NAD metabolism and induction of tyrosine aminotransferase in the rat

The relationship between the NAD-metabolism and the induction of the tyrosine aminotransferase was studied. The content of NAD+ + NADH differs markedly from organ to organ. The highest values can be found in the liver. In intact animals tryptophan leads to an increase of NAD in liver and kidney, but not in brain and spleen. Nicotinamide, on the other hand, induces NAD synthesis in all the organs tested. In adrenalectomized animals, however, there is practically no rise of the NAD content after application of tryptophan contrary to the effect of nicotinamide. The enzyme tyrosine aminotransferase can be induced in intact animals by nicotinamide and tryptophan. This effect is much less pronounced in adrenalectomized animals. In adrenalectomized animals the induction of the tyrosine aminotransferase by tryptophan is markedly elevated by caffeine and theophylline. Under these conditions there is a significant increase of the NAD content as well. The tryptophan promoted induction of the tyrosine aminotransferase is influenced by inhibitors of the ADPR-transferase. The data presented give further evidence that the NAD adenoribosylation metabolism is involved in the induction of the tyrosine aminotransferase.     

A STUDY OF PROPOSED DETERMINANTS OF BRAIN TRYPTOPHAN CONCENTRATION IN RATS AFTER PORTOCAVAL ANASTOMOSIS OR SHAM OPERATION

Liver dysfunction was produced in rats by surgical portocaval anastomosis (PCA), and the time-course of changes in brain tryptophan and 5-HT metabolism studied in relation to plasma changes possibly influencing brain tryptophan concentration. Brain tryptophan and 5-hydroxyindolylacetic acid (5-HIAA) levels were increased greatly and maximally on the day after PCA and remained high. 5-HT changes were less marked but had a similar time-course. Plasma total tryptophan was little changed but plasma free tryptophan was raised. The latter change showed a similar time-course to that of brain tryptophan but was not large enough to account completely for it. Sham operation was followed by significant but transient increases in plasma free tryptophan, brain tryptophan and 5-HIAA but these were much smaller than after PCA. Brain tryptophan did not correlate with plasma total tryptophan either in control or PCA rats but it correlated significantly with plasma free tryptophan in both groups. However brain levels were much higher in PCA rats than in controls with similar plasma free tryptophan levels at all times from the first day after operation. The increase of brain tryptophan in anastomosed rats not accounted for by plasma free tryptophan was explained neither by insulin changes nor by an increase of the insulin/glucagon ratio nor by changes in plasma concentrations of those amino acids which compete with tryptophan for entry into brain. The results therefore indicate an unknown influence on brain tryptophan concentration in PCA rats. As tyrosine changes in brain and plasma after PCA were very similar to those of tryptophan this influence may not be specific to tryptophan. Results suggest that under the conditions used brain tryptophan concentrations of both PCA and control rats are more influenced by changes of plasma free tryptophan concentration than by changes of plasma concentrations of competing amino acids.     

Fetal rat adrenal steroidogenesis and steroid transfer to adrenalectomized mother.

On the 22nd day of gestation in rats, fetuses of acutely adrenalectomized mothers were injected subcutaneously with 0.43 muCi 4-14C-progesterone in 0.05 ml saline. Ten and 20 min after injection to fetuses, samples were taken to determine the 14C-progesterone metabolites in the plasma and adrenal glands. After extraction of the samples taken, the metabolites were separated by two-dimensional thin-layer chromatography and identified by autoradiography. 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 11beta-hydroxyprogesterone were identified in the plasma of injected fetuses, and, in far smaller amounts, in the plasma of their mothers. The plasma of noninjected fetuses also contained very small amounts of these corticoids. The fetal adrenal glands contained far smaller amounts of radioactive steroids than the fetal plasma did. The results obtained show that steroids of fetal origin can cross the placenta in and out, constituting evidence that the fetal adrenal glands are the only source of the plasma corticoids of their adrenalectomized mothers.     

Effects of insulin and streptozotocin-induced diabetes on brain tryptophan and serotonin metabolism in rats

Previous work by other authors has shown hat insulin administration increases brain tryptophan levels and serotonin (5–HT) metabolism. The present study partially replicates these results and tests whether these effects could be due to insulin-induced hypoglycemic stress, since stressers such as immobilization or food deprivation also increase brain tryptophan and 5-HT metabolism. Ingestion of a dextrose solution by rats administered insulin (2 I.U./kg) prevents the extreme fall in blood glucose concentration and rise in plasma corticosterone following insulin injections alone. This treatment, however, produces a larger increase in brain tryptophan (30%) than insulin-injected rats allowed only tap water. The greater accumulation of brain tryptophan may reflect an additive effect of the endogenously released insulin to that exogenously administered, since ingestion of the dextrose solution could trigger insulin secretion. In addition, brain tryptophan and 5-HT metabolism were measured in streptozotocin-diabetic rats maintained on several different feeding schedules to control for the effects of hyperphagia. All groups of diabetics showed significant decreases of approx 30% in brain tryptophan concentrations, while 5-HT metabolism was unchanged. This deficit in brain tryptophan is reversed within 2 h after insulin administration (2 I.U./kg). These results indicate that changes in brain tryptophan and 5-HT metabolism following insulin injections are not due to hypoglycemic stress, and that brain tryptophan is low in diabetics but increases above normal after administration of insulin. The results are discussed with respect to the effects of insulin on plasma levels of the neutral amino acids and a possible direct effect of insulin on the uptake of tryptophan by brain.     

Effects of clofibrate on plasma tryptophan, growth hormone, and prolactin and on brain tryptophan and serotonin in prepubertal rats

The effects of clofibrate administration (200 mg/kg, po) on somatic growth, plasma levels of lipids, tryptophan, growth hormone (GH), and prolactin (PRL), as well as on brain concentrations of tryptophan and 5-hydroxytryptamine (5-HT) were studied in prepubertal male rats. The drug did not significantly alter ponderal growth, but an appreciable reduction of tail length was observed in rats treated for 30 days. Triglyceride concentrations in plasma showed a 43% diminution after 30 days of treatment, whereas free fatty acid (FFA) levels were not modified. Clofibrate administration for 7, 15, or 30 days caused a fall in total tryptophan and a significant increase of the free fraction in plasma with no change in brain tryptophan levels. Brain 5-HT was generally unaffected but a marked elevation of this parameter was noted in rats treated for 15 days. Plasma GH and PRL concentrations remained unaltered. It may be concluded from these findings that the slight reduction of somatic growth, the diminution of triglycerides, and the increase of free tryptophan in plasma, induced by chronic clofibrate treatment, are not associated with variations in brain tryptophan and 5-HT levels or with modifications of plasma GH and PRL titers.     

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.     

Injected tryptophan increases brain but not plasma tryptophan levels more in ethanol treated rats

In previous studies, long term treatment with ethanol has been shown to enhance brain 5-hydroxytryptamine 5-(HT) metabolism by increasing the activity of the regulatory enzyme tryptophan hydroxylase and or availability of circulating tryptophan secondarily to an inhibition of hepatic tryptophan pyrrolase. In the present study ethanol treatment given for two weeks decreased hepatic apo-tryptophan pyrrolase but not total tryptophan pyrrolase activity in rats. Tryptophan levels in plasma and brain did not increase significantly. But there was a marked increase of 5-HT but not 5-hydroxyindoleacetic acid (5-HIAA) concentration in brain, suggesting a possible increase in the activity of tryptophan hydroxylase. The effect of a tryptophan load on brain 5-HT metabolism was therefore compared in controls and ethanol treated rats. One hour after tryptophan injection (50 mg/kg i.p.) plasma concentrations of total and free tryptophan were identical in controls and ethanol treated rats, but the increases of brain tryptophan 5-HT and 5-HIAA were considerably greater in the latter group. The results are consistent with long term ethanol treatment enhancing brain serotonin metabolism and show that brain uptake/utilization of exogenous tryptophan is increased in ethanol treated rats and may be useful to understand the role and possible mechanism of tryptophan/serotonin involvement in mood regulation.     

Injected tryptophan increases brain but not plasma tryptophan levels more in ethanol treated rats

In previous studies, long term treatment with ethanol has been shown to enhance brain 5-hydroxytryptamine 5-(HT) metabolism by increasing the activity of the regulatory enzyme tryptophan hydroxylase and or availability of circulating tryptophan secondarily to an inhibition of hepatic tryptophan pyrrolase. In the present study ethanol treatment given for two weeks decreased hepatic apo-tryptophan pyrrolase but not total tryptophan pyrrolase activity in rats. Tryptophan levels in plasma and brain did not increase significantly. But there was a marked increase of 5-HT but not 5-hydroxyindoleacetic acid (5-HIAA) concentration in brain, suggesting a possible increase in the activity of tryptophan hydroxylase. The effect of a tryptophan load on brain 5-HT metabolism was therefore compared in controls and ethanol treated rats. One hour after tryptophan injection (50 mg/kg i.p.) plasma concentrations of total and free tryptophan were identical in controls and ethanol treated rats, but the increases of brain tryptophan 5-HT and 5-HIAA were considerably greater in the latter group. The results are consistent with long term ethanol treatment enhancing brain serotonin metabolism and show that brain uptake/utilization of exogenous tryptophan is increased in ethanol treated rats and may be useful to understand the role and possible mechanism of tryptophan/serotonin involvement in mood regulation.     

The effect of stress on the activity of hepatic tryptophan pyrrolase, of tyrosine aminotransferase in various organs and on the level of tryptophan in the liver and plasma of rats.

In rats subjected to 400 revolutions in Noble-Collip drums, hepatic tryptophan pyrrolase activity increases and plasma tryptophan level decreases. After bilateral adrenalectomy, the alterations of plasma tryptophan are even more pronounced and liver tryptophan increases in contrast to tryptophan pyrrolase activity which remains unchanged after injury. The possible significance of the posttraumatic increase of tryptophan pyrrolase in intact animals for brain serotonin metabolism and hepatic gluconeogenesis is underlined. The activity of tyrosine aminotransferase in liver, brain, adrenal, kidney and muscle tissue of rats was determined with special reference to the possible effect of the before-mentioned stress procedure. Organ homogenates were centrifuged at 15000 x g and both supernatants and pellets were investigated for enzyme activity with the exception of the liver, where only the supernatant fraction was used. Tyrosine aminotransferase activity in the liver supernatant considerably exceeded the corresponding values in both supernatant and pellet of the remaining organs, in which a prevalence of the mitochondrial enzyme was obvious. In contrast to the clear-cut increase of the hepatic enzyme during stress, essentially no changes were noted in the brain, the adrenals, kidney or muscle under similar conditions...     

Influence of tryptophan on the activity of acetylcholinesterase in the brain of well-fed normal and adrenalectomized rats

Eight hours after a single tube-feeding of tryptophan the activity of acetylcholinesterase in the cerebral hemisphere of well-fed (non-fasted) normal and adrenalectomized rats was 28 and 53% higher, respectively, compared to the corresponding water-fed control. On the other hand, the enzyme activity in the cerebellum of both normal and adrenalectomized rats remained essentially unchanged following tryptophan administration. Pretreatment of adrenalectomized rats with actinomycin-D totally abolished the tryptophan-mediated stimulation of cerebral acetylcholinesterase activity. The pattern of response of cerebral acetylcholinesterase in well-fed adrenalectomized rats over a period of 24-hr following a dose of tryptophan was found to be biphasic.     

Relationship Between Plasma and Brain Tryptophan in Pigs During Experimental Hepatic Coma Before and After Hernodialysis with Selective Membranes

Experimental acute liver ischemia in pigs induces an increment in plasma free tryptophan with decreased total tryptophan. Brain tryptophan is elevated in all brain areas. A slight, but significant increase of brain serotonin is demonstrated in the striatum only, while 5-HIAA (5-hydroxyindoleacetic acid) is significantly lower in the hypothalamus. Other brain areas do not show significant changes in serotonin and 5-HIAA levels. Neither the high plasma free tryptophan levels, nor the decreased sum of neutral competitive amino acids are consistent with such an elevation of brain tryptophan. Hemodialysis was carried out with two different kinds of membranes: cuprophan (with an efficient removal of molecules up to molecular weight 1300) and AN 69 polyacrylonitrile (efficient removal up to 15,000). Ammonia and aminoacid clearance are similar for both membranes. After AN 69, plasmatic free tryptophan and brain tryptophan are lower than after liver devascularization, but still higher than normal. Serotonin significantly increases in the cortex, midbrain and hypothalamus without concomitant rise of 5-HIAA levels. After cuprophan hemodialysis, plasma total tryptophan is lower than in normal and even comatose animals, whereas free tryptophan is normal. Intracerebral tryptophan is similar to AN 69 dialysed animals, but in the hypothalamus it is similar to nondialysed animals. Brain serotonin levels are not modified. 5-HIAA decreases in the hypothalamus. This finding suggests that middle molecules (which are not cleared out with cuprophan hemodialysis) are involved in the intracerebral transfer of tryptophan and the metabolism of serotonin, mainly in the hypothalamus.     

Potential role for nonesterified fatty acids in beta-adrenoceptor-induced increases in brain tryptophan

We tested the hypothesis that beta2- and beta3-adrenergic receptor-mediated increases in brain tryptophan are due to the liberation of fatty acids, which in turn displace tryptophan from its albumin-binding site and thus facilitate its entry into the brain. Male CD-1 mice were injected with subtype-selective beta-adrenergic agonists 1h before brain samples were collected for analysis of tryptophan content by HPLC with electrochemical detection, and blood samples were collected for analysis of total and free tryptophan and nonesterified fatty acid (NEFA) concentrations. The beta2-selective agonist, clenbuterol (0.1 mg/kg), increased concentrations of tryptophan in all brain regions studied and decreased plasma total tryptophan, but had no effect on plasma free tryptophan or NEFAs. The beta3-selective agonists, BRL 37344 (0.2 mg/kg) or CL 316243 (0.01 mg/kg), increased brain tryptophan, plasma NEFAs and free tryptophan. Pretreatment with nicotinic acid (500 mg/kg), an inhibitor of lipolysis, almost completely prevented the increase in plasma free tryptophan and NEFAs, and attenuated the increase in brain tryptophan induced by CL 316243. These results suggest that beta2- and beta3-adrenergic agonists increase brain tryptophan by a mechanism other than the liberation of NEFAs. Nonetheless, beta3-adrenergic agonists appear to increase brain tryptophan by a mechanism that may depend partially on elevations of plasma NEFAs.     

CORRELATION OF PLASMA AND BRAIN AMINO ACID AND PUTATIVE NEUROTRANSMITTER ALTERATIONS DURING ACUTE HEPATIC COMA IN THE RAT

During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.     

Indolic Substances in Plasma, Cerebrospinal Fluid, and Frontal Cortex of Human Subjects Infused with Saline or Tryptophan

Psychiatric patients undergoing the psychosurgical operation of stereotactic subcaudate tractotomy were infused intravenously with either saline or L-tryptophan (15 mg/kg/h). Plasma, lumbar cerebrospinal fluid (CSF), ventricular CSF and a specimen of frontal cortex were collected. The relationships of plasma concentrations of substances claimed to influence brain tryptophan concentration (total tryptophan, free tryptophan, large neutral amino acids) with the concentration of tryptophan in the cortex and CSF were investigated. Tryptophan infusion resulted in plasma tryptophan values comparable to those found after oral doses used in treating depression or insomnia, and about sixfold increases of tryptophan in the cerebral cortex. Increased brain 5-hydroxytryptamine synthesis was indicated by significant rises of CSF 5-hydroxyindoleacetic acid. The concentration of plasma free tryptophan was a better predictor than plasma total tryptophan of cortex tryptophan concentration. As all correlation coefficients of plasma versus brain or plasma versus ventricular CSF tryptophan concentrations were decreased when allowance was made for differences of concentration of large neutral amino acids, the results suggest that the role of these substances within their physiological range as inhibitors of tryptophan transport to the brain may previously have been overemphasised.     

THE EFFECTS OF REPEATED-LYSERGIC ACID DIETHYLAMIDE INJECTIONS ON CATECHOLAMINE LEVELS AND TYROSINE HYDROXYLASE ACTIVITY IN RAT BRAIN REGIONS

Daily injections of 100 μg/kg of d -lysergic acid diethylamide (LSD) for 14 days produced a significant decrease in the dopamine level in rat brain corpus striatum which was still apparent 15 days after the last LSD treatment. Further LSD injections did not change the amount of dopamine depletion. In cerebral cortex, 14 days of LSD injections produced a significant decrease in the norepinephrine level and a significant increase in tyrosine hydroxylase activity. The elevated tyrosine hydroxylase activity was still present 15 days after the final LSD injection but only in those animals receiving daily vehicle injections during this period. Pre-treatment of rats with daily saline injections for 2 weeks before the 2 week period of LSD treatment prevented both the reduced norepinephrine content and elevated tyrosine hydroxylase activity usually found 24 h after the last LSD injection.     

Tryptophan force-feeding: Changes in the activities of acetylcholinesterase in various tissues of well-fed normal and adrenalectomized rats

The activity of acetylcholinesterase in the liver, heart, spleen, lungs and kidneys of well-fed normal and adrenalectomized rats was measured following a single tube-feeding of tryptophan. In well-fed normal rats, 30 min after tryptophan force-feeding, the enzyme activity in the heart and lungs was stimulated by 28 and 25% as compared to the water-fed control while in well-fed adrenalectomized rats acetylcholinesterase acticity in the heart, liver spleen and lungs was 40, 31, 22 and 15% increased, respectively over that of the corresponding control. In both groups of rats the enzyme activity in the kidney was unaffected by tryptophan. In the liver, spleen and heart of well-fed adrenalectomized rats the pattern of response for acetylcholinesterase to a tryptophan dose, over a period of 24-hr. was found to be biphasic. In well-fed adrenalectomized rats the tryptophan-mediated stimulation of acetylcholinesterase activity in the heart was found to be insensitive to actinomycin-D. The tryptophan-mediated stimulation of acetylcholinesterase activity in the heart of well-fed normal and adrenalectomized rats could not be related to the presence of an activator.     

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.     

The role of tryptophan 2,3-dioxygenase in the hormonal control of tryptophan metabolism in isolated rat liver cells. Effects of glucocorticoids and experimental diabetes.

The metabolism of L-tryptophan by isolated liver cells prepared from control, adrenalectomized, glucocorticoid-treated, acute-diabetic, chronic-diabetic and insulin-treated chronic-diabetic rats was studied. Liver cells from adrenalectomized rats metabolized tryptophan at rates comparable with the minimum diurnal rates of controls, but different from rates determined for cells from control rats 4h later. Administration of dexamethasone phosphate increased the activity of tryptophan 2,3-dioxygenase (EC 1.13.11.11) 7-8-fold, and the flux through the kynurenine pathway 3-4-fold, in cells from both control and adrenalectomized rats. Increases in flux through kynureninase (EC 3.7.1.3) and to acetyl-CoA can be explained in terms of increased substrate supply from tryptophan 2,3-dioxygenase. The metabolism of tryptophan was increased 3-fold in liver cells isolated from acutely (3 days) diabetic rats, with a 7-8-fold increase in the maximal activity of tryptophan 2,3-dioxygenase. The oxidation of tryptophan to CO2 and metabolites of the glutarate pathway increased 4-5-fold, consistent with an increase in picolinate carboxylase (EC 4.1.1.45) activity. Liver cells isolated from chronic (10 days) diabetic rats metabolized tryptophan at rates comparable with those of cells from acutely diabetic rats, but with a 50% decrease in the activity of tryptophan 2,3-dioxygenase. The proportion of flux from tryptophan 2,3-dioxygenase to acetyl-CoA, however, was increased by 50%; this was indicative of further increases in the activity of picolinate carboxylase. Administration of insulin partially reversed the effects of chronic diabetes on the activity of tryptophan 2,3-dioxygenase and flux through the kynurenine pathway, but had no effect on the increased activity of picolinate carboxylase. The role of tryptophan 2,3-dioxygenase in regulating the blood tryptophan concentration is discussed with reference to its sensitivity to the above conditions.     

Tryptophan and serotonin metabolism after sustained tryptophan infusion

In an attempt to elucidate the effects of sustained administration of tryptophan on serotonin synthesis and turnover in mammalian brain, mini-osmotic pumps containing tryptophan or vehicle were implanted in albino mice for 24 and 96 h. Despite the extremely low dose of tryptophan administered by these pumps (8–12 mg/kg-day) statistically significant treatment effects were apparent with both treatment durations. Plasma and brain tryptophan concentrations varied in unison, and were inversely related to the tryptophan degradative capabilities of the liver as reflected in tryptophan pyrrolase activity. After 24 h of tryptophan infusion the hepatic enzyme activity was elevated and tryptophan values were no different from controls, and after 96 h the hepatic enzyme activity was reduced and tryptophan values in treated animals were greater than controls. Serotonin was elevated in treated animals after 24 h, but not after 96 h despite the elevated tryptophan concentration at this time. The turnover of serotonin, as evidenced by 5-hydroxyindoleacetic acid concentrations, was not significantly affected by either treatment.Hepatic degradation of tryptophan thus seemed to be an important determinant of total plasma tryptophan, and brain tryptophan values paralleled plasma tryptophan. It appears that serotonin biosynthesis is regulated by factors other than tryptophan availability when the latter is chronically elevated.     

TRYPTOPHAN AND 5-HYDROXYTRYPTAMINE METABOLISM IN THE IMMATURE RAT BRAIN DURING RECOVERY FROM ASPHYXIA

Abstract— The turnover of cerebral 5-HT in the 5-day-old rat during recovery from asphyxia was assessed in the brain by determining 5-hydroxyindoleacetic acid concentration, and by following the accumulation of 5-HT after inhibition of MAO. Marked increases in turnover were found to persist for up to 3 h after resuscitation and this increase was accompanied by increases in brain tryptophan, plasma total tryptophan and the fraction of the plasma total tryptophan that was not bound to albumin. None of these parameters were different from those in control animals 24 h after resuscitation. Brain and plasma tyrosine levels remained unaltered by the treatment at all times investigated.