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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS -1963
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ORNU=7 3466
NOV 13 1957
Die
Gunma Symposium on Endocrinology
Volume 5
Cent-670829-01;
Hormonal Regulation of Enzyme Synthesis*
Francis T. Kenney, Darold Holten** and Chester B. Hager***
Biology Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
USA
LEGAL NOTICE
Thio report was prepared as an account of Government sponsored work. Nolther the United
States, nor the Commission, nor any person acting on behalf of the Commission:
A. Makes any warranty or representation, expressed or implied, with respect to the accu.
racy, compieteness, or usefulness of the information contained in this report, or that the use
of any information, apparatus, method, or procese disclosed in this report may not Infringe
privately owned righta; or
B. Ascumes ady llabilities with respect to lise use of, or for damages resulting from the
use of any information, apparatus, method, or process disclosed in this report.
As used in the above, "person acting on behalf of the Commission includes any em-
ployee or contractor of the Commission, or employee of such contractor, to the extent that
auch employee or contractor of the Commission, or employee of such contractor prepares,
disseminates, or provides access to, any information pursuant to do employment or contract
with the Commission, or his employment with such contractor.
LIORIBUTION OF THIS DOCUMENT IS UNLIMITED
11
FOOTNOTES
Rönearen gunported jointly by the National Cancer Institute, National
Institutes of Health, and ty the U. s. Atomic Energy Commission under
contract with Union Carbide Corporation.
American Cancer Scciety Postdoctorate Fellow, Present address:
Biochemistry Department, Armour Pharmaceutical Company, Kankakee, Illinois.
***
American Cancer Society Postdoctorate Fellow.
TEXT
Running title:
Reculation of enzyme synthesis
Send nroofs to:
Dr. Francis T. Kenney
Biology Division
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
The mechanism by which clucocorticoid hormones Act to regulate
the synthesis of specific liver proteins has been of central interest
for several years in our laboratory. It has slowly become apparent
that synthesis of the glucocorticoid-inducible tyrosine transaminase could
be altered in the absence of adrenal steroids, for the level of this
enzyme could be changed after adrenalectomy by a number of agents in an
actinomycin- and puromycin-sensitive fashion. The diversity of the agents
found to be effective (e.g., pyridoxine, benzoic acid, celite, bentonite,
amino acid mixtures) indicated that direct effects were unlikely, and
suggested the involvement of one or more non-adrenal hormones whose
circulating levels might be changed by these agents. The studies described
here show that synthesis of tyrosine trans aminase can be altered in vivo
by three protein hormones. Two of these effect an induction (i.e., elevate
the rate of enzyme synthesis) by a mechenism which is clearly distinct
from that sensitive to glucocorticoids, while the third stops synthesis
or brings about repression. Details of the experiments described can be
found in the original publicationstos.
An important feature of the model enzyme we have employed in these
analyses is the unusually rapid turnover it undergoes in vivo. Half-life
of the tyrosine transaminase of rat liver is about 1.5 Hours", in contrast
to that of the bulk of the soluble proteins which undergo turnover with
a half-life of about 3 days. Thus,7 a change in the rate of synthesis
of this transaminase 18 quickly reflected in a change in the emount of
enzyme in the liver, which can be readily detected by standard assay
techniques. However, since enzyme activity could be altered without
change in the rate of enzyme synthesis, it is essential that hormonally-
effected changes in enzyme activity be checked by direct measurement
of synthetic rate. For this we use en isotonic-immunochemical procedure,
in which the extent of incorporation of labeled amino acids into the
enzyme during a brief "pulse" is a direct (although only relative)
estimate of the rate of its synthesis. The labeled enzyme is isolated
by treatment of lịver extracts with antitransaminase serum as described
earlier8,9
When adrenalectomized rats are subjected to severe stress' or
given moderate amounts of the hypophyseal growth hormone, the hepatic
level of tyrosine transaminase falls markedly after a variable initial
phase lasting 1 or 2 hours (Figure 1). This drop in activity continues
until only the 4th or 5th hour after a single treatment, but it growth
hormone is given agair. at that time the low enzyme level is maintained.
However this cannot be maintained indefinitely, for the enzyme level
ultimately returns to the rormai "basal" level even if multiple injections
of growth hormone are given (Figure 2). During the phase in which the
enzyme level falls, the rate of decay Approximates the normal turnover
time of the enzyme (t 1/2 = 1.5 hr). This is consistent with a growth
hormone-mediated inhibition of synthesis of the enzyme, which is confirmed
(Table 1) when synthesis is measured by the Immunochemical method described
above. The isotope measurements show that synthesis is reduced essentially
to zero in one experiment and to about 20% of the normal level in a
second experiment; several other analyses yielded results falling between
these extremes. These data also show the well-known' stimulation
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of the synthesis of the bulk of the soluble liver proteins due to growth
hormone. Thus it is clear that treatment of adrenalectomized rats with
this protein hormone leads to a selective repression of the synthesis of
tyrosine transaminase, albeit a temporary one.
The important question, whether or not this reflects a direct effect
of growth hormone in the liver, prae tested in the 1solated liver perfused
by the technique of Miller et al.12,13. Addition of growth hormone to
the perfusing blood in amounts sufficient to bring about repression in vivo
had no effect on the transaminase level of the isolated liver (Figure 3).
This result suggests that the repression response in the liver 18 secondary
to an hormonal effect on some extrahepatic factor. When the blood from rats
treated with growth hormone was used in the perfusion system, there was
some indication of a possible repression, but this has not yet been examined
further.
In these repression studies it was noted that the transaminas e level
often underwent an initial increase after growth hormone treatment, and
this increase was sometimes of considerable magnitude (Figure 1). A study
of the nature of this phase of the response suggested thet it reflects a
brief increase in transaminase synthesis (induction) that could not be due to
residual glucocorticoida. Accordingly we investigated other hormones thet
might sometimes be released as a result of growth hormone treatment. Insulin
falls into this category, and when tested in adrenalectomized rate, was
found to effect a rapid increase in the level of tyrosine transaminase (Figure
4). A survey of other hormones yielded the surprising result that the insulin
antagonist, glucagon, causes an essentially identical change in this enzyme
(Figure 5). Other hormones are without effect, for which we are thankful
since the task of interpreting the regulation of synthesis of a single
enzyme by four hormones, one a steroid and the others polypeptides,
www
e
is sufficiently complex for our tastes.
-.
.
The dose of insulin required for this response (optimal, 0.8 unit/100 €)
is a reasonably physiological one, but maximal effects of glucaçon are
observed only at very high doses (optimal, 150 ve/100p). Preparations of
each of these hormones that are virtually free of the other were fully
effective, but the possibility that one of these acted secondarily by promoting
release of the other could not be excluded by experimentation in vivo. lle
turned again to the isolated, rerfused liver and found that each c? these
hormones induced enzyme synthesis in this system; the response was virtually
identical to that observed in vivo (Figure 6). In these perfusion analyses
the hormones were infused continuously throughout the experiments. Despite
the continual presence of either insulin or glucagon, the induction response
ceased after 2 to 3 hours, in sharn contrest to the hydrocortisone-mediated
induction, which continued as long as 11 hours in the same experimental system
(Figure 6). A similar difference can be seen by repeated hormone treatments
In vivo, i.e., the steroid-mediated elevation in enzyme synthesis is maintained
as long as the steroid is present, but the induction response to either insulin
Conclusive evidence for the existence of two discrete induction
mechanisms was obtained by measurements of hormonal effects on the rate of
enzyme synthesis in vivo. The effects of the various inducing hormones,
singly and in combination, were tested at an optimal time for each
hormone, i.e., a time when each can be seen (ty kinetic analyses) to
be exerting a maximal effect on synthesis. These Analyses showed (1)
that each hormone effects an initial increase in transaminase synthesis
of comparable magnitude, and (2) that the effects of the two pancreatic
hormones are not additive with each other but are additive with the
effect of hydrocortisone (Table 2). We conclude that we are observing
two induction mechanisms operating in the liver, one being sensitive to
Hydrocortisone (and other glucocorticoids) and the other to either of the
pancreatic protein hormones.
We can only speculate about the mechanisms involved in these hormonal
regulations of enzyme synthesis. The hormonal effects on enzyme synthesis
are all blocked if RNA synthesis 18 stopped by moderate doses of actinomycin
3. We interpret this to mean that the nuclear transcription apparatus must
remain essentially intact for the hormones to be effective, and we consider
it reasonable to assume that the hormones must bring about some specific
change in the operation of transcrintional events and related processes.
In sceking out a reasonable explanation for the bewildering variety of
hormonal effects on the synthesis of tyrosine transaminase, we are struck
by a similarity in the induction response to either insulin or glucagon and
the repression response to growth hormone. Roth of these responses, one
ana elevation and the other an inhibition of enzyme synthesis, are temporary
and do not continue even in the presence of the effective hormone, Now we
know that the response to growth hormone 18 a secondary one and cannot be
due to a direct action of this hormone on any component of the protein
synthesizing machinery of the liver. Although the pancreatic hormones
induce enzyme synthesis by direct action in the liver, the fact that the
response is not obligatorily linked to the presence of these hormones leads
us to suggest that they, too, act secondarily and not by direct interaction
with the components of protein synthesis. Each of these "secondary"
hormonal effectors are known to affect permeability of cells, end we may
23
assume that they could affect the properties of intracellular membranes as
well. The passage of information-bearing RNA from the nucleus to the sites
of enzyme synthesis in the cytoplasm may be blocked by actinomycin?'. A
regulation by the "secondary" hormones of the properties of this transmission
across the nuclear membrane would be consistent with our results. As the
nuclear store of transaminas e mRNA would be limited, elevated enzyme
synthesis due to the "secondary" inducers would be temporary, and synthesis
would return to the normal rate as the excess mRNA is translated and degraded.
Transaminuse synthesis could be temporarily stopped in growth hormone-
treated animals by an inhibition of this transmission.
In contrast to these "secondary" effectors, hydrocortisone promotes
an increased transaminase synthesis which continues As long as the steroid
OT
hormone is present. Thus we consider the steroid to be a "primary" inducer
in the sense that it is quite likely that this hormone does act directly
on some component of protein synthesis. Hydrocortisone brings about a
large increase in the synthesis of all types of hepatic RNA in vivo),16.
However this 'burst in general RNA synthesis appears not to be a necessary
part of induced enzyme synthesis, for in culture systems transaminase
induction proceeds without a detectable change in the overall rate of
synthesis of RNA,19. A selective hormonal effect on the synthesis of
a few species of mRNA would probably not be detected by these labeling
experiments.
Indeed, using the technique of competitive hybridization,
a qualitative change in the hybridizable fraction of nuclear RNA was found
to occur as a result of hydrocortisone treatment. While the validity
of this technique has been questioned, it remains nossible that specific
hormonally-induced changes in the mRNA have been found. The available data
are thus not inconsistent with the concept that the primary inducer,
hydrocortisone, induces enzyme synthesis by a selective stimulation of the
transcription of genetic sites coding for the enzymes being induced.
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References
1. Kenney, P.T. (1967) Regulation of tyrosine-a-ketoglutarate
trandaminane in rat zver. V. Menubandon in growth hormone tro AB
rats. J. Biol. Chem. 242, 4367 - 4371.
2. Holten, D. and Kenney, F. T. (1967) Regulation of tyrosine-a-
ketoglutarate transaminase in rat liver. VI. Induction by pancreatic
hormones. J. Biol. Chem. 242, 3272 - 4377.
3. Hager, C. B. and Kenney, F. T. Manuscript in preparation.
4. Kenney, F. T. (1967). Turnover of rat liver tyrosine transeminase :
stabilization after inhibition of protein synthesis. Science 156,
525 - 528.
5. Schimke, R. T., Brown, M. B. and Smallman, E. T. (1963) Turnover of
rat liver arginase. Ann. N. Y. Acad. Sci. 102, 587 - 601.
6. Segal, H. L. and Kim, Y. S. (1965) Environmental control of enzyme
synthesis and degradation. J. Cellular Comp. Physiol. 66, Suppl. 1,
11 - 22.
7. Berlin, C. M. and Schimke, R. T. (1965) Influence of turnover rates
on the responses of enzymes to cortisone. Molec. Pharmacol. 1, 149 - 156.
8. Kenney, F. T. (1962) Induction of tyrosine-a-ketoglutarate transaminase
in rat liver. III. Immunochemical analysis. J. Biol. Chem. 237, 1610 -1614.
9. Kenney, f. T. (1962) Induction of tyrosine-a-ketoglutarate transeminase
in rat liver. IV. Evidence for an increase in the rate of enzyme
synthesis. J. Biol. Chem. 237, 3495 - 3498.

10. Kenney, F. T. and Albritton, W. L. (1965) Repression of enzyme
synthesis at the translational level and its hormonal control.
Proc. Natl. Acad. Sci. USA. 54, 1693 - 1698.
11. Korner, A. (1965) Growth hormone effects on RNA and protein synthesis
in liver. J. Cellular Comp. Physiol. 66, Suppl. 1, 153 - 162.
Miller, L. L., Bly, C. G., Watson, M. L. and Bale, W. F. (1951)
The dominant role of the liver in plasma protein synthesis. J. Exp.
Med. 24, 431 - 453. .
13. Miller, L. L., Burke, W. T. and liaft, D. E. (1955) Studies of the
nitrogen-sparing action of carbohydrate with the 180lated perfused
rat liver. Federation. Proc. 14, 707 - 716.
14. Georgiev, G. P., Samarina, 0. P., Lerman, M. I., Smirnov, M. N. and
Severtsok, A. N. (1963) Biosynthesis of messenger and ribosomal
ribonucleic acids in the nucleochromosomal apparatus of animal cells.
Nature 200, 1291 - 1294.
15. Wicks, W. D., Greenman, D. L. and Kenney, F. T. (1965) Stimulation of
ribonucleic acid synthesis by steroid hormones. I. Transfer RNA. J.
Biol. Chem. 240, 4414 - 4419.
16. Greenman, D. L., Wicks, W. D. and Kenney, F. T. (1.965) Stimulation of
ribonucleic acid synthesis by steroid hormones. II. High molecular
weight components. J. Biol. Chem. 240, 4420 - 11426.
17. Tomkins, G. M., Thompson, E. B., Hayashi, S., Gelehrter, T., Granier,
D, and Peterkofsky, B. (1966) Tyrosine transaminase induction in
mammalian cells in tissue culture. Cola Spring Harbor Symp. Quant. Biol.
XXXI, 349 - 360.
-
-
.

T
S
18. Wicks, W. D. Induction of tyrosine-a-ketoglutarate transaminase
in fetal rat liver. Submitted to J. Biol. Chem.
19. Drews, . and Drawerman, G. (1967) Alterations in the nature of
ribonucleic acid synthesized in rat liver during regeneration and
after cortisol administration. J. Biol. Chem. 242, 801 - 808.
20. Birnboim, H. C., pene, J. J. and Darnell, J. E. (1967) Studies
on Hela cell nuclear DNA-like RNA by RNA-DNA hybridization. Proc.
Natl. Acad. Sci. U.S.A. 58, 320 - 327.
Table 1
Immunochemical analysis of repression by growth_hormone.
Data are the mean
standard orror for
animals in each experiment.
The rats were pulse-labeled with + C-Amino acids 4 hours and 3 hours
after growth hormone treatment in experiments 1 and 2, respectively.
From Kenney?.
Expt, no. Treatment
Transaminase
Activity
Radioactivity in
transaminase soluble proteins
units/me protein
сон
com/me.
None
37.7 + 2.3
18.7 + 1.2
579 $ 106
115 $ 19
803 £ 91
1187 $ 125
STI
None
51.2 £ 5.7
109 £
9
259 4 13
465 4 41
STH
25.3
2.7

Table 2
Additivity of inducing hormones
!!
Data are the mean t standard error for 2 to be animals in each
group. The rats were pulse-labeled with c-amino acids 3 hours after
hydrocortisone treatment and 1.5 hours after insulin or plucaron.
:-23:577. rzeci!T117 is the eczyze radioactivity (1 10%, divided to
the radioactivity of the total soluble proteins. Adapted from Holten
and renney?
Hormone treatment
Transaminase
activity
Pelative radioactivity
units/mg protein
None
idrocortisone (F)
Insulin (1)
Glucagon (G)
F+ I
F+G
I + 0
F + I +
31 $ 3
144 1 13
105 - 11
98 2
269 + 1.9
216 4 24
109 + 6
225 + 1
0.01 + 0.14
6.5€ + 2.5
6.86 $ 0.78
6.32 $ 0.88
14.62 € 0.84
13.71 $ 3.10
5.66 $1.0
14.32 + 1.5
Lepends to Figures
Figure 1. Repression of tyrosine transaminase by growth hormone
in vivo.
In A hypophysectomized-adrenaflectomized rats were given
sheep growth hormone (NIH:G1:87, 0.6 mg/100 g) at zero time.
The broken line indicates those given a second treatment 4
hours after the first. In B hypophysectomized rats were
given bovine growth hormone (NIH:CH:BLO, 0.72 mg/100 g). In
C adrenalectomized (pathogen-free) rats were given bovine
frowth hormone (0.2 mg/100 g) at zero time and again at 4 hours.
Each point represents an analysis on a single animal. From
Kenney?.
Figure 2. Repression after multiple injections of growth hormone.
Bovine growth hormone (NIH:61:B10, 100 ug/100 g) was given
at zero time and again at 3, 5 and 9 hours as indicated by the
arrows. Data are the mean $ standard error for 3 animals at
each point.
Figure 3. Repression studies in isolated, perfused livers.
The blood in "control" and "STI" experiments was from
adrenalectomized donors and was diluted with an equal volume
of Krebs-Ringer bicarbonate buffer containing glucose and an
growth hormone (180 ve/100 & liver donor) was added at zero time
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and an additional 100 ug was infused per hour. When the
blood was from STH-treated donors it was taken 3 hours after
a single done or 200 ve/100. Nach curve represents a single
perfusion experiment. Adapted from Hager and Kenney
Figure 4. Induction by insulin in vivo.
The rats were adrenalectomized and were given 0.75 insulin
units/100 g at zero time. They also received 2 ml of 10%
glucose at houriy intervals when necessary to combat hypoglycemic
shock. Data are the mean t standard error for 3 animals at
each point. From Holten and Kenney'.
Figure 5. Induction by glucagon in vivo.
The glucagon dose was 100 ug/100 g. Data are the meant
standard error for 3 animals at each point. From Holten and
Kenney?
Figure 6.
Induction studies in isolated, perfused livers.
The blood used was from adrenalectomized rats and was diluted
--
as in Figure 3. Hormone dosages : hydrocortisone, 2 mg/100 g
1:
-
-.
.
donor at zero time and 1 mg infused per hour; insulin, 2.5 units/
100 g donor at zero time and 1 unit infused per hour; glucagon,
300 ug/100 g donor at zero time and 125 ug infused per hour.
The pancreatic hormones were each essentially free of the other
hormones. Each curve represents a single perfusion experiment.
Adapted from Hager and Kenney.
-
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