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Mechanism of allylisopropylacetamide-induced increase of δ-aminolevulinate synthetase in liver mitochondria
ARCHIVES
OF
BIOCHEMISTRY
Mechanism
AND
BIOPHYSICS
126, 530-538 (1968)
of Allylisopropylacetamide-Induced
6-Aminolevulinate
Synthetase
in Liver
Increase
of
Mitochondria
III. Effects of Triiodothyronine and Hydrocortisone on the Induction Process TOM10 Department
MATSUOKA,l of Biochemistry, Received
BINKOH
YODA,
Tohoku University
December
AND
GORO
School of Medicine,
29, 1967; accepted
February
KIKUCHI Sendai, Japan
14, 1968
The AIA-induced increase of &aminolevulinate synthetase in rat-liver mitochondria was greatly stimulated when rats were given hydrocortisone or triiodothyronine, while the enzyme level was not increased by the administration of those hormones alone. Kinetic studies of the hormone-stimulated induction process suggested that a closely related synergistic action of AIA2 and hormones is required to bring about the intensive induction of the enzyme. Also evidence was obtained indicating that possibly a “refractory phase” appears right after the period of intensive enzyme induction, and the development of “refractory phase” may also be stimulated by hormones. The possibility was considered that intensive synthesis of S-aminolevulinate synthetase might induce subsequently the synthesis of an inhibitor which interferes with the synthesis or fun&ion of messenger RNA for &aminolevulinate synthetase.
The level of &aminolevulinate synthetase in liver mitochondria greatly increases by the administration of various chemicals to animals (l-9). This is unique as an example of the induction of mitochondria-bound enzyme. Previous study (4) in our laboratory on the process of the AIA-induced increase of d-aminolevulinate synthetase in rat-liver mitochondria revealed the occurrence of two distinctive phases with different sensitivities toward inhibitors of DNA synthesis. The first phase of the induction process was not inhibited by mitomycin C or 5-fluorouridine deoxyribose, whereas the second phase, which appeared to be brought about only after administration of larger amounts of AIA, was strongly inhibited by inhibitors of suggesting the possible DNA synthesis, involvement of synthesis or turnover of DNA in the second phase of induction. Also it was observed that, although the level of 1 Present address: Department of Surgery, Tohoku University School of Medicine, Sendai, Japan. 2Abbreviation: AIA, allylisopropylacetamide.
&aminolevulinate synthetase in rat-liver was not increased by the administration of triiodothyronine or hydrocortisone alone, the level of &aminolevulinate synthetase was considerably higher when both AIA and triiodothyronine or hydrocortisone were simultaneously administered than when AIA alone was administered (cf. ref. 3 and 4). The stimulatory effect of hydrocortisone was also studied by Marver et al. (6), using adrenalectomized rats, and the “permissive” role of hydrocortisone in the AIA-mediated induction of b-aminolevulinate synthetase was verified. The present paper is a report of the study of unique features of the hormonal effect on the induction process of d-aminolevulinate synthetase, particularly in the mitomycininsensitive first phase of the induction. MATERIALS
AND
CONDITIOXING
METHODS OF RATS
Wistar strain male albino rats (150-200 g) were fasted for 24 hours, after which AIA and hormones were administered by subcutaneous injection. 530
&AMINOLEVULINATE
SYNTHETASE
INDUCTION
Unless otherwise stated, 15 mg AIA were given per 100 g body wt. of rats at each administration. This dose of AIA was sufficient maximally to induce the first, phase, but hardly induced the second phase, and hence this dose of AIA was taken as most suitable for the present investigation. Inhibitors, when employed, were given intraperitoneally 1 hour prior to the administration of AIA. Hormones and inhibitors were dissolved in 0.9% saline solution containing 0.01 M sodium phosphate buffer of pH 7.4. Rats were maintained fastinq throughout the experiments.
ASSAY OF ENZYME ACTIVITIES Liver
levulinate
mitochondria
for
synthetase
the
activity
assay
of &amino-
were
prepared
essentially in the similar manner to those described by Narisawa and Kikuchi (4). Protein amounts of mitochondrial preparations were determined by a biuret method (lo), employing the calibration curve standardized according to the Kjeldahl method. The assay system for &aminolevulinate synthetase contained, in a final volume of 2 ml: potassium succinate, 60 pmoles; glycine, 100 pmoles; ATP, 10 pmoles; CoA, 0.05 bmole; pyridoxal phosphate, 0.2 rmole; MgC12, 5 pmoles; mercaptoethanol, 5 pmoles; potassium phosphate buffer (pH 7.4), 100
rmoles; sucrose, 500 rmoles, mg protein of mitochondria.
and approx. 20-25 The mixture was
incubated at 37” in a shaking water bath incubator. Amounts of &aminolevulinate formed were determined by the procedures a.s described in the previous paper (4). Namely, &aminolevulinate was converted to the pyrrole compound by the condensation with acetylacetone and was isolated by a column of Dowex-I. The color was developed by addition of Ehrlich’s reagent and the absorbancy at 553 mp was read against reagent control. The millimolar extinction coefficient at 553 rnp was assumed to be 53. Preliminary experiments showed that the synthesis of &aminolevulinate proceeded linearly for about 40 minutes and that, the reaction rate was proportional to the amount of mitochondrial protein used. Enzyme activities were estimated by employing values obtained at 30 minutes of incubation.
REAGENTS AIA was a gift from F. Hoffmann-LaRoche
&
Co., Basle; mitomycin C, from Kyowa Hakko Co., Tokyo; actinomycin D, from Lederle Laboratories, U.S.A., and hydrocortisone succinate, from Nippon Upjohn Co., Tokyo, respectively. Cycloheximide was obtained from Nutritional Biochem. Corp.,
U.S.A.
Other
AND reagents
531
HORMONES were
obtained
commer-
cially. RESULTS
KINETICS OF ENZYME INCREASE IN THE EARLY STAGE OF INDUCTION As shown
in Fig.
1, the level
of d-amino-
levulinate synthetase in liver mitochondria increased
after
the
administration
of AIA,
with a lag period of about, one hour and reaching a maximum level at about 3 hours. The increase in the enzyme level was not appreciably inhibited by mitomycin C, and the enzyme level remained high for as long as 12 hours in either caseswith or without mitomycin. These represent the characteristics of the first phase of the induction (cf. ref. 4). Triiodothyronine, when administered simultaneously with AIA, greatly stimulated the increase of the enzyme level. The triiodothyronine-stimulated increase of &aminolevulinate synthetase at this stage was practically insensitive to mitomycin (Fig. 1). The stimulation by triiodothyronine, however, did not last for long periods; the highly increased enzyme level soon declined rapidly to the level similar to those observed without triiodothyronine. The results with hydrocortisone were essentially similar to those obtained with triiodothyronine (Fig. 2), except that the highest enzyme level was attained earlier than when triiodothyronine was employed. In Table I are given some examples showing the ranges of deviation of the enzyme level obtained in individual experiments. The hormone-stimulated increase of 6aminolevulinate synthetase became apparent earlier when triiodothyronine or hydrocortisone was injected 1 hour prior to AIA administration (Figs. 3 and 4). Earlier increase of the enzyme level could be observed even when hormones were injected to rats 3-6 hours before AIA. In contrast, when hormones were given 1 hour after AIA, the magnitudes of stimulation by hormones were far smaller, and when hormones were injected to rats 2 hours or later after AIA, practically no stimulation could be observed (Figs. 3 and 4). It appears that a closely related synergistic action of AIA and hor-
532
MATSUOKA,
YODA,
AX11
KIKUCHI
None AIA+TS+AcD D 0
3
4 TIME
FIG. 1. Effect of synthetase. Amounts mitomycin C (MiC) wt. of rats at each values obtainedwith simultaneously with to rats 1 hour prior
I
I
2
I
5
6
7
6
I
9
(hours)
triiodothyronine on the early stage of induction of Saminolevulinate of hormone and inhibitors used were: triiodothyronine (T3) 50 pg, 0.4 mg, and actinomycin I> (A&) 0.1 mg, respectively, per 100 g body administration. The values presented in the figure are the averages of 4 to 14 animals tested, respectively. Triiodothyronine was given to rats the administration of AIA. Inhibitors, when employed, were injected to the administration of AIA.
manesis required to bring about the stimulation of the induction. FURTHER EXAMIXATIOX OF THE EARLY STAGE OF ENZYME INDUCTION
0
I
2
3 TIME
4
5
6
(hours)
FIG. 2. Effect of hydrocortisone on the early stage of induction of 6-aminolevulinate synthetase. 5 mg hydrocortisone succinate (HC) per 100 g body wt. of rats were injected simultaneously with AIA. Each value in the figure is the average of values obtained with not less than 4 animals. Other conditions and symbols were similar to those for Fig. 1.
It was shown in Figs. 3 and 4 that when rats were given hormones 1 hour prior to AIA, the once increased enzyme level began to decline earlier than when hormones and AIA were given simultaneously. With the aim to obtain further insight to these phenomena, we examined the effect of second administration of AIA, with or without hormones, on the subsequent process of enzyme induction. In the experiments shown in Fig. 5, rats were given another dose of AIA at 3 hours after the first AIA administration. The enzyme level in these rats increased significantly after the second AIA administration. When rats were given both AIA and triiodothyronine or hydrocortisone at 3 hours after the first AIA, the enzyme levels at 4.5 hours (namely 1.5 hours after the second AIA) were considerably higher than those in rats which did not receive hormones. However, the enzyme levels in
&AMINOLEVULINATE TABLE AVER-AGE
VALIJES
VIATION
AND
OF
SYNTHET~SE
SYNTHETASE
I RANGES
LEVELS
OF
OBTAINED
IN
OF STANDARD &AMINOLEVULIN.~TE INDIVIDUAL
INDUCTION
AND
533
HORMONES
DE-
EXPERI-
MENTS
The values were calculated from the date obtained in the experiments shown in Fig. 1. Numbers in brackets represent the numbers of animals tested.
Drugs
injected time
at 0
&Aminolevulinate synthetase activity (m~moles &aminolevulinate formedlmg protein/hour)
I-
At 3 hours after drug administration
AIA AIA and mitomycin C AIA and triiodothyronim AIA, triiodothyronine and mitomycin C
At 6 hours after drug administration
6.1 i 1.06 5.5 zk 0.85
(14) 5.0 (5) 4.8
12.1 f
3.08
L1.2 f
3.06
I
0
i f
1.75 0.55
(8) (6)
(9)
6.3 f
1.91
(10)
(6)
4.8 zt 1.46
-I
(4)
2 TIME
I
0
3 (hours)
4
I
I
5
6
FIG. 4. Effect of hydrocortisone given at different times before or after the AIA administration. Solid line, without hydrocortisone; (H), hydrocortisone succinate was injected 1 hour prior to AIA; (- - -0)) hydrocortisone was injected simultaneously with AIA; (- - --0), hydrocortisone was injected a,t 1 hour after AIA; (- - -A), hydrocortisone was injected at 2 hours aft,er AIA; (a), hydrocortisone was injected at 4.5 hours after AIA. Each value in the figure is the average of vallles obtained with not less than 4 animals.
t -I
0
I
2 TIME
3 (hours)
4
5
6
FIG. 3. Effect of triiodothyronine given at different times before or after the AIA administration. Rats were given 15 mg AIA per 100 g body wt. at 0 time. Single injection of triiodothyronine (50 pg per 100 g body wt.) was given to respective groups of rats at different times. Solid line, without triiodothyronine; (W), triiodothyronine was injected 1 hour prior to the AIA administration; (- - -0)) triiodothyronine was injected simultaneously with AIA; (Of, triiodothyronine was injected at 1 hour after AIA; (A), triiodothyronine was injected at 2 hours after AIA. Each value in the figure is the average of values obtained with 4 to 10 animals tested. Other conditions were similar to those for Fig. 1.
.A
0 0
I I
I 2
I 3
I 4
TIME
(hours)
I 5
1 6
FIG. 5. Effect of hormones on the induction after second administration of AIA. Rats were given the first administration of AIA at 0 time, and at 3 hours of the experiment, rats were given the second administration of AIA (- - -0) or the combined administration of AIA and triiodothyronine (- - -0) or AIA and hydrocortisone (---a), and killed for enzyme assay at 6 hours of experiment. Amounts of AIA and hormones injected at 3 hours were the same as those in Figs. 1 and 2. The numbers in the brackets in the figure represent the number of animals tested.
534
MATSUOKA,
YODA,
AXD
KIKUCHI
rats which received both AIA and hormones at 3 hours after the first AIA soon declined rapidly, so that the enzyme levels at 6 hours were distinctly lower than those in rats which did not receive hormones. In another series of experiments, shown in Fig. 6a, rats were injected both AIA and triiodothyronine
essentially similar to those observed with triiodothyronine. It is noteworthy in Fig. 6a, that when hormones were given t,ogether
at 0 time,
zyme levels in rats which received hormones at the time of second AIA injection decreased at almost same rates to those in rats which did not receive the second AIA. These results suggest the possibility that once an intensive induction occurs in rats, then it is
with
the
second
AIA,
the
enzyme
levels
found at 4.5 and 6 hours were rather lower than the corresponding values obtained without
and after 3 hours they were given another dose of AIA with or without hor-
mones. In these experiments, however, the enzyme level was no more increased by the second injection of AIA. The same type of experimenOs but with hydrocortisone were also performed, and as shown in Fig. 6b, the features observed with hydrocortisone were
hormones,
followed
by some
and
moreover,
unreactive
the en-
or refractory
phase where the inducing machinery in rats
(a)
(b)
(8)
I
I
I
I
2
3
4
I
5
4
I
6
TIME
2
3
4
5
(hours)
FIG. 6. Effect of second AIA administration on the hormone-stimulated induction of 6aminolevulinate synthetase. (a). (-•), time course of the induction after the first simultaneous administration of AIA and triiodothyronine at 0 time; (o), b-aminolevulinate synthetase activities in rats which received the second administration of AIA at the time indicated by arrow in the figure; (O), i-aminoleuvlinate synthetase activities in rats which received the second administration of both AIA and triiodothyronine; (A), &aminolevulinate synthetase activity in rats whichreceived the second administrationof AIA together with hydrocortisone. Amounts of AIA, triiodothyronine and hydrocortisone injected were the same to those in Figs. 1 and 2. (b). (---A), time course of the induction after the first simultaneous administration of AIA and hydrocortisone at 0 time; (- - -A), L-aminolevulinate synthetase activities in rats which received the second administration of AIA at the times indicated by arrows in the figure. Amounts of AIA and hydrocortisone injected were same to those for Fig. 2. The numbers in the brackets in the figure represent the number of animals tested.
6
&AMINOLEVULINATE
SYNTHETASE
does not show appreciable response to either AIA or hormones, and the development of “refractory phase” may also be stimulated by hormones. The apparent “refractory phase”, however, seems to continue only for relatively short periods, for it was observed in an independent experiment that when rats, which had been given triiodothyronine and AIA at 0 time, were given the second dose of AIA at 6 hours after the first AIA, the enzyme level in these rats increased thereafter significantly. Cycloheximide, a specific inhibitor of protein synthesis (ll), inhibited the synthesis of 6aminolevulinate synthetase in any systems tested, and when cycloheximide was given to rats at 3 hours after AIA, it gave rise to a sharp drop of the enzyme level (Table II). This confirms a high rate of t,urnover of inducibly formed d-aminolevulinate synthetase (cf. ref. 9). The induction of &aminolevulinate synthetase is also completely abolished if TABLE
II
EFFECTS OF ADMINISTRATION OF ACTINOMYCIN D AND CYCLOHEXIMIDE IN THE MIDDLE OF INDUCTION OF &AMINOLEVULINATE SYNTHETASE Amounts of AIA, triiodothyronine, hydrocortisone and actinomycin D were the same as those given in Figs. 1 and 2. 5 mg cycloheximide were used per 100 g body wt. of rats. The values given in the Table are the averages of values obtained with the numbers of rats shown in brackets, respectively. -
Drugs injected at 0 time
6-Aminolevulinate SJmthetase activity at 6 hours after AM (1.nfimoles I-aminole,vulinate formed/ Ing protein/hour)
Drugs injected at 3 hours after AIA’= --
None None AIA AIA AIA AIA and triiodo, thyronine AIA and triiodo. thyronine AIA and triiodo. thyronine 0 For the values refer to Table I.
None Cycloheximide None Actinomycin Cycloheximide None Actinomycin Cycloheximide
at 3 hours
D
D
2.0 0.5 5.2 4.9 1.1 6.3
(6) (6) (16) (6) (6) (10)
6.3
(6)
1.5
(3)
of the experiments,
INDUCTION
AND
HORMONES
535
actinomycin D is injected simultaneously with AIA (cf. Fig. 1 and ref. 4 and 9). As can be seen from Table II, however, when actinomycin D was injected at not 0 time, but 3 hours after AIA, it did not appreciably enhance the enzyme decrease during the subsequent period of 3 hours. Since the turnover rate of &aminolevulinate synthetase has been evidenced to be very high, the results obtained would indicate that &aminolevulinate synthetase was being synthesized continuously even after the actinomycin injection, probably being supported by existing messenger RNA. On the other hand, we observed that when rats were killed at 4.5 hours after actinomycin D (namely 7.5 hours after AIA), the enzyme activities in these rats were less than half of those observed without actinomycin D. These results suggest that the life-time of the messenger RNA for d-aminolevulinate synthetase may be about 3 hours, in agreement with the estimation by Marver et al. (9. Similar experiments were made also with triiodothyronine-treated rats, and again it was found that actinomycin exterted practically no effect, on the rate of enzyme decrease in these rats during the 3 hours after actinomycin D (Table II). As discussed before, a “refractory phase” developes at. about 3 hours after AIA in triiodothyroninetreated rats, and in the “refractory phase” the enzyme level decreases rapidly. Considering these circumstances together, it seems likely that the rate of messenger RNA synthesis in these rats should have been reduced to an extremely low level within 3 hours after AIA. or, even if the messenger RNA was exist~ing, it might not be functioning in the “refractory phase”. EFFECT OF HORMONES THE INDUCTION IN ADRENALECTOMIZED RATS
0~
Rats were bilaterally adrenalectomized by dorsal approach under general anaesthesia with ether, and fasted for 36 hours before being used. Adrenalectomized rats were allowed to take 1% saline solution ud libifum throughout the experiments. The level of I-aminolevulinate synthetase in
536
MATSUOKA, TABLE
EFFECT
OF
HORMONES
S-AMINOLEVULINBTE
YODA,
III ON
THE
SYNTHETASE ECTOMIZED RUTS
INDUCTION IN
OF
ADRENAL-
25 mg AIA per 100 g body wt. of rats were given to each group of rat. Amounts of triiodothyronine and hydrocortisone succinate employed were 50 pg and 5 mg per 100 g body wt. of rats, respectively. Hormones were injected simultaneously with AIA. Rats were killed at 3 hours after the AIA administration. The values in the table represent the averages of values obtained with 4-10 animals tested. &Amin;~~$inate Drugs
administered
None AIA AIA plus triiodothyronine AIA plus hydrocortisone AIA plus triiodothyronine hydrocortisone
(mpmoles/m protein/hour
and
F
2 5.0 5.8 7.7 10.2
liver mitochondria was not affected by adrenalectomy and by fasting for 36 hours. After 36 hours of fasting, the rats were given 25 mg AIA per 100 g body wt. and killed for enzyme assay at 3 hours after AIA. As shown in Table III, the level of d-aminolevulinate synthetase in adrenalectomized rats was increased by more than two fold by the administration of AIA, although magnitudes of the enzyme increase in adrenalectomized rats were smaller than those in intact rats under comparable experimental conditions. The increase of 6aminolevulinate synthetase in adrenalectomized rats was more pronounced when rats were given triiodothronine or hydrocortisone simultaneously with the administration of AIA, thus confirming the results of Marver et al. (9) obtained with hydrocortisone. Hydrocortisone exerted more significant effect than triiodothyronine. When both triiodothyronine and hydrocortisone were given to adrennlectomized rats simultaneously with AIA, the 6aminolevulinate synthetase activity increased to almost the same level as observed in intact rats which were given both triiodothyronine and AIA. DISCUSSIOr\’
The experimental that triiodot#hyronine
results indicated and hydrocortisone
AND
KIKUCHI
strongly stimulated the AIA-induced synthesis of &aminolevulinate synthetase in rat-liver, while the administration of these hormones alone did not give rise to the increased synthesis of the enzyme. il closely related synergistic action of AIA and hormones appeared to be required to bring about the stimulation of the induction, although apparently AIA played a primary role in the induction of &aminolevulinate synthetase and roles of hormones are only auxilliary. In in vitro system with isolated liver mitochondria, as well as with a soluble enzyme preparation obtained from acetone powder of liver mitochondria, the B-aminolevulinate synthetase activity was not influenced by addition of various concentrations of triiodothyronine or hydrocortisone. There have been several lines of evidence that hormones may affect fairly directly RNA synthesis at the gene level (12-20). The observed hormonal stimulation of the AIA-induced synthesis of b-aminolevulinate synthetase seems to be due principally to the hormone-stimulated increase of messenger RNA synthesis. The effect of triiodothyronine on the induction of 6aminolevulinate synthetase seems to be in the similar sense as also “permissive” effect assumed for the hydrocort’isone (cf. ref. 6). It remains to be clarified, however, whether the “permissive” effects of hydrocortisone and triiodothyronine are independent or interrelated each other. The “permissive” effect does not appear to be due to effects of hormones on the level of succinyl-CoA synthetase, for we observed that the level of this enzyme was not appreciably influenced by either the combined or separate administration of hormones and AIA. Labbe et al. (21) reported that the total succinyl-Coil synthetase activity in liver from AIA-induced porphyria mouse was increased considerably and the inducible fraction of the enzyme was the isozyme of the non-inducible constitutive enzyme. However, we were unable to confirm the reported situations with respect to either mouse or rat (to be published). Hormones may also regulate protein synthesis at the RNA template (22-24). With respect to the &aminolevulinate
s-AMINOLEVULINATE
SYNTHETASE
synthetase induction, however, the contribution, if any, of the stimulation by hormones at the translation level seems to be minor, since hormones, when given 1 hour or later after the AIA administration (namely in the middle of active synthesis of the enzyme) did not significantly increase the &aminolevulinate synthetase level (cf. Figs. 3 and 4). It is noteworthy t’hat some “refractory phase” seemed to appear right after the period of intensive enzyme induction particularly in hormone-treated rats. Also in “refractory phase” the enzyme synthesis, if any, appeared to be extremely small as judged by the very rapid decline of the enzyme level in the ment,ioned period. This suggests that the amount of messenger RNA for b-aminolevulinate synthetase had been reduced to a extremely low level in “refractory phase,” or, even if the messenger RNA was existing, its function might have been inhibited in some unknown way. On the other hand, it was observed that, as shown in Fig. 1, the b-aminolevulinate synthetase level in hormone-untreated rats was maintained considerably high and fairly constant for as long as 12 hours after the single administration of AIA, while the turnover rate of the inducibly formed &aminolevulinate synthetase was evidenced t’o be very high. It is conceivable that in these rats the messenger RNA was being synthesized continuously for as long as 12 hours at nearly constant and considerably higher rates than those in control rats n-ithout AIA. These circumstances suggest the occurrence of complicated mechanisms which may participate in the regulat,ion of synthesis of &aminolevulinate synt’hetase so as to maintain a certain level of the enzyme in rat liver. Various possibilities can be considered to account for the observed “refractory phase” of the &aminolevulinate synthet,ase induction. The simplest hypothesis may be that some “inhibitor” appears as a consequence of intensive enzyme induction. One candidate for the suspected “inhibitor” may be heme. Several reports (7, 8, 2.5, 26) indicated that the synthesis of &aminolevulinate synthetase is strongly suppressed by the administration of hemin. Granick
INDUCTION
AND
HORMONES
537
(7) proposed a hypothesis assuming that the level of &aminolevulinate synthetase in liver is controlled by feedback repression in which heme may be a corepressor and that heme and an inducing chemical compete for each other on the same site on a repressor. Evidence was obtained in our laboratory that hemin might exert its inhibitory action at a step of synthesis of messenger RNA for d-aminolevulinate synthetase (26). Moreover, hemoglobin and even bilirubin were effective in inhibiting the induction of b-aminolevulinate synthetase (26), suggesting that bilirubin, too, might share the role of heme as the corepressor. Granick’s hypothesis was favorably adopted by Waxman et al. (8) and by Marver et al. (27) to interpret their experimental findings. Levere and Granick (28) further suggested that a repressor mechanism controlling the rate of synthesis of &aminolevulinate synthetase is operative in erythroid precursor cells of chick blastoderm. However, the idea of reciprocal regulation mechanism, as proposed by Granick (7) and exbended by Waxman et al. (8), seems to be not enough to account for the observed sharp decline of the enzyme level as well as the apparent “refractory phase” of the induction, for, as shown in Fig. 6, the sharp decline of the b-aminolevulinate synthetase level was not appreciably prevent’ed by the second administ’ration of AIA, although there remains an alternate possibility that the hormones may also stimulate the synthesis of enzymes which metabolize AIA. The temporary presence of high levels of such enzymes taken in combination with Granick’s mechanism would also account for the observed features of the hormonal effects upon the &aminolevulinate synthetase induction. Further studies are required t’o examine these possibilities. The second candidate for the “inhibitor” could be a repressor of protein nature which inhibits further enzyme synthesis by inhibiting the function of existing messenger RNA. This type of regulation of enzyme synthesis was first postulated by Garren et al. (29) for the hydrocortisone-stimulated synthesis of tryptophan pyrrolase (EC 1.13.1.12) and tyrosine-a!-ketoglutaratc
538
MATSUOKA,
YODA,
transaminase (EC 2.6.1.5) in rat liver, and the synthesis of this repressor was supposed to be very sensitive to actinomycin action. The work by Garren et al. (29), however, was questioned recently by Mishkin and Shore (30) who claimed to have failed to find any apparent stimulation by actinomycin D of the hydrocortisone-induced increase of tryptophan pyrrolase. We also failed to observe the stimulation by actinomycin D of the AIA-induced increase of d-aminolevulinate synthetase (cf. Table II). Thus, although the inhibitor protein theory as postulated originally by Garren el al. (29) looks very attractive as a hypothesis to account for our observations on the stimulation and inhibition of the induction of &aminolevulinate synthetase, it is premature at present to put much emphasis on this hypothesis. Naturally the artificial administration of hormones may bring about a disturbance of hormonal homeostasis in rats and this may also exert some influences on the feature of the enzyme induction. ACKNOWLEDGMENT The authors acknowledge F. HoffmannLaRoche & Co., Basle ; Kyowa Hakko Co., Tokyo; Nippon Upjohn Co., Tokyo, and Lederle Laboratories, U.S.A., for their kind supply of reagents. This investigation was supported in part by P.H.S Research Grant AM 08016-03 and 04 from the National Institute of Arthritis and Metabolic Diseases, U.S.A. (G.K). REFERENCES 1. GRANICK, S., AND URATA, G., J. Biol. Chem. 238, 821 (1963). 2. MIYAKOSHI, T., AND KIKUCHI, G., Tohoku J. Exptl. Med. 79, 199 (1963). 3. NARISAWA, K., AND KIEUCHI, G., Biochim. Biophys. Acta 99, 580 (1965). 4. NARISAWA, K., AND KIKUCHI, G., Biochim. Biophys. Acta 138, 596 (1966). 5. MARVER, H. S., TSCHUDY, D. P., PERLROTH, M. G., AND COLLINS, A., J. Biol. Chem. 241, 2803 (1966).
AND
KIKUCHI
6. MBRVER, H. S., COLLIXS, A., .\SD TSCHUDY, D. P., Biochem. J. 99, 31C (1966). 7. GRANICK, S., J. Biol. Chem. 241, 1359 (1966). 8. WAXM.4~, A. D., COLLINS, A., AND TSCHUDY, D. P., Biochem. Biophys. Res. Commun. 24, 675 (1966). 9. MARVER, H. S., COLLINS, A., TSCHUDY, D. P., BND RECHCIGL, M., JR., J. Biol. Chem. 241, 4323 (1966). 10. GORNALL, A. G., BARDAWILL, C. S., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). 11. YOUNG, C. W., ROBINSON, P. F., AND SACKTOR, B., Biochem. Pharmacol 12, 855 (1963). 12. KARLSON, P., Perspectives Biol. Med. 6, 203 (1963). 13. DAVIDSON, E. H., Sci. American 212,36 (1965). 14. DAHMUS, M. E., AND BONNER, J., Proc. N&Z. Acad. Sci. 64, 1370 (1965). 15. DUKES, P. P., AND SEKERIS, C. E., 2. Physiol. Chem. 341, 149 (1965). 16. LIAO, S., BARTON, R. W., AND LIN, A. H., Proc. Natl. Acad. Sci. 66, 1593 (1966). 17. KIM, K. H., AND COHEN, P. P., Proc. Null. Acad. Sci. 66, 1251 (1966). 18. MEANS, A. R., AND HAMILTON, T. H., Proc. Nutl. Acud. Sci. 66, 1594 (1966). 19. LUKACS, I., AND SEKERIS, C. E., Biochim. Biophys. Acta 134, 95 (1967). 20. KIDSON, C., Nature 213,779 (1967). 21. LABBE, R. F., KURUMADA, T., AND ONISAWA, J., Biochim. Biophys. Acta 111, 403 (1965). 22. SOKOLOFF, L., FRANCIS, C. M., AND CAMPBELL, P. L., Proc. Nutl. Acud. Bci. 62, 728 (1964). 23. GARREN, L. D., NEY, R. L., AND DAVIS, W. W., Proc. Natl. Acad. Sci. 63, 1443 (1965). 24. GORSKI, J., AND PADNOS, D., Arch. Biochem. Biophys. 113, 100 (1966). 25. WADA, O., J. Jap. Sot. Int. Med. 64, 673 (1965) (in Japanese). 26. HAYASHI, N., YODA, B., .&ND KIKUCHI, G., J. Biochem., 63,446 (1968). 27. MARVER, H. S., TSCHUDY, D. P., PERLROTH, M. G., AND COLLINS, A., Science 164, 501 (1966). 28. LEVERE, R., AND GRANICK, S., J. Biol. Chem. 242, 1903 (1967). 29. GARREN, L. D., HOWELL, R. R., TOMKINS, G. M., AND CROCCO, R. M., Proc. Natl. Acud. Sci. 62, 1121 (1964). 30. MISHKIN, E. P., .&ND SHORE, M. L., Biochim. Biophys. Acta 138, 169 (1967).
OF
BIOCHEMISTRY
Mechanism
AND
BIOPHYSICS
126, 530-538 (1968)
of Allylisopropylacetamide-Induced
6-Aminolevulinate
Synthetase
in Liver
Increase
of
Mitochondria
III. Effects of Triiodothyronine and Hydrocortisone on the Induction Process TOM10 Department
MATSUOKA,l of Biochemistry, Received
BINKOH
YODA,
Tohoku University
December
AND
GORO
School of Medicine,
29, 1967; accepted
February
KIKUCHI Sendai, Japan
14, 1968
The AIA-induced increase of &aminolevulinate synthetase in rat-liver mitochondria was greatly stimulated when rats were given hydrocortisone or triiodothyronine, while the enzyme level was not increased by the administration of those hormones alone. Kinetic studies of the hormone-stimulated induction process suggested that a closely related synergistic action of AIA2 and hormones is required to bring about the intensive induction of the enzyme. Also evidence was obtained indicating that possibly a “refractory phase” appears right after the period of intensive enzyme induction, and the development of “refractory phase” may also be stimulated by hormones. The possibility was considered that intensive synthesis of S-aminolevulinate synthetase might induce subsequently the synthesis of an inhibitor which interferes with the synthesis or fun&ion of messenger RNA for &aminolevulinate synthetase.
The level of &aminolevulinate synthetase in liver mitochondria greatly increases by the administration of various chemicals to animals (l-9). This is unique as an example of the induction of mitochondria-bound enzyme. Previous study (4) in our laboratory on the process of the AIA-induced increase of d-aminolevulinate synthetase in rat-liver mitochondria revealed the occurrence of two distinctive phases with different sensitivities toward inhibitors of DNA synthesis. The first phase of the induction process was not inhibited by mitomycin C or 5-fluorouridine deoxyribose, whereas the second phase, which appeared to be brought about only after administration of larger amounts of AIA, was strongly inhibited by inhibitors of suggesting the possible DNA synthesis, involvement of synthesis or turnover of DNA in the second phase of induction. Also it was observed that, although the level of 1 Present address: Department of Surgery, Tohoku University School of Medicine, Sendai, Japan. 2Abbreviation: AIA, allylisopropylacetamide.
&aminolevulinate synthetase in rat-liver was not increased by the administration of triiodothyronine or hydrocortisone alone, the level of &aminolevulinate synthetase was considerably higher when both AIA and triiodothyronine or hydrocortisone were simultaneously administered than when AIA alone was administered (cf. ref. 3 and 4). The stimulatory effect of hydrocortisone was also studied by Marver et al. (6), using adrenalectomized rats, and the “permissive” role of hydrocortisone in the AIA-mediated induction of b-aminolevulinate synthetase was verified. The present paper is a report of the study of unique features of the hormonal effect on the induction process of d-aminolevulinate synthetase, particularly in the mitomycininsensitive first phase of the induction. MATERIALS
AND
CONDITIOXING
METHODS OF RATS
Wistar strain male albino rats (150-200 g) were fasted for 24 hours, after which AIA and hormones were administered by subcutaneous injection. 530
&AMINOLEVULINATE
SYNTHETASE
INDUCTION
Unless otherwise stated, 15 mg AIA were given per 100 g body wt. of rats at each administration. This dose of AIA was sufficient maximally to induce the first, phase, but hardly induced the second phase, and hence this dose of AIA was taken as most suitable for the present investigation. Inhibitors, when employed, were given intraperitoneally 1 hour prior to the administration of AIA. Hormones and inhibitors were dissolved in 0.9% saline solution containing 0.01 M sodium phosphate buffer of pH 7.4. Rats were maintained fastinq throughout the experiments.
ASSAY OF ENZYME ACTIVITIES Liver
levulinate
mitochondria
for
synthetase
the
activity
assay
of &amino-
were
prepared
essentially in the similar manner to those described by Narisawa and Kikuchi (4). Protein amounts of mitochondrial preparations were determined by a biuret method (lo), employing the calibration curve standardized according to the Kjeldahl method. The assay system for &aminolevulinate synthetase contained, in a final volume of 2 ml: potassium succinate, 60 pmoles; glycine, 100 pmoles; ATP, 10 pmoles; CoA, 0.05 bmole; pyridoxal phosphate, 0.2 rmole; MgC12, 5 pmoles; mercaptoethanol, 5 pmoles; potassium phosphate buffer (pH 7.4), 100
rmoles; sucrose, 500 rmoles, mg protein of mitochondria.
and approx. 20-25 The mixture was
incubated at 37” in a shaking water bath incubator. Amounts of &aminolevulinate formed were determined by the procedures a.s described in the previous paper (4). Namely, &aminolevulinate was converted to the pyrrole compound by the condensation with acetylacetone and was isolated by a column of Dowex-I. The color was developed by addition of Ehrlich’s reagent and the absorbancy at 553 mp was read against reagent control. The millimolar extinction coefficient at 553 rnp was assumed to be 53. Preliminary experiments showed that the synthesis of &aminolevulinate proceeded linearly for about 40 minutes and that, the reaction rate was proportional to the amount of mitochondrial protein used. Enzyme activities were estimated by employing values obtained at 30 minutes of incubation.
REAGENTS AIA was a gift from F. Hoffmann-LaRoche
&
Co., Basle; mitomycin C, from Kyowa Hakko Co., Tokyo; actinomycin D, from Lederle Laboratories, U.S.A., and hydrocortisone succinate, from Nippon Upjohn Co., Tokyo, respectively. Cycloheximide was obtained from Nutritional Biochem. Corp.,
U.S.A.
Other
AND reagents
531
HORMONES were
obtained
commer-
cially. RESULTS
KINETICS OF ENZYME INCREASE IN THE EARLY STAGE OF INDUCTION As shown
in Fig.
1, the level
of d-amino-
levulinate synthetase in liver mitochondria increased
after
the
administration
of AIA,
with a lag period of about, one hour and reaching a maximum level at about 3 hours. The increase in the enzyme level was not appreciably inhibited by mitomycin C, and the enzyme level remained high for as long as 12 hours in either caseswith or without mitomycin. These represent the characteristics of the first phase of the induction (cf. ref. 4). Triiodothyronine, when administered simultaneously with AIA, greatly stimulated the increase of the enzyme level. The triiodothyronine-stimulated increase of &aminolevulinate synthetase at this stage was practically insensitive to mitomycin (Fig. 1). The stimulation by triiodothyronine, however, did not last for long periods; the highly increased enzyme level soon declined rapidly to the level similar to those observed without triiodothyronine. The results with hydrocortisone were essentially similar to those obtained with triiodothyronine (Fig. 2), except that the highest enzyme level was attained earlier than when triiodothyronine was employed. In Table I are given some examples showing the ranges of deviation of the enzyme level obtained in individual experiments. The hormone-stimulated increase of 6aminolevulinate synthetase became apparent earlier when triiodothyronine or hydrocortisone was injected 1 hour prior to AIA administration (Figs. 3 and 4). Earlier increase of the enzyme level could be observed even when hormones were injected to rats 3-6 hours before AIA. In contrast, when hormones were given 1 hour after AIA, the magnitudes of stimulation by hormones were far smaller, and when hormones were injected to rats 2 hours or later after AIA, practically no stimulation could be observed (Figs. 3 and 4). It appears that a closely related synergistic action of AIA and hor-
532
MATSUOKA,
YODA,
AX11
KIKUCHI
None AIA+TS+AcD D 0
3
4 TIME
FIG. 1. Effect of synthetase. Amounts mitomycin C (MiC) wt. of rats at each values obtainedwith simultaneously with to rats 1 hour prior
I
I
2
I
5
6
7
6
I
9
(hours)
triiodothyronine on the early stage of induction of Saminolevulinate of hormone and inhibitors used were: triiodothyronine (T3) 50 pg, 0.4 mg, and actinomycin I> (A&) 0.1 mg, respectively, per 100 g body administration. The values presented in the figure are the averages of 4 to 14 animals tested, respectively. Triiodothyronine was given to rats the administration of AIA. Inhibitors, when employed, were injected to the administration of AIA.
manesis required to bring about the stimulation of the induction. FURTHER EXAMIXATIOX OF THE EARLY STAGE OF ENZYME INDUCTION
0
I
2
3 TIME
4
5
6
(hours)
FIG. 2. Effect of hydrocortisone on the early stage of induction of 6-aminolevulinate synthetase. 5 mg hydrocortisone succinate (HC) per 100 g body wt. of rats were injected simultaneously with AIA. Each value in the figure is the average of values obtained with not less than 4 animals. Other conditions and symbols were similar to those for Fig. 1.
It was shown in Figs. 3 and 4 that when rats were given hormones 1 hour prior to AIA, the once increased enzyme level began to decline earlier than when hormones and AIA were given simultaneously. With the aim to obtain further insight to these phenomena, we examined the effect of second administration of AIA, with or without hormones, on the subsequent process of enzyme induction. In the experiments shown in Fig. 5, rats were given another dose of AIA at 3 hours after the first AIA administration. The enzyme level in these rats increased significantly after the second AIA administration. When rats were given both AIA and triiodothyronine or hydrocortisone at 3 hours after the first AIA, the enzyme levels at 4.5 hours (namely 1.5 hours after the second AIA) were considerably higher than those in rats which did not receive hormones. However, the enzyme levels in
&AMINOLEVULINATE TABLE AVER-AGE
VALIJES
VIATION
AND
OF
SYNTHET~SE
SYNTHETASE
I RANGES
LEVELS
OF
OBTAINED
IN
OF STANDARD &AMINOLEVULIN.~TE INDIVIDUAL
INDUCTION
AND
533
HORMONES
DE-
EXPERI-
MENTS
The values were calculated from the date obtained in the experiments shown in Fig. 1. Numbers in brackets represent the numbers of animals tested.
Drugs
injected time
at 0
&Aminolevulinate synthetase activity (m~moles &aminolevulinate formedlmg protein/hour)
I-
At 3 hours after drug administration
AIA AIA and mitomycin C AIA and triiodothyronim AIA, triiodothyronine and mitomycin C
At 6 hours after drug administration
6.1 i 1.06 5.5 zk 0.85
(14) 5.0 (5) 4.8
12.1 f
3.08
L1.2 f
3.06
I
0
i f
1.75 0.55
(8) (6)
(9)
6.3 f
1.91
(10)
(6)
4.8 zt 1.46
-I
(4)
2 TIME
I
0
3 (hours)
4
I
I
5
6
FIG. 4. Effect of hydrocortisone given at different times before or after the AIA administration. Solid line, without hydrocortisone; (H), hydrocortisone succinate was injected 1 hour prior to AIA; (- - -0)) hydrocortisone was injected simultaneously with AIA; (- - --0), hydrocortisone was injected a,t 1 hour after AIA; (- - -A), hydrocortisone was injected at 2 hours aft,er AIA; (a), hydrocortisone was injected at 4.5 hours after AIA. Each value in the figure is the average of vallles obtained with not less than 4 animals.
t -I
0
I
2 TIME
3 (hours)
4
5
6
FIG. 3. Effect of triiodothyronine given at different times before or after the AIA administration. Rats were given 15 mg AIA per 100 g body wt. at 0 time. Single injection of triiodothyronine (50 pg per 100 g body wt.) was given to respective groups of rats at different times. Solid line, without triiodothyronine; (W), triiodothyronine was injected 1 hour prior to the AIA administration; (- - -0)) triiodothyronine was injected simultaneously with AIA; (Of, triiodothyronine was injected at 1 hour after AIA; (A), triiodothyronine was injected at 2 hours after AIA. Each value in the figure is the average of values obtained with 4 to 10 animals tested. Other conditions were similar to those for Fig. 1.
.A
0 0
I I
I 2
I 3
I 4
TIME
(hours)
I 5
1 6
FIG. 5. Effect of hormones on the induction after second administration of AIA. Rats were given the first administration of AIA at 0 time, and at 3 hours of the experiment, rats were given the second administration of AIA (- - -0) or the combined administration of AIA and triiodothyronine (- - -0) or AIA and hydrocortisone (---a), and killed for enzyme assay at 6 hours of experiment. Amounts of AIA and hormones injected at 3 hours were the same as those in Figs. 1 and 2. The numbers in the brackets in the figure represent the number of animals tested.
534
MATSUOKA,
YODA,
AXD
KIKUCHI
rats which received both AIA and hormones at 3 hours after the first AIA soon declined rapidly, so that the enzyme levels at 6 hours were distinctly lower than those in rats which did not receive hormones. In another series of experiments, shown in Fig. 6a, rats were injected both AIA and triiodothyronine
essentially similar to those observed with triiodothyronine. It is noteworthy in Fig. 6a, that when hormones were given t,ogether
at 0 time,
zyme levels in rats which received hormones at the time of second AIA injection decreased at almost same rates to those in rats which did not receive the second AIA. These results suggest the possibility that once an intensive induction occurs in rats, then it is
with
the
second
AIA,
the
enzyme
levels
found at 4.5 and 6 hours were rather lower than the corresponding values obtained without
and after 3 hours they were given another dose of AIA with or without hor-
mones. In these experiments, however, the enzyme level was no more increased by the second injection of AIA. The same type of experimenOs but with hydrocortisone were also performed, and as shown in Fig. 6b, the features observed with hydrocortisone were
hormones,
followed
by some
and
moreover,
unreactive
the en-
or refractory
phase where the inducing machinery in rats
(a)
(b)
(8)
I
I
I
I
2
3
4
I
5
4
I
6
TIME
2
3
4
5
(hours)
FIG. 6. Effect of second AIA administration on the hormone-stimulated induction of 6aminolevulinate synthetase. (a). (-•), time course of the induction after the first simultaneous administration of AIA and triiodothyronine at 0 time; (o), b-aminolevulinate synthetase activities in rats which received the second administration of AIA at the time indicated by arrow in the figure; (O), i-aminoleuvlinate synthetase activities in rats which received the second administration of both AIA and triiodothyronine; (A), &aminolevulinate synthetase activity in rats whichreceived the second administrationof AIA together with hydrocortisone. Amounts of AIA, triiodothyronine and hydrocortisone injected were the same to those in Figs. 1 and 2. (b). (---A), time course of the induction after the first simultaneous administration of AIA and hydrocortisone at 0 time; (- - -A), L-aminolevulinate synthetase activities in rats which received the second administration of AIA at the times indicated by arrows in the figure. Amounts of AIA and hydrocortisone injected were same to those for Fig. 2. The numbers in the brackets in the figure represent the number of animals tested.
6
&AMINOLEVULINATE
SYNTHETASE
does not show appreciable response to either AIA or hormones, and the development of “refractory phase” may also be stimulated by hormones. The apparent “refractory phase”, however, seems to continue only for relatively short periods, for it was observed in an independent experiment that when rats, which had been given triiodothyronine and AIA at 0 time, were given the second dose of AIA at 6 hours after the first AIA, the enzyme level in these rats increased thereafter significantly. Cycloheximide, a specific inhibitor of protein synthesis (ll), inhibited the synthesis of 6aminolevulinate synthetase in any systems tested, and when cycloheximide was given to rats at 3 hours after AIA, it gave rise to a sharp drop of the enzyme level (Table II). This confirms a high rate of t,urnover of inducibly formed d-aminolevulinate synthetase (cf. ref. 9). The induction of &aminolevulinate synthetase is also completely abolished if TABLE
II
EFFECTS OF ADMINISTRATION OF ACTINOMYCIN D AND CYCLOHEXIMIDE IN THE MIDDLE OF INDUCTION OF &AMINOLEVULINATE SYNTHETASE Amounts of AIA, triiodothyronine, hydrocortisone and actinomycin D were the same as those given in Figs. 1 and 2. 5 mg cycloheximide were used per 100 g body wt. of rats. The values given in the Table are the averages of values obtained with the numbers of rats shown in brackets, respectively. -
Drugs injected at 0 time
6-Aminolevulinate SJmthetase activity at 6 hours after AM (1.nfimoles I-aminole,vulinate formed/ Ing protein/hour)
Drugs injected at 3 hours after AIA’= --
None None AIA AIA AIA AIA and triiodo, thyronine AIA and triiodo. thyronine AIA and triiodo. thyronine 0 For the values refer to Table I.
None Cycloheximide None Actinomycin Cycloheximide None Actinomycin Cycloheximide
at 3 hours
D
D
2.0 0.5 5.2 4.9 1.1 6.3
(6) (6) (16) (6) (6) (10)
6.3
(6)
1.5
(3)
of the experiments,
INDUCTION
AND
HORMONES
535
actinomycin D is injected simultaneously with AIA (cf. Fig. 1 and ref. 4 and 9). As can be seen from Table II, however, when actinomycin D was injected at not 0 time, but 3 hours after AIA, it did not appreciably enhance the enzyme decrease during the subsequent period of 3 hours. Since the turnover rate of &aminolevulinate synthetase has been evidenced to be very high, the results obtained would indicate that &aminolevulinate synthetase was being synthesized continuously even after the actinomycin injection, probably being supported by existing messenger RNA. On the other hand, we observed that when rats were killed at 4.5 hours after actinomycin D (namely 7.5 hours after AIA), the enzyme activities in these rats were less than half of those observed without actinomycin D. These results suggest that the life-time of the messenger RNA for d-aminolevulinate synthetase may be about 3 hours, in agreement with the estimation by Marver et al. (9. Similar experiments were made also with triiodothyronine-treated rats, and again it was found that actinomycin exterted practically no effect, on the rate of enzyme decrease in these rats during the 3 hours after actinomycin D (Table II). As discussed before, a “refractory phase” developes at. about 3 hours after AIA in triiodothyroninetreated rats, and in the “refractory phase” the enzyme level decreases rapidly. Considering these circumstances together, it seems likely that the rate of messenger RNA synthesis in these rats should have been reduced to an extremely low level within 3 hours after AIA. or, even if the messenger RNA was exist~ing, it might not be functioning in the “refractory phase”. EFFECT OF HORMONES THE INDUCTION IN ADRENALECTOMIZED RATS
0~
Rats were bilaterally adrenalectomized by dorsal approach under general anaesthesia with ether, and fasted for 36 hours before being used. Adrenalectomized rats were allowed to take 1% saline solution ud libifum throughout the experiments. The level of I-aminolevulinate synthetase in
536
MATSUOKA, TABLE
EFFECT
OF
HORMONES
S-AMINOLEVULINBTE
YODA,
III ON
THE
SYNTHETASE ECTOMIZED RUTS
INDUCTION IN
OF
ADRENAL-
25 mg AIA per 100 g body wt. of rats were given to each group of rat. Amounts of triiodothyronine and hydrocortisone succinate employed were 50 pg and 5 mg per 100 g body wt. of rats, respectively. Hormones were injected simultaneously with AIA. Rats were killed at 3 hours after the AIA administration. The values in the table represent the averages of values obtained with 4-10 animals tested. &Amin;~~$inate Drugs
administered
None AIA AIA plus triiodothyronine AIA plus hydrocortisone AIA plus triiodothyronine hydrocortisone
(mpmoles/m protein/hour
and
F
2 5.0 5.8 7.7 10.2
liver mitochondria was not affected by adrenalectomy and by fasting for 36 hours. After 36 hours of fasting, the rats were given 25 mg AIA per 100 g body wt. and killed for enzyme assay at 3 hours after AIA. As shown in Table III, the level of d-aminolevulinate synthetase in adrenalectomized rats was increased by more than two fold by the administration of AIA, although magnitudes of the enzyme increase in adrenalectomized rats were smaller than those in intact rats under comparable experimental conditions. The increase of 6aminolevulinate synthetase in adrenalectomized rats was more pronounced when rats were given triiodothronine or hydrocortisone simultaneously with the administration of AIA, thus confirming the results of Marver et al. (9) obtained with hydrocortisone. Hydrocortisone exerted more significant effect than triiodothyronine. When both triiodothyronine and hydrocortisone were given to adrennlectomized rats simultaneously with AIA, the 6aminolevulinate synthetase activity increased to almost the same level as observed in intact rats which were given both triiodothyronine and AIA. DISCUSSIOr\’
The experimental that triiodot#hyronine
results indicated and hydrocortisone
AND
KIKUCHI
strongly stimulated the AIA-induced synthesis of &aminolevulinate synthetase in rat-liver, while the administration of these hormones alone did not give rise to the increased synthesis of the enzyme. il closely related synergistic action of AIA and hormones appeared to be required to bring about the stimulation of the induction, although apparently AIA played a primary role in the induction of &aminolevulinate synthetase and roles of hormones are only auxilliary. In in vitro system with isolated liver mitochondria, as well as with a soluble enzyme preparation obtained from acetone powder of liver mitochondria, the B-aminolevulinate synthetase activity was not influenced by addition of various concentrations of triiodothyronine or hydrocortisone. There have been several lines of evidence that hormones may affect fairly directly RNA synthesis at the gene level (12-20). The observed hormonal stimulation of the AIA-induced synthesis of b-aminolevulinate synthetase seems to be due principally to the hormone-stimulated increase of messenger RNA synthesis. The effect of triiodothyronine on the induction of 6aminolevulinate synthetase seems to be in the similar sense as also “permissive” effect assumed for the hydrocort’isone (cf. ref. 6). It remains to be clarified, however, whether the “permissive” effects of hydrocortisone and triiodothyronine are independent or interrelated each other. The “permissive” effect does not appear to be due to effects of hormones on the level of succinyl-CoA synthetase, for we observed that the level of this enzyme was not appreciably influenced by either the combined or separate administration of hormones and AIA. Labbe et al. (21) reported that the total succinyl-Coil synthetase activity in liver from AIA-induced porphyria mouse was increased considerably and the inducible fraction of the enzyme was the isozyme of the non-inducible constitutive enzyme. However, we were unable to confirm the reported situations with respect to either mouse or rat (to be published). Hormones may also regulate protein synthesis at the RNA template (22-24). With respect to the &aminolevulinate
s-AMINOLEVULINATE
SYNTHETASE
synthetase induction, however, the contribution, if any, of the stimulation by hormones at the translation level seems to be minor, since hormones, when given 1 hour or later after the AIA administration (namely in the middle of active synthesis of the enzyme) did not significantly increase the &aminolevulinate synthetase level (cf. Figs. 3 and 4). It is noteworthy t’hat some “refractory phase” seemed to appear right after the period of intensive enzyme induction particularly in hormone-treated rats. Also in “refractory phase” the enzyme synthesis, if any, appeared to be extremely small as judged by the very rapid decline of the enzyme level in the ment,ioned period. This suggests that the amount of messenger RNA for b-aminolevulinate synthetase had been reduced to a extremely low level in “refractory phase,” or, even if the messenger RNA was existing, its function might have been inhibited in some unknown way. On the other hand, it was observed that, as shown in Fig. 1, the b-aminolevulinate synthetase level in hormone-untreated rats was maintained considerably high and fairly constant for as long as 12 hours after the single administration of AIA, while the turnover rate of the inducibly formed &aminolevulinate synthetase was evidenced t’o be very high. It is conceivable that in these rats the messenger RNA was being synthesized continuously for as long as 12 hours at nearly constant and considerably higher rates than those in control rats n-ithout AIA. These circumstances suggest the occurrence of complicated mechanisms which may participate in the regulat,ion of synthesis of &aminolevulinate synt’hetase so as to maintain a certain level of the enzyme in rat liver. Various possibilities can be considered to account for the observed “refractory phase” of the &aminolevulinate synthet,ase induction. The simplest hypothesis may be that some “inhibitor” appears as a consequence of intensive enzyme induction. One candidate for the suspected “inhibitor” may be heme. Several reports (7, 8, 2.5, 26) indicated that the synthesis of &aminolevulinate synthetase is strongly suppressed by the administration of hemin. Granick
INDUCTION
AND
HORMONES
537
(7) proposed a hypothesis assuming that the level of &aminolevulinate synthetase in liver is controlled by feedback repression in which heme may be a corepressor and that heme and an inducing chemical compete for each other on the same site on a repressor. Evidence was obtained in our laboratory that hemin might exert its inhibitory action at a step of synthesis of messenger RNA for d-aminolevulinate synthetase (26). Moreover, hemoglobin and even bilirubin were effective in inhibiting the induction of b-aminolevulinate synthetase (26), suggesting that bilirubin, too, might share the role of heme as the corepressor. Granick’s hypothesis was favorably adopted by Waxman et al. (8) and by Marver et al. (27) to interpret their experimental findings. Levere and Granick (28) further suggested that a repressor mechanism controlling the rate of synthesis of &aminolevulinate synthetase is operative in erythroid precursor cells of chick blastoderm. However, the idea of reciprocal regulation mechanism, as proposed by Granick (7) and exbended by Waxman et al. (8), seems to be not enough to account for the observed sharp decline of the enzyme level as well as the apparent “refractory phase” of the induction, for, as shown in Fig. 6, the sharp decline of the b-aminolevulinate synthetase level was not appreciably prevent’ed by the second administ’ration of AIA, although there remains an alternate possibility that the hormones may also stimulate the synthesis of enzymes which metabolize AIA. The temporary presence of high levels of such enzymes taken in combination with Granick’s mechanism would also account for the observed features of the hormonal effects upon the &aminolevulinate synthetase induction. Further studies are required t’o examine these possibilities. The second candidate for the “inhibitor” could be a repressor of protein nature which inhibits further enzyme synthesis by inhibiting the function of existing messenger RNA. This type of regulation of enzyme synthesis was first postulated by Garren et al. (29) for the hydrocortisone-stimulated synthesis of tryptophan pyrrolase (EC 1.13.1.12) and tyrosine-a!-ketoglutaratc
538
MATSUOKA,
YODA,
transaminase (EC 2.6.1.5) in rat liver, and the synthesis of this repressor was supposed to be very sensitive to actinomycin action. The work by Garren et al. (29), however, was questioned recently by Mishkin and Shore (30) who claimed to have failed to find any apparent stimulation by actinomycin D of the hydrocortisone-induced increase of tryptophan pyrrolase. We also failed to observe the stimulation by actinomycin D of the AIA-induced increase of d-aminolevulinate synthetase (cf. Table II). Thus, although the inhibitor protein theory as postulated originally by Garren el al. (29) looks very attractive as a hypothesis to account for our observations on the stimulation and inhibition of the induction of &aminolevulinate synthetase, it is premature at present to put much emphasis on this hypothesis. Naturally the artificial administration of hormones may bring about a disturbance of hormonal homeostasis in rats and this may also exert some influences on the feature of the enzyme induction. ACKNOWLEDGMENT The authors acknowledge F. HoffmannLaRoche & Co., Basle ; Kyowa Hakko Co., Tokyo; Nippon Upjohn Co., Tokyo, and Lederle Laboratories, U.S.A., for their kind supply of reagents. This investigation was supported in part by P.H.S Research Grant AM 08016-03 and 04 from the National Institute of Arthritis and Metabolic Diseases, U.S.A. (G.K). REFERENCES 1. GRANICK, S., AND URATA, G., J. Biol. Chem. 238, 821 (1963). 2. MIYAKOSHI, T., AND KIKUCHI, G., Tohoku J. Exptl. Med. 79, 199 (1963). 3. NARISAWA, K., AND KIEUCHI, G., Biochim. Biophys. Acta 99, 580 (1965). 4. NARISAWA, K., AND KIKUCHI, G., Biochim. Biophys. Acta 138, 596 (1966). 5. MARVER, H. S., TSCHUDY, D. P., PERLROTH, M. G., AND COLLINS, A., J. Biol. Chem. 241, 2803 (1966).
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