Tryptophan Oxygenase Activity Research Articles

Comparative analysis of tryptophan oxygenase activity and glucocorticoid receptor under the influence of insulin

This investigation addresses the interaction of insulin (INS) and glucocorticoid (GC) signaling in the hepatic regulation of tryptophan oxygenase (TO) enzyme activity in the rat. Male Wistar rats (200-250 g b.w) received an injection of the different doses of INS (10, 25, 50, 70 and 100 microg/200 g b.w., i.p.) and were used for experiments 3 h and 18 h after INS administration. This study shows that maximum of TO activity was found at dose of 50 microg of INS with peak increases observed at 3 h and 18 h after injection of INS, while INS had no effect on TO activity in adrenalectomized rats. The analysis of INS effects on glucocorticoid receptor-complex (GC/GR complex) stability shows that complexes from INS-treated rats are less stable than those from control animals. In addition, INS-stimulated stability of glucocorticoid receptor (GR) protein was significantly increased from the controls. Furthermore, the results show that GC/GR complexes from INS-treated rats could be activated and accumulated at higher rate in cell nuclei of control animals. These data support the involvement of INS in modulation of GC signaling pathway which mediates, in part, the activity of TO.

Electrophysiological characteristics of a population of mushroom body neurons in Apis Mellifera honeybees in kynurenine deficiency

Electrophysiological methods were used to study the neurophysiological characteristics of mushroom body neurons in snow (laranja) mutant and wild-type bees. The snow (laranja) mutation, which produces a sharp reduction in the activity of the enzyme tryptophan oxygenase, thus creating a deficiency of all kynurenines in the body, modifies the bioelectrical properties of mushroom body neurons. The parameters most dependent on the snow (laranja) mutation and, thus, the level of endogenous kynurenines, were the duration of action potential afterdepolarization recorded from neurons in the calyx and the amplitude of the postsynaptic potential of neurons in the calyx evoked by focal stimulation of the antennal lobes. There was also a tendency to an increase in the spontaneous spike frequency. These data lead to the conclusion that the effect of the mutation is recessive in nature and appears only in homozygotes (bearing two mutant alleles).

Basal and glucocorticoid induced changes of hepatic glucocorticoid receptor during aging: relation to activities of tyrosine aminotransferase and tryptophan oxygenase

The characteristics of glucocorticoid receptors, their sensitivity to glucocorticoid as well as the basal and glucocorticoid induced thyrosine aminotranferase (TAT) and tryptophan oxygenase (TO) activities were studied in rat liver during aging. The concentration (N) and dissociation constant (K(d)) of glucocorticoid receptor (GR) significantly change during the aging both in untreated and dexamethasone treated animals. The level of receptors was lower in dexamethasone treated rats of all analyzed aged groups compared to untreated animals. In comparison to untreated groups, there was no correlation between the changes of N and K(d) during the lifespan. According to immunochemical analysis, the decline of receptor protein content occurs during lifespan. Dexamethasone treatment reduced the level of receptor protein compare to respective age group of untreated rats. The glucocorticoid-receptor (G-R) complexes from both untreated and treated animals underwent thermal activation, although the extent of activation was more pronounced in the case of untreated groups compared to treated animals. The magnitude of heat activation of receptor complexes was more pronounced in the liver of the youngest untreated rats compared to elderly ones, while the receptor activation between treated groups of studied ages has shown less significant differences. Besides, basal as well as induced TAT and TO activities after dexamethasone injection also showed age-related alterations. The observed alterations in GR might play a role in the changes of the cell responses to glucocorticoid during the age. This presumption is supported by detected changes in basal and dexamethasone induced TAT and TO activities during aging.

ROUGH AND ROCKY ROAD TO THE RETINOID REVOLUTION

Some of us who were born in the middle of Europe between World Wars I and II had to face quite a few unusual challenges that we all met in different ways. I was born and raised in Prague, Czechoslovakia, a country at the time of my birth that was governed by a Western style of democracy, which was later destroyed by the occupation by Nazi Germany and subsequently by the takeover by the equally cruel Communists. Life required special means of adaptation to the changing living conditions and a great deal of luck to survive. After graduating from the School of Technology, I started working in the Department of Medicine at Charles University in Prague as a clinical chemist in endocrinology. This work was followed with training in basic biochemistry and the study of metabolic changes in stress. This rather diversified research, due to my changing of workplaces, led to the findings that diet can change enzymatic activity of liver tryptophan oxygenase. For a short time I worked on the metabolism of cyclic AMP in Escherichia coli, and at the age of 41, I made a risky move and succeeded in escaping with my family from the "paradise of communism." The reasons for this decision will become clear. After settling in the United States, I worked on the mechanism of activation of liver tryptophan oxygenase by cyclic AMP and eventually moved to the Vanderbilt University School of Medicine. There I initially worked on the mechanism of action of steroid hormones and finally on the molecular mechanism of action of retinoids, retinol, and retinoic acid. Also in cooperation with neonatologists, I initiated studies on prematurely born human neonates which led to successful supplementation of these patients with vitamin A. The work from my laboratory and my coworkers eventually became recognized.

Elevated basal activity of tryptophan oxygenase in alcohol-preferring C57BL mice.

Tryptophan oxygenase activity in alcohol-preferring C57Bl mice and control CBA and DBA/2 mice was studied under nonstressful conditions and after glucocorticoid-induced stress. Elevated basal tryptophan oxygenase activity in C57Bl mice is probably responsible for reduced brain content of tryptophan and serotonin associated with alcohol preference.

Cancer cachexia and depressive states: a neuro-endocrine-immunological disease?

Plasma 5-hydroxytryptamine (serotonin), tryptophan, neopterin and cortisol levels were measured in patients with depressive cancer cachexia and in healthy controls during the same time period. Patients with advanced cancers had significantly raised neopterin, a marker of endogenous gamma-interferon (IFN-gamma) production, and cortisol values, but decreased serotonin and tryptophan levels. Much work has been done to elucidate the possible role of serotonin in depressive states. IFN-gamma induces a high level of indoleamine dioxygenase (IDO), a tryptophan degrading enzyme, and high cortisol levels induce high tryptophan oxygenase activity, which in turn increases metabolism along the tryptophan-nicotinic acid pathway. These results suggest that persistent immune activation and intense adrenal activity occur in patients with cancer cachexia, resulting in disorders involving tryptophan metabolism followed by depression in cancer cachexia.

Conversion ratio of tryptophan to niacin in rats fed a vitamin B1-free diet.

The effect of a vitamin B1-free diet on the conversion ratio of tryptophan to niacin in rats was investigated using the current methods for determination of the intermediate metabolites. Rats were fed with diets with or without B1 for 33 days. The body weight gain and food intake in the B1-free group were almost the same as the control group for 10 days, but, they steeply dropped after that time and the conversion ratio of tryptophan to niacin began to increase. The ratio reached 7-fold that of the control group on the last day of the experiment. This finding seriously differed from the previous reports, which described that B1 was needed in the first part of tryptophan conversion to niacin and that the conversion ratio of tryptophan to niacin decreased in B1-deficient rats. Furthermore, the activity of tryptophan oxygenase, in which it was reported that B1 is required for the tryptophan catalytic reaction, did not decrease but increased even when the B1-free diet was fed. These results suggest that there is a very small possibility of the direct involvement of B1 in the conversion of tryptophan to niacin.

Increase in tryptophan oxygenase activity in alcoholic patients.

This study was conducted in order to assess whether chronic excessive alcohol consumption affects the activity of the liver enzyme tryptophan oxygenase which is rate limiting along the most important pathway of tryptophan catabolism. Five alcoholics were studied twice, once shortly after admission to an inpatient unit and the second time 1 month later. On each study day patients were given a tryptophan load of 50 mg/kg. Kynurenine in the urines (which reflects tryptophan oxygenase activity) was measured for a period of 6 hr following the load and showed a significantly enhanced activity of the enzyme shortly after cessation of drinking. This increased activity could explain the lowered tryptophan levels we have previously reported in alcoholics. The increase in enzyme activity may have been mediated by a rise in glucocorticoid hormones. In all instances, plasma cortisol measured hourly for 6 hr after the start of the experiment, was higher shortly after cessation of drinking than 1 month later.

Purification and characteristics of glucocorticoid antagonizing factor in endotoxemia.

The present study involved the purification of GAF (glucocorticoid antagonizing factor) released in blood of endotoxemic mice, using the inhibition rate of tryptophan oxygenase (TO) activity in the mice liver as a parameter, to determine if this plays a role in metabolic disorders. GAF-rich serum in zymosan-primed and endotoxin-injected mice was subjected to chromatography on DEAE-Sepharose CL-6B, Blue Sepharose CL-6B and Sephadex G-200 superfine columns. Finally, GAF fractions were purified by chromatography on a DEAE-Sepharose CL-6B column. The purified GAF showed a single band in electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. The molecular weight of GAF was estimated to be 90,000. The purified GAF was regarded as glycoprotein. No factor (100 micrograms) exhibited lethal action on mice. The activity of TO in cortisone treated mice after injection of purified GAF was markedly lower than that in cortisone alone treated mice. On the other hand, there were no differences in tyrosine aminotransferase activities between the GAF plus cortisone injected group and cortisone only treated group. The glucose level after injection of GAF in cortisone treated mice initially showed hyperglycemia, but declined toward hypoglycemia 2 hr after injection, and thereafter returned nearly to the normal range by 4 hr. The liver glycogen level in GAF plus cortisone-treated mice was markedly lower than that in cortisone-alone treated mice.

Developmental control of gene expression of tryptophan 2,3-dioxygenase in neonatal rat liver

The developmental change in gene expression of tryptophan 2,3-dioxygenase (EC 1.13.11.11) in rat liver was studied by dot-blot hybridization with cDNA of the enzyme as a probe. The mRNA of tryptophan oxygenase is not expressed in fetal liver, but is expressed very slightly 1 day after birth. Its expression increases first gradually until 12 days after birth and then rapidly, and reaches the adult level about 22 days after birth. On the other hand, mRNA of albumin in the liver, measured with its cDNA, increases rapidly in the late fetal period and reaches almost the adult level at the time of birth. Studies on in vitro transcription by the nuclear run-off technqiue showed that the developmental increases in the mRNAs of tryptophan oxygenase and albumin are caused by an increase in the rates of transcription of their genes. Treatment of rats with cortisol significantly increased the amount of tryptophan oxygenase mRNA in the liver from soon after birth. This treatment did not increase mRNA of albumin. It is suggested from these findings that the gene of tryptophan oxygenase is switched on as early as the first day after birth in the few differentiated hepatocytes present in the liver and that the number of these differentiated cells gradually increases during early postnatal development. Although injected glucocorticoid stimulated transcription of the gene of tryptophan oxygenase precociously during this period, presumably in vivo the activity of tryptophan oxygenase normally increases about 2 weeks after birth, because this is when the plasma concentrations of glucocorticoid and glucagon increase sufficiently to be effective.

The antiglucocorticoid RU486 inhibits the ethanol-induced increase of tryptophan oxygenase

The effect of the glucocorticoid-antagonist RU486 (Roussel-Uclaf, France) on the increased activity of hepatic tryptophan oxygenase (TO) after administration of corticosterone and ethanol in rats was studied. RU486 (40 mg/kg per os) inhibited completely the effect of corticosterone (5–15 mg/kg injected intraperitoneally) on TO. Ethanol (4 g/kg) given intraperitoneally is followed by peak corticosterone concentrations comparable to those seen after the administration of 5–10 mg exogenous corticosterone, and increased the TO activity 3-fold 4 h after the injection. RU486 inhibited completely the ethanol-induced increase of TO, indicating that this increase is mediated by corticosterone.

RU 38486: A potent antiglucocorticoid in vitro and in vivo

The antiglucocorticoid activity of RU 38486, was studied both in vitro and in vivo. In vitro studies, RU 38486 was characterized by a high affinity (3 times higher than that of dexamethasone) for the cytosolic glucocorticoid receptor in rat hepatoma tissue culture (HTC) cells. This high affinity was due to a very low dissociation rate of the complexes formed with the receptor. In whole cells it was a potent full antagonist of dexamethasone-induced tyrosine aminotransferase (TAT) activity: the IC50 was 6–7 times lower than the concentration of the dexamethasone used. It was devoid of any glucocorticoid activity up to a concentration of 10 μM. In in vivo studies using adrenalectomized rats, RU 38486 totally inhibited dexamethasone-induced hepatic tryptophan oxygenase (TO) activity. It is also the first pure antagonist of dexamethasone-induced hepatic TAT. However, doses as high as 5 mg/kg of body weight were required for a 50% inhibition of the effect of dexamethasone at 0.01 mg/kg. RU 38486 did not display any glucocorticoid effect on these two responses up to 50 mg/kg.

Involvement of Protein Synthesis in the Action of Glucocorticoids on the Working Capacity of Adrenalectomized Rats

The administration of cortisol for 3 or 5 days increased the maximal duration of swimming under additional load, equal to 3% of body weight, in adrenalectomized but not in intact Wistar rats. The liver tryptophan oxygenase activity also increased. The blockade of RNA synthesis by actinomycin D excluded both results of cortisol administration. High doses of progesterone (25 or 75 mg/day) decreased the working capacity, rendering the action of cortisol on the working capacity of adrenalectomized rats insignificant. However, the antagonism between two hormones was not striking at the level of liver tryptophan oxygenase induction. The results suggest that the action of glucocorticoids on the working capacity is mediated by RNA and, further, by protein synthesis. The competition for glucocorticoid cytoplasmic receptors, to be found between glucocorticoids and a high level of progesterone, can disturb this mechanism. The synthesis of hepatic enzyme proteins is not responsible for the action of glucocorticoids on the working capacity.

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.

Ethanol increases rat liver tryptophan oxygenase: Evidence for corticosterone mediation

Acute administration of ethanol (4.0 g/kg) intragastrically or intraperitoneally induced rat liver tryptophan oxygenase (TO) activity 3–4 fold 4–5 hr later. Ethanol administration increased the concentration of plasma free tryptophan and free fatty acids. Pretreatment with a beta-receptor blocker, propranolol, modified the latter responses without affecting the TO induction due to ethanol. The rise of the level of plasma free tryptophan due to ethanol was too small to influence TO activity. Liver tryptophan concentration and TO half-life was unchanged after ethanol administration. Ethanol administration increased the concentration of plasma corticosterone sufficiently to increase TO activity. Pretreatment with a glucocorticoid antagonist blocked this TO response to ethanol. The increased TO activities found after ethanol or corticosterone treatment were influenced in the same manner and to the same extent by cycloheximide. Taken together it is concluded that ethanol induces TO through a rise of glucocorticoid hormones and not by a tryptophan-linked mechanism.

Effect of pyridoxine administration on the induction of cytosolic aspartate aminotransferase in the liver of rats treated with hydrocortisone.

The effects of pyridoxine and hydrocortisone administrations on the rate of synthesis of cytosolic aspartate aminotransferase in adrenalectomized pyridoxine-deficient rat livers were examined. Induction of cytosolic aspartate aminotransferase by hydrocortisone was observed 6 to 10 h after the injection. Treatment with pyridoxine daily for 6 days but not for the 2 days, resulted in suppression of the enzyme synthesis. Similar suppression of enzyme synthesis was observed in rats without hydrocortisone treatment. The activity of tryptophan oxygenase, which is known to be induced by glucocorticoid and does not contain pyridoxal phosphates, was higher in the livers of pyridoxine-deficient rats than in that of controls. The possible effects of pyridoxal phosphate on the action of glucocorticoid are discussed based on the results.

Ethanol-induced increase in liver tryptophan oxygenase activity in the starved rat: Evidence against tryptophan mediation

A single oral dose of ethanol (4.0 g/kg) increased the activity of liver tryptophan oxygenase in starved male rats. The peak increase of 340% for the total activity and 400% for the holoenzyme activity occurred 6 hr after ethanol administration. At or after these peaks, the levels of tryptophan in plasma and brain but not in liver, decreased significantly. Plasma total tryptophan and brain tryptophan started to decrease significantly as early as 0.5–1.0 hr after the ethanol treatment, while the activity of liver tryptophan oxygenase was still at the control level. These findings suggest that not all the changes in tissue tryptophan concentrations seen after acute ethanol treatment are caused by increased liver tryptophan oxygenase activity. Prior to the increase in liver tryptophan oxygenase activity, an increase of 104 and 50% in plasma corticosterone and free tryptophan, respectively, were seen 15 min after ethanol treatment. However, the increase in liver tryptophan at this time appeared to be small (13%) and statistically insignificant. With tryptophan treatment, the initial peak levels of liver tryptophan and plasma free tryptophan required to stimulate an increase in tryptophan oxygenase activity were 170 times higher than those caused by ethanol. It was therefore concluded that increases in plasma and liver tryptophan after acute ethanol ingestion, probably mediated by the lipolytic action of ethanol, are too small to cause the increase in liver tryptophan oxygenase activity seen after ethanol administration. However, experiments with different corticosterone doses showed that ethanol-induced increases in plasma corticosterone concentrations are high enough to cause an increase in liver tryptophan oxygenase activity.

Effect of propranolol and cycloheximide on the ethanol-induced increase in liver tryptophan oxygenase activity in starved rats

Increased supply of trytophan to the liver, resulting from the lipolytic action of ethanol, is suggested to be responsible for the increased activity of liver tryptophan oxygenase after ingestion of a single large dose of ethanol. This hypothesis was tested using an antilipolytic drug, propranolol, prior to ethanol treatment. It was found that, while propronolol did inhibit the ethanol-induced increase in blood unesterified fatty acids and free tryptophan concentrations it did not prevent the activation of tryptophan oxygenase by ethanol. In another experiment, where cycloheximide was used to block protein synthesis, it was found that increased protein synthesis rather than decreased protein degradation is probably responsible for the accumulation of liver tryptophan oxygenase after ethanol ingestion.

Utilization of Dietary Tryptophan in Iron-Deficient Rats

Excretion of urinary tryptophan in iron-deficient rats increased threefold while the excretion of N-methyl nicotinamide, a catabolic product of tryptophan, decreased by one-half compared to control rats. The activity of hepatic tryptophan oxygenase, the first enzyme of tryptophan metabolism, was not affected by iron deficiency. It is concluded that tryptophan supplementation in severe iron deficiency would not be beneficial.

Inhibition of tryptophan metabolism by oestrogens in the rat: a factor in the aetiology of pellagra.

The effect of the administration of oestrone sulphate on tryptophan metabolism has been assessed in rats in order to determine whether and to what extent inhibition of tryptophan metabolism by oestrogens may be a factor in the aetiology of pellagra, and might explain the reported twofold excess of females over males in many outbreaks of pellagra. Feeding ovariectomized rats for 1 week on a diet containing 15 mg oestrone sulphate/kg led to significant inhibition of kynurenine hydroxylase (EC 1.14.13.9), kynureninase (EC 3.7.1.3) and 3-hydroxyanthranilate oxidase (EC 1.13.11.6). There was also a significant increase in plasma tryptophan, suggesting decreased activity of tryptophan oxygenase (EC 1.13.11.11). This inhibition of tryptophan metabolism will result in a considerable reduction in the synthesis of the nicotinamide nucleotide coenzymes (NAD and NADP) from tryptophan. When ovariectomized rats were maintained for 4 weeks on a diet providing no preformed niacin and only a marginally adequate amount of tryptophan (1030 mg/kg), the addition of sulphate to the diet led to a significant reduction in the liver content of nicotinamide nucleotides and the urinary excretion of the end-product of NAD metabolism, N1-methyl nicotinamide. It is suggested that when the diet is only marginally adequate in tryptophan and niacin, inhibition of tryptophan metabolism by endogenous or administered oestrogens may be an additional factor in the development of pellagra.