Induction of Hepatic Tyrosine Aminotransferase by Indole Amines*

SUMMARY L-Tryptophan, L-S-hydroxytryptophan, serotonin, and other indole amines increase tyrosine aminotransferase activity in mice liver.

L-5-Hydroxytryptophan was as effective as serotonin, whereas L-tryptophan was far less effective at low doses.
If inhibitors of aromatic L-amino acid decarboxylase were injected prior to the administration of indole compounds, the increase of tyrosine aminotransferase activity by Shydroxytryptophan or tryptophan was completely blocked, whereas the increase by serotonin was not reduced.
On the other hand, a monoamine oxidase inhibitor enhanced the increase of tyrosine aminotransferase activity by serotonin or 5-hydroxytryptophan.
These results indicate that indole amines are the active agents in the increase of enzyme level. Among various indole amines, serotonin, Smethoxytryptamine, and tryptamine were effective in increasing the enzyme activity.
Although the increase in the enzyme activity was partially mediated by adrenal hormones, these indole amines could increase tyrosine aminotransferase activity in adrenalectomized mice. Cycloheximide blocked the increase of tyrosine aminotransferase activity by serotonin, indicating that the increase is due to induction of new enzyme. The results suggest a possible role of indole amines in the regulation of metabolism.
Swiss-Webster male mice, weighing 20 to 22 g, were supplied from Simonsen Laboratories, Gilory, California. Twenty mice were housed in one cage, and kept under controlled light (light on from 7:00 a.m. to 7:00 p.m.).
All mice were deprived of food from 9:00 a.m. Water was available ad libitum.
Each group consisted of 5 to 10 mice, and the experiments were repeated two or three times.
Adrenalectomized mice were bilaterally adrenalectomized under a Luxo lamp and the animals were maintained on a normal diet and 0.9% NaCl solution drinking water.
Adrenalectomized mice were used for experiments 6 or 7 days after adrenalectomy. At the time of killing, it was confirmed that no residual adrenal remained.
Since tyrosine aminotransferase activity shows circadian variations, all mice were killed between 1:00 p.m. and 3:00 p.m., and the livers were quickly removed and frozen on Dry Ice. Tyrosine aminotransferase activity was measured within 24 hours after killing.
Liver was homogenized in 5 volumes of 0.14 M KC1 containing 0.02 M potassium phosphate buffer (pH 7.0), and centrifuged at 35,000 X g for 20 min. Tyrosine aminotransferase activity was assayed by the method of Diamondstone (10) using the supernatant.
For the measurement of serotonin, brain or liver was homogenized in 5 volumes of 4% perchloric acid containing 0.2% EDTA, 7218 Tgrosine Aminofransjerase and Indole Amines vol. 246, iSo. 23 and centrifuged at 35,000 X g for 20 min. The supernatant was RESULTS neutralized by the addition of 2 N KOH drop by drop, and the supernatant was analyzed for serotonin by the method of Increase of Tyrosine Aminotransjerase by Indole Ccmpounds-Serotonin or n-5-hydroxytryptophan was injected ntraueri-Erdelyi, Angwin, and Burchas (11).
toneally into mice, and the mice were killed 2, 4, 6, and 9 hours after the injection. As shown in Fig. 1, tyrosine ,aminotransferase activity increased &fold 2 hours after injection of serotonin, reaching a maximum value at 4 hours. At the maximum, tyrosine aminotransferase activity was 4-fold higher than the basal level. Nine hours after the injection, tyrosine aminotransferase activity returned to the initial level. An identical pattern was obtained with n-5-hydroxytryptophan.
In the following experiments, therefore, tyrosine aminotransferase activity was measured 4 hours after injection of indole compounds .   O  I  I  I  I  0  2  4  6 6 Hourfs the mean. i0 The dose response of tyrosine aminotransferase to serotonin, L-5-hydroxytryptophan, and L-tryptophan is shown in Fig. 2. At a dose of 0.05 mmole of serotonin, tyrosine aminotransferase activity increased a-fold, with a 4-fold increase at a dose of 0.5 mmole. r+Hydroxytryptophan was less effective at doses lower than.,O.5 mmole. At a dose of 0.5 or 1.0 mmole of n-5hydroxytryptophan, tyrosine aminotransferase activity increased 4-fold to the same level as with serotonin. On the other hand, L-tryptophan was far less effective than serotonin or L-5-hydroxytryptophan At a dose of 0.5 mmole or less, no significant increase was observed. At a dose of 1.0 mmole, tyrosine aminotransferase activity increased 2-fold, which was statistically significant. At a dose of 2 mmoles, tyrosine aminotransferase activity increased 4-fold as shown in Fig. 3.
Various indole compounds were injected at a dose of 0.5 mmole per kg body weight, and tyrosine aminotransferase activity was measured. As shown in Table I, serotonin creatinine sulfate, serotonin hydrogen oxalate, and free serotonin resulted in the same amount of increase of tyrosine aminotransferase activity, indicating that the increase was due to serotonin moiety. 5-Serotonin creatinine sulfate (MIT) or n-5-hydroxytryptophan Methoxytryptamine and tryptamine increased tyrosine amino-(L-I-HTP) was injected intraperitoneally at a dose of 0.5 mmole per kg of body weight, and the mice were killed at various time intervals as indicated. Vertical  or n-tryptoally at the doses indicated. Four hours after injection of the phan (L-TP), at the doses indicated. The mice were killed 4 compounds, the mice were killed and tyrosine aminotransferase hours after the injection of indole compounds, and tyrosine aminoactivity was measured. Vertical bars indicate standard errors of transferase activity was measured. Vertical bars indicate standthe mean.
ard errors of the mean.  I compounds by themselves or some metabolites of these com-E$ect of various indole compou.ncls pounds are responsible for the increase of the enzyme activity.
Four hours after the injection, the mice were killed and n-tryptophan is decarboxylation of these amino acids to the and tyrosine aminotransferase activity was measured.
Mela-corresponding amines. The effect of inhibitors of aromatic tonin or 6-hydroxymelatonin was injected as a suspension. Sero-n-amino acid decarboxylase,'therefore, was investigated. Among this' study, because these two inhibitors are the most potent and long lasting inhibitors, and because the pattern of inhibition  (12,13). MK-486 inhibits aromatic L-amino acid decarboxylase in the peripheral tissues, but not in the brain, whereas Ro 4-4602 inhibits aromatic n-amino acid decarboxylase both in the peripheral tissues and in the brain. As shown in Fig. 3 Four hours after injection of serotonin or intraperitoneal injection of n-5-hydroxytryptophan (0.5 mmole n-5-hydroxytryptophan, the mice were killed, and tyrosine amino-per kg). After various time intervals, the mice were killed and transferase activity was measured.
Vertical bars indicate stand-serotonin levels in the liver or in the brain were measured. Verard errors of the mean.
tical bars indicate standard errors of the mean.

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Tyrosine Aminotrctnsjerase and Indole Amines Vol. 246, No. 23 Ro 4-4602 has no effect on the increase of the enzyme activity by serotonin. Ro 4-4602 showed a different effect on decarboxylaserotonin, whereas it completely blocked the increase of the en-tion of 5-hydroxytryptophan in the brain. Fifteen minutes zyme activity by L-5-hydroxytryptophan.
after. the injection of 5-hydroxytryptophan, serotonin levels in The in sivo effect of these inhibitors on decarboxylation of 5-the brain did not increase, with a moderate increase 1 and 4 hydroxytryptophan was shown in Fig. 5. After the injection of hours after the injection of 5-hydroxytryptophan. 5-hydroxytryptophan, a large amount of serotonin accumulated The results shown above are taken to show that decarboxylain the liver, decreasing to the basal level 4 hours after the injec-tion of n-5-hydroxytryptophan or ntryptophan is essentially tion. On the other hand, MK-486 or Ro 4-4602 completely required for the intirease of tyrosine aminotransferase activity blocked decarboxylation of 5-hydroxytryptophan in the liver. by these amino acids. In the brain, MK-486 slightly increased the accumulation of  4.3.4). An inhibitor of monoamine oxidase, pargyline, was injected prior to injection of indole compounds. As shown in Fig. 6, pargyline has no effect on the basal level of tryosine aminotransferase activity. Pargyline, however, greatly enhanced the increase of the enzyme activity by serotonin or n-5-hydroxytryptophan, indicating that serotonin, but not its metabolites derived via monoamine oxidase is responsible for the increase of the enzyme activity. The level of the enzyme activity after the injection of n-tryptophan alone or n-tryptophan plus pargyline was not significantly dBerent from the level in the control groups, although there was a tendency for an elevation after pretreatment with monoamine oxidase inhibitor. Increase of Tyrosine Aminotransjerase Activity in Adrenulectomized Mice-Serotonin as well as tryptamine increased plasma corticosterone level a-fold 30 min after injection. NaCl (0.9%) injections also increased plasma corticosterone level by 50%. Adrenalectomized mice, therefore, were used in the following experiments. Indole amino acids and amines were tested for the increase of tyrosine aminotransferase activity and the re-  a Differs from NaCl group at p < 0.01.
On the other hand, L-tryptophan was less effective in increasing the enzyme activity. The increase of the enzyme activity was, however, statistically significant at a dose of 1.0 mmole of L-tryptophan (p < 0.05). The effect of the aromatic L-amino acid decarboxylase inhibitor, Ro 4-4602, was again investigated with adrenalectomized mice. As shown in Fig. 7, the increase of the enzyme activity by L-5-hydroxytryptophan or L-tryptophan was completely blocked by the pretreatment with the inhibitor. In order to study whether or not the increase of tyrosine aminotransferase activity by serotonin is due to net synthesis of the enzyme molecule, the effect of cycloheximide or actinomycin D was investigated.
As shown in Table III, cyclohexirnide completely blocked the increase of tyrosine aminotransferase activity, indicating that the increase of tyrosine aminotransferase act,ivity by serotonin is due to the synthesis of new enzyme.
On the other hand, actinomycin D could not block the increase of tyrosine aminotransferase activity, suggesting that RNA synthesis is not involved in this induction. DISCUSSION The results reported in this paper indicate that indole amino acids must be decarboxylated to the corresponding amines to induce tyrosine aminotransferase, and that indole amines are possible agents for induction of tyrosine aminotransferase in mouse liver.
Since all N-substituted indole amines except N-acetylserotonin were ineffective, the free amino group of indole amines is necessary for the induction of tyrosine aminotransferase. N-Acetylserotonin might be deacetylated to serotonin in vivo. The results also indicate that the effect of indole amines on tyrosine aminotransferase is a direct peripheral effect, and is not due to an effect on the brain.
In our experiments, L-tryptophan was far less effective as an inducing agent than L-5-hydroxytryptophan, in good agreement with the results by Kenney and Flora (8). At a dose of 0.5 mmole or less, L-tryptophan failed to increase tyrosine aminotransferase activity in both intact or adrenalectomized mice. This phenomenon might be related to the different K, values of aromatic L-amino acid decarboxylase for L-trgptophan and L&hydrosytryptophan.
The K, value for L&hydroxytryptophan is low (2 X 10e5 M), whereas the K, value for L-tryptophan is high (3 X 10e3 M) (14). Because of the high K, value, a high dose of L-tryptophan would be necessary for the induction of tyrosine aminotransferase.
Yatvin and Pitot (15) reported that tyrosine aminotransferase activity increased 5-fold at a low dose of L-tryptophan (5 mg/lOO g, body wt). These investigators, however, administered tryptophan orally.
It might be possible that tryptophan is converted to active rnetabolites by gut flora. Although our results support the conclusion that indole amines, but not other indole compounds, are responsible for the induction of tyrosine aminotransferase in liver, we should be very cautious in evaluating the effects of metabolic inhibitors, because rnany inhibitors might have unknown metabolic effects. Rosen and Milholland (9) showed that n-tryptophan or indole was as effective as L-tryptophan or serotonin in increasing tyrosine aminotransferase activity in rat liver, which can not be explained from our results.
The result wit.h cycloheximide indicates that serotonin induces synthesis of new enzyme, although the possibility that indole amines increased tyrosine aminotransferase activity by derreasing the degradat,ion of the enzyrne has not been excluded.
Actinomycin 11 did not block the increase of tyrosine aminotransferase activity.
This result is inconsistent with the reports by other investigators (15,16) that the induction of tyrosine aminotransferase by L-tryptophan WBS completely blocked by actnomycin D. It might be possible that sctinomycin D blocks t'he induction by L-tryptophan by other unknown processes. Some of the effect of indole compounds is mediated by adrenal hormones, because these indole compounds elevate plasma corticosterone level, and because adrenalectomy reduces the amount of increase of tyrosine aminotransferase activity by indole cornpounds.
These indole compounds, however, could increase tyrosine aminotransferase activity in adrenalectomized mice, suggesting that some other mechanism than adrenal hormones is also involved in t,he induction.
Several possibilities are conceivable.
(a) Is it mediated by pancreatic hormones such as glucagon or insulin? (b) Is it mediated by cyclic AMP in the liver?
(c) Does serotonin act directly on the liver? These questions still remain to be elucidated. If serotonin acts directly or via adenyl cyclase in the liver, this might suggest a reg-ulatory mechanism of indole amines in peripheral tissues. We are not sure yet whether the phenomenon reported in this paper represents a physiological role of indole amines, or it simply represents a pharmacological effect of serotonin exogenously administered. Indole amines have been shown to exhibit a number of actions; such compounds may induce or suppress some enzymes in tissues, resulting in metabolic changes. Such a possibility would represent another biochemical role of the biogenic amines.