted in near maximal induction of tryptophan oxygenase; a smaller amount of endotoxin reduced significantly the level of enzyme than that required when 5 mg of hormone was injected. The smallest amount of endotoxin that prevented tryptophan oxygenase induction was given intravenously to adrenalectomized mice in which 25 mug of cortisone was administered. The amount (0.01 mug) is 1/40,000th of the ld(50). The other metabolic processes subject to alteration by endotoxin required at least 100 to 400 times as much. This property of endotoxin can serve as a sensitive bioassay, although the dose-response curve is steep.     

Effect of high- and low-protein diets on the regulation of rat liver tyrosine aminotransferase and tryptophan oxygenase activities by l-tyrosine and l-tryptophan

Induction of rat liver tyrosine aminotransferase by l-tyrosine and tryptophan oxygenase by l-tryptophan was studied in groups of rats fed on diets containing 18 or 5% protein. The basal activity of hepatic tyrosine aminotransferase of rats receiving 5% protein gradually increased with the age of the animals but that of rats receiving 18% protein did not. l-Tyrosine induced hepatic tyrosine aminotransferase in rats receiving 18% protein when tested at ages from 4 to 20 weeks. When induction by l-tyrosine was carried out in rats receiving the 5% protein diet, significant induction of tyrosine aminotransferase occurred only in 4- or 6-week-old rats. Induction by l-tryptophan of tryptophan oxygenase in liver or the basal activity of this enzyme in liver did not differ between the groups fed on 5 and 18% protein. On changing the diet from 0 to 18% protein, the above-mentioned effects on the induction of hepatic tyrosine aminotransferase were reversed.