Reprinted from ScrENcE, February 27, 1959, Vol. 129, No. 3348, pages 569-570.

Effects of Thyroxin on Amino
Acid Incorporation into Protein

Abstract. The effect of thyroxin on the
in vitro incorporation of pL-leucine-1-C™
into the protein of rat liver homogenates
has been investigated. Both thyroxin pre-
treatment in vivo and thyroxin in vitro at
a concentration of 1 x 10°°M were found
to increase the rate of amino acid incor-
poration. The increased activity following
the thyroxin pretreatment in vivo was
found to be localized in the mitochondrial
fraction. It is suggested that the accelera-
tion of metabolic rate characteristic of
thyroxin action may be secondary to the
stimulation of energy-requiring reactions
such as protein synthesis.

Recent concepts of the mechanism of
action of thyroxin have emphasized its
uncoupling effect on oxidative phospho-
rylation (1, 2). This effect, however, is
observed only with relatively high con-
centrations of thyroxin, occurs equally
well with both the p- and L- forms (/),
and, except for changes in oxidative
metabolism, explains few of the physio-
logical effects of the thyroid hormone.
Many clinical features of thyroid disease
suggest a major, if not primary, role of
the thyroid hormone in protein metabo-
lism. In immature animals it is involved
in growth; in adults it causes pronounced
changes in nitrogen metabolism. Fur-
thermore, in the adult brain and testis—
organs in which the quantities of protein
and lipid turned over per unit time are
apparently negligible compared with
turnover of carbohydrate, as evidenced
by a respiratory quotient of approxi-
mately 1 (3)—the characteristic accel-
eration of metabolic rate that is observed
in almost all other tissues is absent in
hyperthyroidism (4).

Previous observations (5) have indi-
cated that thyroxin pretreatment in vivo
stimulates amino acid uptake into the
protein of rat liver slices. To investigate
further the apparent relationship between
thyroid function and protein synthesis,
studies were undertaken to determine the
effects of in vivo and in vitro thyroxin
administration on the in vitro incorpora-
tion of pt-leucine-1-C#* into the proteins
of rat liver homogenates, Livers from
90- to 150-g fasting, male Sprague-
Dawley rats were homogenized by means
of glass homogenizers in 5 ml of 0.25M
sucrose solution per gram of tissue.
Homogenization was performed at 0°
to 2°C, and tissue fractions were main-
tained at that temperature through all
subsequent operations until final incuba-
tion. Intact cells, nuclei, and cell debris
were removed by centrifugation at 700g
for 10 minutes. The supernatant fluid
was spun at 54,000g for 60 minutes in a

cpm per mg protein per hour

 

a Mitochondria N H N H N H H N
Supernatant N H N H H N N H
Microsomes N H 85 N NH NH

Uniform Microsomes Supernatant Mitochondria
reversed reversed reversed

-- Normal (at least 2 components normal)
a Hyperthyroid (at least 2 components hyperthyroid)
Fig. 1. Localization of increased amino acid incorporating activity in fractions of liver
homogenates from rats pretreated with thyroxin. Results are representative of seven such

experiments.

Spinco model L ultracentrifuge. The
sediment, containing both mitochondrial
and microsomal fractions, was suspended
in appropriate amounts of 0.25M sucrose
and supernatant fluid to yield a suspen-
sion containing particulate fractions and
supernatant fluid equivalent to approxi-
mately 200 mg and 30 mg of liver, re-
spectively, per 0.45 ml, the quantity of
homogenate added to each of the experi-
mental flasks.

In eight experiments rats were paired
for age and weight; one received almost
daily intraperitoneal injections of 100
ug of sodium thyroxin in 1 ml of 0.01N
NaOH; the other received equivalent
amounts of the NaOH solution alone.
After at least six doses in 7 days, homog-
enates were prepared simultaneously
from both animals as described above,
and pt-leucine-1-C** incorporation activ-
ity in both was measured in parallel
flasks in a single combined experiment.
Flask contents and incubation procedure
are described in the title of Table 1. The
reaction was terminated with 12-percent
trichloroacetic acid, and the precipitated
protein was purified and plated on filter
paper by a modification of the method
of Siekevitz (6). Sample weights were
determined from difference in planchet
weights before and after plating. Radio-
activity was measured with a thin-win-
dow Geiger-Mueller counter; total
counts collected were sufficient to yield
a 3-percent coefficient of variation.
Counting rates were corrected for back-
ground, self-absorption, and zero time
controls. The results are summarized in
Table 1. Although protein nitrogen con-
centrations, as determined by the micro-
Kjeldahl technique, were identical in

Table i. Effects of thyroxin on pi-leucine-
1-C™ incorporation into protein of rat-
liver homogenates. To each flask (25-ml
Erlenmeyer) were added 5 pmole of ade-
nosine-5’-monophosphate, 20 ywmole of
potassium phosphate (pH 7.4), 5 pmole
of MgCls, 50 umole of potassium a-keto-
glutarate, 0.8 ywmole of pi-leucine-1-C*
(specific activity, 5.33 pc/uwmole), and
0.45 ml of the appropriate homogenate
prepared in 0.25M sucrose, as described in
the text. In in vitro studies, 0.022 ymole
of sodium thyroxin contained in 0.1 ml
of 0.01N NaOH was added to the experi-
mental flasks; all other flasks received
equivalent amounts of the NaOH solution
alone. The reaction mixture was brought
to a final volume of 1.7 ml with 0.25M
sucrose. Incubation in air was carried out
with shaking in a water bath at 37°C for
1 hour. Zero time controls were included
in all experiments.

 

Activity (count/min
mg of protein

Item per hr)

 

Standard

M
se error

 

Thyroxin pretreatment in vivo
(8 rat pairs)

Normal rat 29.0 +19
Hyperthyroid rat 42.3 +3.0

Difference 13.8* £3.2
Effect (%) +46

Treatment with 1.3 x 10°'M thyroxin
in vitro (7 experiments)

 

Control 26.9 +18

Thyroxin-treated 31.9 $2.3
Difference 50° t14

Effect (%) +19

* Denotes statistical significance; p <.02 (de-

termined by method of paired comparison).

Reprinted with permission by the
U. S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE

Public Health Service
both groups (2.17 mg per flask), leucine
incorporation was substantially greater
in the homogenates from thyroxin-
treated rats,

In order to localize the source of the
increased activity, mitochondria and mi-
crosomes were prepared separately from
livers of both types of animals by cen-
trifugation at 15,000g for 15 to 20
minutes and 105,000g for 60 minutes,
respectively. All possible combinations of
mitochondria, microsomes, and super-
natant derived from both homogenates
were incubated as described above. Rep-
resentative results of an experiment of
this type are illustrated graphically in
Fig. 1. It is clear that most, if not all,
of the increased activity is localized in
the mitochondrial (15,000g) fraction.

In Table 1 are also summarized the
results of seven experiments in which the
effects of 1.3 x 10°5M thyroxin added in
vitro to normal rat liver homogenates
were studied under the conditions speci-
fied. Although less pronounced, the ef-
fects were just as consistent as those ob-
served with thyroxin administration in
vivo, a stimulation occurring in every
one of the experiments. Similar effects

2

have been observed in several experi-
ments with slightly altered conditions.
The effect is erratic with more highly
concentrated homogenates and is com-
pletely climinated by doubling the Mg**
concentration. Preliminary observations
indicate that increasing graded effects
occur with thyroxin concentrations be-
tween 1x10 and 1x10*M. At 1x
10-3M, the effects of the uncoupling of
oxidative phosphorylation supersede, and
a marked inhibition of amino acid incor-
poration occurs, indicating a qualita-
tively different phenomenon.

To test the possibility that the thy-
roxin effects may be preservative rather
than stimulatory, a few short-term incu-
bations have been performed. The effects
of thyroxin pretreatment in vivo are
clearly not preservative; they are as great
during the linear period of amino acid
incorporation as at 60 minutes. The ef-
fects of thyroxin in vitro are less clear;
they are distinctly present during the
linear period but become more pro-
nounced with longer incubation.

The results of these studies (7) sug-
gest that uncoupling of oxidative phos-
phorylation is not a physiological action

of thyroxin. They support, rather, the
hypothesis that thyroxin stimulates en-
ergy-requiring processes, such as protein
synthesis, and that its characteristic ac-
celeration of oxygen consumption is sec-
ondary to the increased demand.
Louis SoxoLorr

Seymour KaurMAN
Laboratory of Clinical Science,
Laboratory of Cellular Pharmacology,
National Institute of Mental Health,
Bethesda, Maryland

References and Notes

1. G. F. Maley and H. A. Lardy, J. Biol. Chem.
204, 435 (1953).

2. F. L. Hoch and F, Lipmann, Proc. Natl. Acad.
Sci. U.S. 40, 909 (1954).

3. S. §. Kety and C. F. Schmidt, J. Clin: Invest.
27, 476 (1948); H. E. Himwich and L, H. Na-
hum, Am. J. Physiol. 88, 680 (1929).

4. L. Sokoloff, R. L. Wechsler, R. Mangold, K.
Balls, S. S. Kety, J. Clin. Invest. 32, 202
(1953); E. S. Gordon and A. E. Heming,
Endocrinology 34, 353 (1944).

5. C. H. DuToit. in A Symposium on Phosphorus
Metabolism, W. D. McElroy and B. Glass, Eds.
(Johns Hopkins Press, Baltimore, Md., 1952),
vol. 2, p. 597.

6. P. Siekevitz, J. Biol. Chem. 195, 549 (1952).

. We wish to express our appreciation to Mrs.

G. B. Deibler and Miss P. Campbell for their
outstanding technical assistance.

21 August 1958

ST