Acute effects of thyroid hormones on the production of adrenal
cAMP and corticosterone in male rats
Ming-Jae
Lo1,
Mei-Mei
Kau1,
Yen-Hao
Chen2,
Shiow-Chwen
Tsai1,
Yu-Chung
Chiao1,
Jiann-Jong
Chen1,
Charlie
Liaw1,
Chien-Chen
Lu1,
Bu-Pian
Lee1,
Si-Chih
Chen2,
Victor S.
Fang3,
Low-Tone
Ho3, and
Paulus S.
Wang1
1 Department of Physiology,
National Yang-Ming University, Taipei 112;
2 Department of Biology,
Fu-Jen Catholic University, Taipei 242; and
3 Department of Medical Research
and Education, Veterans General Hospital-Taipei, Taipei 112, Taiwan, Republic of China
 |
ABSTRACT |
The acute effects of
thyroid hormones on glucocorticoid secretion were studied. Venous blood
samples were collected from male rats after they received intravenous
3,5,3'-triiodothyronine
(T3) or thyroxine
(T4). Zona
fasciculata-reticularis (ZFR) cells were treated with
adrenocorticotropic hormone (ACTH),
T3,
T4, ACTH plus
T3, or ACTH plus
T4 at 37°C for 2 h.
Corticosterone concentrations in plasma and cell media, and also
adenosine 3',5'-cyclic monophosphate (cAMP) production in
ZFR cells in the presence of 3-isobutyl-1-methylxanthine, were
determined. The effects of thyroid hormones on the activities of
steroidogenic enzymes of ZFR cells were measured by the amounts of
intermediate steroidal products separated by thin-layer chromatography. Administration of T3 and
T4 suppressed the basal and the
ACTH-stimulated levels of plasma corticosterone. In ZFR cells, both
thyroid hormones inhibited ACTH-stimulated corticosterone secretion,
but the basal corticosterone was inhibited only with
T3
>10
10 M or
T4
>10
8 M. Likewise,
T3 or
T4 at
10
7 M inhibited the basal-
and ACTH-stimulated levels of intracellular cAMP. Physiological doses
of T3 and
T4 decreased the activities of
3
-hydroxysteroid dehydrogenase, 21-hydroxylase, and
11
-hydroxylase. These results suggest that thyroid hormones
counteract ACTH in adrenal steroidogenesis through their inhibition of
cAMP production in ZFR cells.
3,5,3'-triiodothyronine; thyroxine; zona
fasciculata-reticularis cells; P450c11 activity; adenosine
3',5'-cyclic monophosphate
 |
INTRODUCTION |
PHYSICIANS AND PHYSIOLOGISTS have long hypothesized
connections between hypothyroidism and adrenocortical dysfunction. The interaction of pituitary-thyroid and pituitary-adrenal functions has
been studied, and the influences of thyroid hormones on adrenocortical function have been demonstrated (5, 29, 34). Chronic administration of
3,5,3'-triiodothyronine
(T3) at high concentrations (40 µg; 3-36 days) in male rats increases plasma and adrenal
corticosterone, as well as the induction of hypertrophy, in the gland
(7). Nevertheless, opposite results have been found in experiments in
which administration of physiological
T3 at 8 µg significantly depressed plasma and adrenal corticosterone levels during a 36-day interval (7). Plasma corticosterone and pituitary adrenocorticotropic hormone (ACTH) concentrations may remain normal in rats given T3 (15). The in vitro production
of adrenal corticoids remains unchanged after thyroglobulin feeding
(34). ACTH-induced increases in plasma-free corticoids are exaggerated
in hyperthyroid rats (15, 34), but this may be accounted for by the
reduction in volume of corticosterone distribution to peripheral
tissues (34). Hypothyroid males have higher 24-h mean serum
concentrations of total plasma cortisol in the normal circadian
rhythmicity and cortisol production rate, with no change in serum
cortisol-binding globulin concentrations compared with normal subjects
(12).
It has been shown that thyroid hormones modulate adenylate cyclase
activity in rat liver and heart (35), as well as human adipocytes (37)
and luteinized granulosa cells (10). Rubio et al. (28) found that in
brown adipose tissue the
1,2-adrenergic receptor number
and capacity to generate adenosine 3',5'-cyclic monophosphate (cAMP) are reduced in hypothyroidism. Neri et al. (23)
indicated that thyrotropin-releasing hormone markedly inhibits glucocorticoid secretion of rat adrenocortical cells, which selectively impairs the late steps of corticosterone synthesis (i.e., 11- and
18-hydroxylation).
Because of the conflicting results of previous studies regarding the
role of thyroid hormones on adrenocortical function, as well as the
lack of data on thyroid hormone regulation of adrenocortical function
via cAMP production and steroidogenesis enzyme activity, the present
study was designed to evaluate 1)
the acute effects of thyroid hormones on the secretion of
corticosterone both in vivo and in vitro;
2) the possible positive correlation
between corticosterone and cAMP production under the influence of
thyroid hormones; and 3) the
possible correlation between corticosterone secretion and
postpregnenolone steroid enzyme activity under the influence of thyroid
hormones.
 |
MATERIALS AND METHODS |
Animals.
Male Sprague-Dawley rats weighing 300-350 g were housed in a
temperature-controlled room (22 ± 1°C) with 14 h of artificial illumination daily (0600-2000). Food and water were given ad
libitum. All animal experimentation has been conducted humanely and in conformance with the policy statement of the Committee of National Yang-Ming University.
In vivo experiments: effects of a single injection of thyroid
hormones.
All rats were anesthetized with ether and catheterized via the right
jugular vein (38). They were injected 20 h after the catheterization
with saline, T3 (5 µg · ml
1 · kg
body wt
1, Sigma Chemical,
St. Louis, MO), thyroxine (T4; 20 µg · ml
1 · kg
body wt
1; Sigma Chemical),
ACTH (5 µg · ml
1 · kg
body wt
1), ACTH plus
T3, or ACTH plus
T4. Blood samples (0.3 ml each) were collected from the jugular catheter 0, 30, 60, 90, 120, and 180 min after the challenge between 0800 and 1200. The lost blood volume
was replenished with heparinized saline immediately after each
bleeding.
Plasma was separated by centrifugation at 10,000 g for 1 min and stored at
20°C. The concentrations of total
T3 and total T4 in rat plasma were measured by
radioimmunoassay (RIA). To measure corticosterone, 0.1 ml plasma was
mixed with 1 ml diethyl ether (10 × vol), shaken for 20 min,
centrifuged at 1,000 g for 5 min, and
then quickly frozen in a mixture of acetone and dry ice. The organic
phase was collected, dried, and reconstituted in a buffer solution
[0.1% gelatin in phosphate-buffered saline (PBS), pH 7.5]
before the concentration of corticosterone was measured by RIA.
Preparation of zona fasciculata-reticularis cells for cell culture.
An adrenocortical preparation enriched with zona
fasciculata-reticularis (ZFR) cells for culture was performed following
a method described by Purdy et al. (26) in 1991 with minor
modifications. Male Sprague-Dawley rats were decapitated. The adrenal
glands were rapidly excised and stored in an ice-cold 0.9% NaCl
solution. The adipose tissues were removed. The encapsulating glands
were separated into capsule (mainly zona glomerulosa) and inner zone (mainly ZFR) fractions with forceps. The fractions of inner zone from
10-20 adrenals were incubated with collagenase (2 mg/ml, Sigma
Chemical) at 37°C in a shaking water bath, 100-110
strokes/min, for 60 min. The collagenase was dissolved in 2-4 ml
of Krebs-Ringer bicarbonate buffer (3.6 mmol K+/l, 11.1 mmol glucose/l)
with 0.2% bovine serum albumin (BSA) medium (KRBGA), pH 7.4. ZFR cells
were dispersed by repeated pipetting and filtered through a nylon mesh. After centrifugation at 200 g for 10 min, the cells were washed in KRBGA medium and centrifuged again.
Erythrocytes were eliminated from ZFR cells by washing with 4.5 ml
distilled water for a few seconds. The ZFR cells were then mixed with
0.5 ml of 10× Hanks' balanced salt solution (pH 7.4). After
centrifugation at 200 g for 10 min,
the supernatant was discarded, and the pellet was resuspended in 3 ml
of KRBGA solution. An aliquot (20 µl) was used for cell counting in a
hemocytometer after staining with 0.05% nigrasin stain. Cells in
culture medium were further diluted to a concentration of 5-10 × 104 cells/ml and divided
into the test tubes.
In vitro experiments.
The ZFR cells were incubated with or without hormones dissolved in 1 ml/tube of KRBGA medium for 120 min at 37°C under 95% O2-5%
CO2. To measure the effects of
T3 or
T4 on the 11
-hydroxylase activity, ZFR cells were incubated for 60 min in KRBGA medium. After
preincubation, the cells were incubated in tubes containing 0.5 ml
deoxycorticosterone (DOC,
10
8 M, Sigma Chemical) in
the presence or absence of T3
(10
11-10
9
M) or T4
(10
9-10
7
M). For studying the in vitro effect of hormones on adenylyl cyclase
and the accumulation of cAMP, cells were incubated for 60 min with a
medium containing forskolin
(10
6 M) or 0.5 mM
3-isobutyl-1-methylxanthine (IBMX). After cells were primed with
forskolin or IBMX, they were incubated for 120 min in tubes containing
0.5 ml KRBGA in the presence or absence of hormones, such as
ACTH-(1
24) (10
8 M, Sigma
Chemical), T3
(10
11-10
7
M), T4
(10
10-10
7
M), ACTH plus T3, or ACTH plus
T4. At the end of the incubation period, the concentration of corticosterone in cultured media was
measured by RIA. Cells were homogenized in 500 µl of 65% ice-cold ethanol by polytron (PT-3000, Kinematica, Lucerne, Switzerland) and
centrifuged at 200 g for 10 min. The
supernatants of the cell extracts and cultured media were lyophilized
in a vacuum concentrator (SpeedVac, Savant) and reconstituted with an
assay buffer (0.05 M sodium acetate buffer with 0.01% azide, pH 6.2)
before the concentration of cAMP was measured by RIA.
RIA of total T3 and total
T4.
Plasma of total T3 and total
T4 was determined by RIA using the
kits provided by Amersham International, Buckinghamshire, UK.
RIA of corticosterone.
An antiserum to the corticosterone was generated by immunizing rabbits
with 4-pregnen-11
,21-diol-3,20-dione 3-carbozymethyloxime-BSA conjugate (Steraloids). With this antiserum (PSW4-9) an RIA was established for the measurement of plasma corticosterone levels. In
this RIA system, a known amount of unlabeled corticosterone, an aliquot
of plasma extract, or media samples adjusted to a total volume of 0.2 ml by a buffer solution (0.1% gelatin-PBS, pH 7.5) were incubated with
0.1 ml corticosterone antiserum (1:16,000 dilution) diluted with 0.1%
gelatin-PBS and 0.1 ml
[3H]corticosterone
[~8,000 counts/min (cpm); Amersham International] at
4°C for 24 h. Duplicate standard curves with 6 points ranging from
2.5 to 1,200 pg of corticosterone were included in each assay. An
adequate amount (0.2 ml) of 0.25% dextran-coated charcoal (Sigma Chemical) was then added with further incubation in an ice bath for 15 min. At the end of the incubation period, the assay tubes were
centrifuged at 1,000 g for 15 min. The
supernatant was mixed with 3 ml of liquid scintillation fluid (Ready
Safe, Beckman) before the radioactivity was counted in an automatic
beta counter (Wallac 1409, Pharmacia, Turku, Finland). The
sensitivity of corticosterone RIA was 5 pg/assay tube. The inhibition
curves produced by ether-extracted rat plasma and the incubation medium
of rat adrenal glands were parallel to the curve of unlabeled
corticosterone (Fig. 1). The cross-reactivities were 12% with 11-DOC, 1% with
11-dehydrocorticosterone, 0.3% with aldosterone, and <0.2% with
18-hydroxydeoxycorticosterone, progesterone, estradiol, and
testosterone. The intra- and interassay coefficients of variation were
3.3% (n = 5) and 9.2%
(n = 4), respectively.

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Fig. 1.
Dose-response curve for corticosterone standard, incubation medium of
zona fasciculata-reticularis (ZFR) cells, and extract of rat plasma
after log-logit transformation. B/Bo, ratio of binding of
[3H]corticosterone in the presence of unlabeled
corticosterone to maximal binding of [3H]corticosterone
with anticorticosterone antibody.
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RIA of cAMP.
The concentration of adrenal cAMP was determined by RIA as described
elsewhere (17, 36). With the anti-cAMP serum no. CV-27 pool, the
sensitivity of cAMP was 2 fmol/assay tube. The intra- and interassay
coefficients of variability were 6.9%
(n = 5) and 11.9%
(n = 5), respectively.
Activities of 3
-hydroxysteroid dehydrogenase,
21-hydroxylase, and 11
-hydroxylase.
ZFR cells (1 × 105 per tube)
were preincubated for 60 min at 37°C in 95%
O2-5%
CO2 in 1 ml KRBGA medium. After
centrifugation at 200 g for 10 min,
the supernatant was discarded, and the cells were incubated for 60 min
in tubes in 0.2 ml KRBGA containing pregnenolone
(10
9 M) and
[3H]pregnenolone
(8,000-10,000 cpm, 4.5~5.0 pmol, NEN-Du Pont) or DOC
(10
9 M) and
[14C]DOC
(18,000-20,000 cpm, 1.8-2.0 nmol; NEN-Du Pont) in the
presence or absence of hormones, such as
T3
(10
11-10
9
M) or T4
(10
9-10
7
M). At the end of incubation, the medium containing radioactive products was removed from cultures by centrifugation at 200 g for 10 min. The media were extracted
with 5 volumes of diethyl ether, shaken for 30 min, centrifuged at 200 g for 3 min, and then quickly frozen
in a mixture of acetone and dry ice. The organic phase was collected,
dried, and reconstituted in 100% ethanol. Aliquots of 50 µl of each
sample and 5 µl of unlabeled carrier steroids (1 mg/ml) were spotted
on silica gel G sheets containing a fluorescent indicator
(Macherey-Nagel, Düren, Germany) and chromatographed
in a carbon tetrachloride-acetone (4:1, vol/vol) solution. The sheets
were dried, and steroid-containing spots were located under ultraviolet
light. The Rf
values were as follows: progesterone = 0.95; DOC = 0.7;
corticosterone = 0.3. The spots were cut off and transferred into vials
containing 1 ml of liquid scintillation fluid (Ready Safe, Beckman)
before the radioactivity was counted using an automatic beta counter
(Wallac 1409, Pharmacia). The recovery of
[14C]DOC after ether
extraction and thin-layer chromatography (TLC) was 60%.
The activity of 11
-hydroxylase was defined as the ratio of
[14C]corticosterone
and [14C]DOC in the
medium samples after incubation of ZFR cells with [14C]DOC for 60 min.
In the experiment of the incubation of ZFR cells with
[3H]pregnenolone, the
activities of 3
-hydroxysteroid dehydrogenase (3
-HSD), 21-hydroxylase, and 11
-hydroxylase were expressed as the
radioactivities of
[3H]progesterone,
[3H]DOC, and
[3H]corticosterone,
respectively. The recovery of
[3H]corticosterone
after ether extraction and TLC was 54%.
Statistical analysis.
The treatment means of both in vivo and in vitro studies were tested
for homogeneity using analysis of variance (ANOVA), and the difference
between specific means was tested for significance using Duncan's
multiple range test (33). A difference between two means was considered
statistically significant when P was <0.05.
 |
RESULTS |
Effects of intravenous injection of
T3 and T4 on
plasma total T3 and total
T4.
A single intravenous injection of
T3 or
T4 increased plasma concentrations
of T3 (35-fold) or
T4 (9-fold) at 30 min after injection compared with the basal level in the same group
(P < 0.01; Fig.
2, top and
bottom). The levels of plasma
T3 or
T4 in T3- or
T4-injected rats increased
significantly from 30 to 180 min after injection compared with the
saline-injected animals (P < 0.01),
respectively (Fig. 2, top and
bottom).

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Fig. 2.
Effects (means ± SE) of a single iv injection of
3,5,3'-triiodothyronine
(T3), adrenocorticotropic
hormone (ACTH), or ACTH plus T3 on
concentration of plasma total T3
(top), and those of thyroxin
(T4), ACTH, or ACTH plus
T4 on concentration of plasma
total T4
(bottom). Male rats were iv injected
with saline, T3 (5 µg/kg),
T4 (20 µg/kg), ACTH (5 µg/kg),
ACTH plus T3, or ACTH plus
T4 via the right jugular vein.
Blood samples were collected through a jugular catheter at times
indicated. ** P < 0.01 vs.
saline-injected rats. +, ++ P < 0.05, P < 0.01 vs. value at 0 min, respectively.
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ACTH plus T3 or ACTH plus
T4 significantly increased plasma
T3 or
T4 concentration at 30, 60, 120, and/or 180 min after injection compared with the corresponding
basal levels in the same group (P < 0.01; Fig. 2, top and
bottom). After injection of ACTH
plus T3 or ACTH plus
T4, plasma
T3 or
T4 concentrations from 30 to 180 min were significantly higher than those in ACTH-injected animals
(P < 0.01; Fig. 2,
top and
bottom).
Effects of intravenous injection of T3
and T4 on plasma corticosterone.
A single intravenous injection of
T3 significantly decreased plasma
corticosterone at 30, 120, and 180 min after injection compared with
the basal level in the same group (P < 0.01; Fig. 3,
top). Three hours after injection of
T3, plasma corticosterone diminished significantly compared with the saline-injected animals (P < 0.01; Fig. 3,
top).

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Fig. 3.
Effects (means ± SE) of a single iv injection of
T3 on concentration of plasma
corticosterone (top) and on response
of plasma corticosterone to ACTH
(bottom). Male rats were iv injected
with saline, T3 (5 µg/kg), ACTH
(5 µg/kg), or ACTH plus T3 via
the right jugular vein. Blood samples were collected through a jugular
catheter at times indicated.
** P < 0.01 vs.
saline-injected rats. ++ P < 0.01 vs. value at 0 min. #,
## P < 0.05 and
P < 0.01 vs. ACTH-injected rats,
respectively.
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Thirty minutes after a single injection of ACTH, the plasma
corticosterone levels responded with a 4.3-fold increase (from 20.6 ± 3.6 to 90.5 ± 8.4 ng/ml; Fig. 3,
bottom). Administration of both ACTH
and T3 significantly reduced
(P < 0.05 or
P < 0.01) the corticosterone
response between 30 and 120 min after injection compared with the
ACTH-stimulated group (Fig. 3,
bottom).
One hundred eighty minutes after intravenous injection of
T4, the plasma corticosterone
levels responded with a 2.5-fold decrease (from 17 ± 1.9 to 6.7 ± 1.4 ng/ml; Fig. 4,
top) compared with the basal level
in the same group. A single intravenous injection of
T4 significantly decreased plasma
corticosterone at 120 and 180 min after injection compared with the
saline-injected group (P < 0.05 or
P < 0.01; Fig. 4,
top).

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Fig. 4.
Effects (means ± SE) of a single iv injection of
T4 on the concentration of plasma
corticosterone (top) and on response
of plasma corticosterone to ACTH
(bottom). Male rats were iv injected
with saline, T4 (20 µg/kg), ACTH
(5 µg/kg), or ACTH plus T4 via
the right jugular vein. Blood samples were collected through a jugular
catheter at times indicated. *,
** P < 0.05 and P < 0.01 vs. saline-injected rats, respectively.
++ P < 0.01 vs. value at 0 min. ## P < 0.01 vs.
ACTH-injected rats.
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From 30 to 90 min after injection of ACTH plus
T4, significantly diminished
plasma corticosterone was noted compared with the level of the group
treated with ACTH alone (P < 0.05 or
P < 0.01; Fig. 4,
bottom).
Effects of T3 and
T4 on the release of corticosterone in
vitro.
ACTH stimulated the production of corticosterone for 120 min in ZFR
cells in a dose-dependent manner (Fig. 5).
The increase was already significant (6.6-fold) at a dose of
10
10 M and reached an
~15.5-fold increase at a dose of
10
8 M.

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Fig. 5.
Effects (means ± SE) of ACTH
(10 10-10 8
M) with 5 × 104 ZFR
cells/tube for 2 h on corticosterone production in male rats.
** P < 0.01 vs. ACTH = 0 M.
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Incubation of either T3
(10
9-10
7
M), T4
(10
7 M) alone, or
T3 or
T4 in combination with ACTH
(10
8 M) significantly
(P < 0.01) decreased the release of
corticosterone from ZFR cells compared with the vehicle or ACTH-treated
groups, respectively (Fig. 6).

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Fig. 6.
Effects (means ± SE) of T3
(10 10-10 7
M) and T4
(10 10-10 7
M) on basal and ACTH-stimulated release of corticosterone from ZFR
cells in vitro. ** P < 0.01 vs. control. ++ P < 0.01 vs.
ACTH = 0 M.
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Forskolin (10
6 M) caused a
3.5-fold rise in corticosterone production. Administration of
T3
(10
11-10
9
M) or T4
(10
8 M,
10
7 M) significantly
lowered the forskolin-stimulated production of corticosterone in ZFR
cells (Fig. 7).

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Fig. 7.
Effects (means ± SE) of T3
(10 11-10 9
M) and T4
(10 9-10 7
M) on deoxycorticosterone (DOC)- and forskolin-stimulated release of
corticosterone from ZFR cells for 2 h.
## P < 0.01 vs. control.
*,** P < 0.05 and
P < 0.01 vs. DOC = 10 8 M, respectively.
++ P < 0.01 vs. forskolin = 10 6 M.
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Administration of ZFR cells for 120 min by DOC
(10
8 M) in combination with
T3
(10
9 M) or
T4
(10
9-10
7
M) significantly (P < 0.05 or
P < 0.01) decreased the release of
corticosterone compared with the DOC-treated group (Fig. 7).
Effects of T3 and
T4 on the in vitro production of cAMP in
response to IBMX.
The levels of extracellular (i.e., medium) and intracellular (i.e.,
cell) cAMP after incubation of rat ZFR cells with 0.5 mM IBMX are
illustrated in Fig. 8.
T3 and
T4 did not alter the basal levels
of extracellular cAMP (Fig. 8, top).
T3 and
T4 at 10
7 M decreased the basal
levels of intracellular cAMP (Fig. 8,
bottom) and the stimulatory effect
of ACTH on the levels of both extra- and intracellular cAMP. Low doses
of T3
(10
9 and
10
8 M) or
T4
(10
8 M) did not alter the
basal levels but attenuated the ACTH-stimulated levels of both extra-
and intracellular cAMP.

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Fig. 8.
Effects (means ± SE) of T3
(10 9-10 7
M) and T4
(10 9-10 7
M) on basal and ACTH (10 8
M)-stimulated levels of extracellular
(top) and intracellular
(bottom) cAMP after incubation of
rat ZFR cells with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). *,
** P < 0.05 and
P < 0.01 vs. control. +,
++ P < 0.05 and
P < 0.01 vs. group treated with IBMX
alone. Please note log scale on
y-axis.
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Effects of T3 and
T4 on the activities of
3
-HSD, 21-hydroxylase, and
11
-hydroxylase.
Incubation of both T3
(10
10 M or
10
9 M) and
T4
(10
9-10
7
M) in combination with DOC
(10
9 M) and
[14C]DOC (1.8~2.0
nmol) for 60 min markedly decreased 11
-hydroxylase activity
(expressed as the ratio of
[14C]corticosterone/[14C]DOC)
from 22 to 63% compared with the control group (Fig.
9).

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Fig. 9.
Effects (means ± SE) of T3
(10 11-10 9
M) and T4
(10 9-10 7
M) on activity of 11 -hydroxylase in rat ZFR cells for 1 h. Cells
were incubated with 200 µl DOC
(10 9 M) and
[14C]DOC (1.8~2.0
nmol) in the presence or absence of
T3 or
T4. Radioactive products in the
medium were extracted with ether and then analyzed by thin-layer
chromatography (TLC). ** P < 0.01 vs. control.
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Administration of ZFR cells for 60 min with
T3
(10
10 M) or
T4
(10
8 M) in combination with
pregnenolone (10
9 M) and
[3H]pregnenolone
(4.5~5.0 pmol) resulted in a decline [between 49 and 66% in
3
-HSD activity (Fig. 10,
top) and 28-30% in both
3
-HSD and 21-hydroxylase activities (Fig. 10,
middle)].

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Fig. 10.
Effects (means ± SE) of T3
(10 10 M) and
T4
(10 8 M) on the activities
of 3 -hydroxysteroid dehydrogenase (3 -HSD,
top), 21-hydroxylase
(middle), and 11 -hydroxylase
(bottom) in rat ZFR cells. Cells
were incubated with 200 µl pregnenolone
(10 9 M) and
[3H]pregnenolone
(4.5~5.0 pmol) in the presence or absence of
T3 or
T4 for 1 h. Radioactive products
in the medium were extracted with ether and then analyzed by TLC.
** P < 0.01 vs. control.
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T3 and
T4 caused about 14% inhibition
(P < 0.01) in 3
-HSD,
21-hydroxylase, and 11
-hydroxylase activities (Fig. 10,
bottom).
 |
DISCUSSION |
It has been demonstrated that chronic administration of
T3 in low doses (8 µg) decreases
the levels of plasma and adrenal corticosterone (7). Our data indicated
that a single intravenous injection of
T3 or
T4 decreased the level of plasma
corticosterone at 180 min compared with both the saline-injected group
and the basal level in the same group (Figs. 3 and 4,
top). Furthermore, the
administration of T3 or
T4 reduced the stimulatory effect of ACTH on corticosterone secretion. These results reflect that the
thyroid hormones, T3 and
T4, exert an acute inhibitory
effect on both the basal and the ACTH-induced secretion of
corticosterone. These data are in agreement with the observation by
Iranmanesh et al. (12) and Boler and Moore (4), but not with the
findings by Sanchez-Franco et al. (29) in propylthiouracil
(PTU)-treated rats. The reasons for these differences are unclear.
Because the plasma ACTH remained unchanged in the PTU-treated rats and
no data regarding T4 replacement
in PTU-induced hypothyroid rats were presented (29), we suspect that
the reduction of plasma corticosterone may be due to the toxic effect
of PTU on the adrenal gland. Recently, we found that the administration
of PTU (1 mg/ml) inhibited both basal and ACTH-stimulated production of
corticosterone in rat ZFR cells by 57 and 94%, respectively
(unpublished data).
Although high post-ACTH levels of plasma corticosteroid found in
thyroid-treated animals compared with controls have been attributed to
a reduction in the volume of distribution of corticosterone in the
thyroid-treated animals (34), our results indicated that the thyroid
hormones exert an acute inhibitory effect on the basal and the
ACTH-stimulated secretion of corticosterone. The disagreement between
past and present findings might be due to the different methods for
hormone measurement (the previous fluorometric assay vs. the present
RIA) and/or the duration for observations (the previous chronic
vs. the present acute). Chronic administration of
T3 at high concentrations
(25-40 µg) in rats induces an increase in plasma corticosterone
and adrenal hypertrophy, indicating intense stimulation of adrenal
cortical function in chronic, severe hyperthyroidism (7). It has been
reported that the effect of in vivo
T4 on adrenocortical secretion is
related to the duration of treatment (4). Thyroid hormones have also
been proposed to play a role in the maintenance of biological rhythms
(32). It has been found that the amplitude of the circadian rhythm of
blood corticosterone levels gradually decreases with time after
thyroidectomy, and daily treatment with
T3 or
T4 for 2 wk restores the amplitude of the circadian adrenocortical rhythm to prethyroidectomized levels
(22). Moreover, the plasma corticosterone response to corticotropin-releasing hormone (CRH) stimulation is increased, even
though the response to ACTH is decreased, in rats administered chronically with pharmacological doses of
T4 compared with euthyroid rats
(14). These results reflect the fact that chronic deficiency or
administration of thyroid hormones causes a complicated effect on the
hypothalamus-pituitary-adrenal (HPA) axis.
It has been known that PTU-induced hypothyroidism causes a significant
reduction in CRH gene transcripts in the paraventricular nucleus and
reduces both anterior pituitary proopiomelanocortin expression and
circulating corticosterone in the rat (30). The circulating levels of
thyroid hormones have a major effect on the central regulation of the
HPA axis (30). Our data indicate that acute administration of
T3 or
T4 evokes an inhibitory rather than a stimulatory effect on corticosterone secretion. In humans, hypercortisolemia in primary hypothyroidism has been attributed to the
decreased metabolic clearance rate (MCR) of cortisol (12). The
prolonged half-life of endogenously secreted cortisol shown in
hypothyroid subjects is consistent with the decreased disappearance rates of exogenously administered labeled cortisol in hypothyroid subjects (2, 11, 39). It has been shown that the MCR of cortisol is
increased in hyperthyroid males (9) and decreased in hypothyroid males
(12). Therefore, a rapid clearance rate provides one explanation for
the suppression of total plasma corticosterone concentrations observed
in our T3- and
T4-injected rats.
It has been shown that feeding of thyroglobulin suppresses adrenal
corticoid production in vitro in ACTH-maintained hypophysectomized rats
(34). The present in vitro data provide evidence that
T3 and
T4 diminish the release of rat
corticosterone by acting directly on the adrenal ZFR cells (Fig. 6).
These findings are in agreement with the observations by Moore and
Callas (19), who found that drastic mitochondrial alterations
characterized the zona fasciculata of hyperthyroid rats, suggesting
that thyroid hormones may act directly on adrenal fasciculata cells.
Boler and Moore (4) observed that the suppression of adrenocortical
steroidogenesis produced by thyroid hormone is related to a
mitochondrial effect, although the mechanism is unknown. The inhibition
of postpregnenolone steroidogenic enzymes in response to
T3 and
T4 (Figs. 9 and 10) reflects a
strong correlation between decreased steroidogenesis (including
3
-HSD, 21-hydroxylase, and 11
-hydroxylase activities) and the
inhibition of corticosterone production in ZFR cells after administration of T3 or
T4.
It has been shown that the rat genome contains four
P450c11 genes (CYP11
1,
CYP11
2, CYP11
3, CYP11
4). One of these (CYP11
1) encodes
P450c11
, which is the steroid
11
-hydroxylase found solely in ZFR cells and is responsible for the
conversion of 11-DOC to corticosterone (18). However, the regulation of
P450c11
1 mRNA expression by
thyroid hormones in rats is not known. Our results indicated that
T3 and
T4 inhibit the stimulatory effect of DOC on corticosterone release (Fig. 7) and the 11
-hydroxylase activity (Figs. 9 and 10) in ZFR cells. During the last decade, the
specific T3 receptors have been
identified in rat FRTL 5 thyroid follicular cells (1), in anterior
pituitary GH cells (6), and in human luteinized granulosa cells (10).
Although the thyroid hormone receptor has not been identified in rat
adrenocortical cells, it is probable that
T3 or
T4 acts on the ZFR cells via specific thyroid hormone receptors.
Simonian demonstrated that T3
alone had no effect on 3
-HSD activity in human fetal adrenal cell
cultures for 48 h (31). However, treatment with maximal concentrations
of 10 nM ACTH plus 1 nM T3
increased the 3
-HSD activity an additional 59-115% over that
for ACTH alone (31). Our results indicated that thyroid hormones alone
inhibit the 3
-HSD activity of adrenocortical cells in the adult rat.
The reasons for the different observations are not known at the present
time, but they may be attributable to development, species, or
treatment duration. It has been shown that use of adrenal homogenates
or mitochrondria from euthyroid animals indicates that
T4 also suppresses some phases of
corticoid conversion (13) and inhibits 11
-hydroxylation (25) and the adrenal transhydrogenase enzyme (24). It has been reported that thyroidectomy for 8 wk slowed down the activity of microsomal 21-hydroxylase and mitochondrial 11
-hydroxylase by ~30% (3). The
absence of thyroid hormone may decrease the transmembrane gradient of
the H ions that drive ATP and NADPH synthesis, which are both coupled
to electron transport chain function (8). This observation, however,
was not in agreement with the results by Freedland and Murad (8), which
showed a significant increase in mitochondrial malic enzyme activity
after in vitro T3 administration. The direct effect of thyroid hormones on adult rat ZFR cells and the
activities of steroidogenic enzymes have not been previously investigated. We found a marked inhibitory effect of
T3 and
T4 on postpregnenolone
steroidogenic enzymes, including 3
-HSD, 21-hydroxylase, and
11
-hydroxylase activities. These findings are in agreement with the
results reported by Peron et al. (25).
ACTH regulates glucocorticoid production by acting on specific
receptors in the adrenal cortex. The number of ACTH binding sites in
adrenocortical cells is increased by exposure of these cells to the
activators of the cAMP pathway, e.g., dibutyryl cAMP or forskolin (20).
In the present study, we found that the stimulatory effects of ACTH on
both plasma corticosterone and corticosterone production in vitro were
diminished by T3 and
T4.
T3 and
T4 attenuated the stimulatory
effects of corticosterone release in ZFR cells by adenylyl cyclase
agonist and forskolin and decreased the stimulatory effects of ACTH on
cAMP production, indicating that cAMP mediates this regulatory
mechanism. Because T4 at
10
9 M inhibited
ACTH-induced release of corticosterone (Fig. 6) but did not alter the
level of extracellular cAMP (Fig. 8), we suggest that the cAMP response
element was not the only pathway of the inhibition of corticosterone
production by thyroid hormones. ACTH receptor genomic DNA has been
isolated in the human (21), bovine (27), and mouse (16). Whether
thyroid hormones alter the gene expression of the ACTH receptor in rats
is not known but is worth investigating.
In summary, these findings suggest that acute administration of thyroid
hormones 1) inhibits the secretion
of corticosterone, both in vivo and in vitro;
2) attenuates the stimulatory
effects of ACTH on the secretion of corticosterone via a decrease of
cAMP production in ZFR cells; and 3)
decreases the activities of 3
-HSD, 21-hydroxylase, and
11
-hydroxylase in ZFR cells. These results may contribute to the
characterization of the regulatory mechanisms of adrenocortical
function by thyroid hormones. Although the in vitro effect of thyroid
hormones is fast, whether the inhibition of thyroid hormones on
steroidogenesis in ZFR cells is mediated by nuclear receptor mechanisms
is not clear at the present time. Furthermore, the inhibitory effects
of T3 and
T4 on corticosterone secretion
might be of interest in the therapy of patients with hypercortisolemia
caused by primary hypothyroidism.
 |
ACKNOWLEDGEMENTS |
The authors greatly appreciate Dr. C. Weaver's English editing.
 |
FOOTNOTES |
This study was supported by Grant NRICM-85104 from the National
Research Institute of Chinese Medicine; Grant VGHYM- 86-S4-19 from the VGH-NYMU Joint Research Program, Tsou's Foundation, ROC; a
grant from the Veterans General Hospital-Taipei; NSC
86-2314-B-010-074 from the National Science Council; and an
award from the Medical Research and Advancement Foundation in memory of
Dr. Chi-Shuen Tsou, ROC, to P. S. Wang.
Address for reprint requests: P. S. Wang, Dept. of Physiology, National
Yang-Ming Univ., Shih-Pai, Taipei, Taiwan, Republic of China.
Received 18 March 1997; accepted in final form 29 October 1997.
 |
REFERENCES |
1.
Akiguchi, I.,
K. Strauss,
M. Borges,
J. E. Silva,
and
A. C. Moses.
Thyroid hormone receptors and 3,5,3'-triiodothyronine biological effects in FRTL 5 thyroid follicular cells.
Endocrinology
131:
1279-1287,
1992[Abstract].
2.
Beisel, W. R.,
V. C. Diraimondo,
P. Y. Chao,
J. M. Rosner,
and
P. H. Forsham.
The influence of plasma protein binding on the extra-adrenal metabolism of cortisol in normal, hyperthyroid and hypothyroid subjects.
Metabolism
13:
942-951,
1964.
3.
Benelli, C.,
O. Michel,
and
R. Michel.
Effect of thyroidectomy on the rat adrenal cortex enzyme activities involved in corticosterone and aldosterone biosynthesis.
J. Steroid Biochem.
16:
755-761,
1982[Medline].
4.
Boler, R. K.,
and
N. A. Moore.
Depression of adrenocortical function by pharmacologic dose of thyroxine in intact and unilaterally adrenalectomized rats.
Horm. Res.
16:
209-218,
1982[Medline].
5.
Bray, G. A.,
and
H. S. Jacobs.
Thyroid activity and other endocrine glands.
In: Handbook of Physiology. Endocrinology. Washington, DC: Am. Physiol. Soc., 1974, sect. 7, vol. III, chapt. 24, p. 413-433.
6.
Childs, G. V.,
K. Taub,
K. E. Jones,
and
W. W. Chin.
Triiodothyronine receptor beta-2 messenger ribonucleic acid expression by somatotropes and thyrotropes: effect of propylthiouracil-induced hypothyroidism in rats.
Endocrinology
129:
2767-2773,
1991[Abstract].
7.
D'Angelo, S. A.,
and
J. M. Grodin.
Experimental hyperthyroidism and adrenocortical function in the rat.
Endocrinology
74:
509-514,
1964.
8.
Freedland, R. A.,
and
S. Murad.
Effect of thyroid hormones on metabolism. III. Effect of thyroxine and thyroidectomy on adrenal gland enzyme activities.
Endocrinology
84:
692-694,
1969[Medline].
9.
Gallagher, T. F.,
L. Hellman,
J. Finkelstein,
K. Yoshida,
E. D. Weitzman,
H. D. Roffwarg,
and
D. K. Fukushima.
Hyperthyroidism and cortisol secretion in man.
J. Clin. Endocrinol. Metab.
34:
919-927,
1972[Medline].
10.
Goldman, S.,
M. Dirnfeld,
H. Abramovici,
and
Z. Kraiem.
Triiodothyronine (T3) modulates hCG-regulated progesterone secretion, cAMP accumulation and DNA content in cultured human luteinized granulosa cells.
Mol. Cell Endocrinol.
96:
125-131,
1993[Medline].
11.
Ichikawa, Y.,
K. Yoshida,
and
M. Kawagoe.
Altered equilibrium between cortisol and cortisone in plasma in thyroid dysfunction and inflammatory diseases.
Metabolism
26:
989-997,
1977[Medline].
12.
Iranmanesh, A.,
G. Lizarralde,
M. L. Johnson,
and
J. D. Veldhuis.
Dynamics of 24-hour endogenous cortisol secretion and clearance in primary hypothyroidism assessed before and after partial thyroid hormone replacement.
J. Clin. Endocrinol. Metab.
70:
155-161,
1990[Abstract].
13.
Jao, J.,
and
S. B. Koritz.
The in vitro effects of thyroxine on corticoid synthesis in rat adrenal homogenates.
Metabolism
11:
1302-1309,
1962.
14.
Kamilaris, T. C.,
C. R. Debold,
E. O. Johnson,
E. A. Mamalaki,
S. J. Listwak,
A. E. Calogero,
K. T. Kalogeras,
P. W. Gold,
and
D. N. Orth.
Effects of short and long duration hypothyroidism and hyperthyroidism on the plasma adrenocorticotropin and corticosterone responses to ovine corticotropin-releasing hormone in rats.
Endocrinology
128:
2567-2576,
1991[Abstract].
15.
Kawai, A.
Pituitary adrenocorticotropic activity in altered thyroid function.
Endocrinol. Jpn.
9:
113-120,
1962.
16.
Kubo, M.,
T. Ishizuka,
H. Kijima,
M. Kakinuma,
and
T. Koike.
Cloning of a mouse adrenocorticotropin receptor-encoding gene.
Gene
153:
279-280,
1995[Medline].
17.
Lu, S.-S.,
C.-P. Lau,
Y.-F. Tung,
S.-W. Huang,
Y.-H. Chen,
H.-C. Shih,
S.-C. Tsai,
C.-C. Lu,
S.-W. Wang,
J.-J. Chen,
E. J. Chien,
C.-H. Chien,
and
P. S. Wang.
Lactate stimulates progesterone secretion via an increase in cAMP production in exercised female rats.
Am. J. Physiol.
271 (Endocrinol. Metab. 34):
E910-E915,
1996[Abstract/Free Full Text].
18.
Mellon, S. H.,
S. R. Bair,
and
H. Monis.
P450c11
3 mRNA, transcribed from a third P450c11 gene, is expressed in a tissue-specific, developmentally, and hormonally regulated fashion in the rodent adrenal and encodes a protein with both 11-hydroxylase and 18-hydroxylase activities.
J. Biol. Chem.
270:
1643-1649,
1995[Abstract/Free Full Text].
19.
Moore, N. A.,
and
G. Callas.
The effects of hyperthyroidism on the fine structure of the zona fasciculata of the rat adrenal cortex.
Anat. Rec.
174:
451-468,
1972[Medline].
20.
Mountjoy, K. G.,
I. M. Bird,
W. E. Rainey,
and
R. D. Cone.
ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines.
Mol. Cell Endocrinol.
99:
R17-R20,
1994[Medline].
21.
Mountjoy, K. G.,
L. S. Robbins,
M. T. Mortrud,
and
R. D. Cone.
The cloning of a family of genes that encode the melanocortin receptors.
Science
257:
1248-1251,
1992[Medline].
22.
Murakami, N.,
C. Hayafuji,
and
K. Takahashi.
Thyroid hormone maintains normal circadian rhythm of blood corticosterone levels in the rat by restoring the release and synthesis of ACTH after thyroidectomy.
Acta Endocrinol. (Copenh.)
107:
519-524,
1984[Medline].
23.
Neri, G.,
L. K. Malendowicz,
P. Andreis,
and
G. G. Nussdorfer.
Thyrotropin-releasing hormone inhibits glucocorticoid secretion of rat adrenal cortex: in vivo and in vitro studies.
Endocrinology
133:
511-514,
1993[Abstract].
24.
Oldham, S. B.,
J. J. Bell,
and
B. W. Harding.
Role of the bovine adrenal cortical pyridine nucleotide transhydrogenase in 11
-hydroxylation.
Arch. Biochem. Biophys.
123:
469-506,
1968.
25.
Peron, F. G.,
F. Guerra,
and
J. L. McCarthy.
Further studies on corticosteroidogenesis. IV. Inhibition of utilization of biological substrates for corticoid synthesis by high calcium concentrations. Possible role of transhydrogenase in corticosteroidogenesis.
Biochim. Biophys. Acta
117:
450-469,
1966[Medline].
26.
Purdy, S. J.,
B. J. Whitehouse,
and
D. R. E. Abayasekara.
Stimulation of steroidogenesis by forskolin in rat adrenal zona glomerulosa preparations.
J. Endocrinol.
129:
391-397,
1991[Abstract].
27.
Raikhinstein, M.,
M. Zohar,
and
I. Hanukoglu.
cDNA cloning and sequence analysis of the bovine adrenocorticotropic hormone (ACTH) receptor.
Biochim. Biophys. Acta
1220:
329-332,
1994[Medline].
28.
Rubio, A.,
A. Raasmaja,
A. L. Maia,
K. R. Kim,
and
J. E. Silva.
Effects of thyroid hormone on norepinephrine signaling in brown adipose tissue. I. Beta 1- and beta 2-adrenergic receptors and cyclic adenosine 3',5'-monophosphate generation.
Endocrinology
136:
3267-3276,
1995[Abstract].
29.
Sanchez-Franco, F.,
L. Fernandez,
G. Fernandez,
and
L. Cacicedo.
Thyroid hormone action on ACTH secretion.
Horm. Metab. Res.
21:
550-552,
1989[Medline].
30.
Shi, Z. X.,
A. Levy,
and
S. L. Lightman.
Thyroid hormone-mediated regulation of corticotropin-releasing hormone messenger ribonucleic acid in the rat.
Endocrinology
134:
1577-1580,
1994[Abstract].
31.
Simonian, M. H.
ACTH and thyroid hormone regulation of 3
-hydroxysteroid dehydrogenase activity in human fetal adrenocortical cells.
J. Steroid Biochem.
25:
1001-1006,
1986[Medline].
32.
Simpkins, C.
Thyroid hormone in biological rhythms.
Med. Hypotheses
12:
179-184,
1983[Medline].
33.
Steel, R. D.,
and
J. H. Torrie.
Principles and Procedures of Statistics. New York: McGraw-Hill, 1960.
34.
Steinetz, B. G.,
and
V. L. Beach.
Some influences of thyroid on the pituitary-adrenal axis.
Endocrinology
72:
45-58,
1963.
35.
Sundaresan, P. R.,
and
S. P. Banerjee.
Differential regulation of beta-adrenergic receptor-coupled adenylate cyclase by thyroid hormones in rat liver and heart: possible role of corticosteroids.
Horm. Res.
27:
109-118,
1987[Medline].
36.
Tsai, S. C.,
C. C. Lu,
C. P. Lau,
G. H. Hwang,
H. Y. Lee,
S. L. Chen,
S. W. Huang,
H. C. Shih,
Y. H. Chen,
Y. C. Chiao,
S. W. Wang,
and
P. S. Wang.
Progesterone stimulates in vitro release of prolactin and thyrotropin involving cAMP production in rat pituitary.
Chin. J. Physiol.
39:
245-251,
1996[Medline].
37.
Wahrenberg, H.,
A. Wennlund,
and
P. Arner.
Adrenergic regulation of lipolysis fat cells from hyperthyroid and hypothyroid patients.
J. Clin. Endocrinol. Metab.
78:
898-903,
1994[Abstract].
38.
Wang, P. S.,
J. Y. Liu,
C. Y. Hwang,
C. Hwang,
C. H. Day,
C. H. Chang,
H. F. Pu,
and
J. T. Pan.
Age-related differences in the spontaneous and thyrotropin-releasing hormone-stimulated release of prolactin and thyrotropin in ovariectomized rats.
Neuroendocrinology
49:
592-596,
1989[Medline].
39.
Zumoff, B.,
H. L. Bradlow,
J. Levin,
and
D. K. Fukushima.
Influence of thyroid function on the in vivo cortisol-cortisone equilibrium in man.
J. Steroid Biochem.
18:
437-440,
1983[Medline].
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