1 Third Department of Internal Medicine, Gifu University School of Medicine, Gifu 500-8705; and 2 Gifu Prefectural Hospital, Gifu 500-8717, Japan
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ABSTRACT |
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To evaluate the effects of altered
corticosteroid metabolism on the hypothalamic-pituitary-adrenal axis,
we examined rats treated with glycyrrhizic acid (G rats) or rifampicin
(R rats) for 7 days. The half-life of exogenously administered
hydrocortisone as a substitute for corticosterone was longer in G rats
and shorter in R rats, with no differences in basal plasma levels of
ACTH or corticosterone. The ACTH responses to human
corticotropin-releasing factor (CRF) or insulin-induced
hypoglycemia were greater in G rats and tended to be smaller in R rats
compared with those in the control rats, whereas the corticosterone
response was similar. No difference was observed in the content and
mRNA level of hypothalamic CRF among the groups. The number and mRNA
level of CRF receptor and type 1 11-hydroxysteroid
dehydrogenase (11-HSD1) mRNA level in the pituitary were increased in G
rats but not changed in R rats, suggesting that chronically increased
intrapituitary corticosterone upregulates pituitary CRF receptor
expression. In contrast, CRF mRNA levels in the pituitary were
increased in R rats. Our data indicate novel mechanisms of
corticosteroid metabolic modulation and the involvement of pituitary
11-HSD1 and CRF in glucocorticoid feedback physiology.
corticotropin-releasing factor; corticotropin-releasing factor
receptor; type 1 11-hydroxysteroid dehydrogenase; glucocorticoid
feedback
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INTRODUCTION |
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IT IS WELL KNOWN
that endogenous corticosteroid levels regulate the
hypothalamic-pituitary-adrenal axis, whereas the effects of altered
corticosteroid metabolism with unchanged peripheral levels on the axis
remain unclear. Cortisol and corticosterone, the active glucocorticoid
in humans and in rodents, respectively, are metabolized via similar
pathways (21). Among other conversions, both undergo
oxidation and/or oxoreduction at C-11 by the type 1 and type 2 isozymes
of 11-hydroxysteroid dehydrogenase (11-HSD1 and 11-HSD2,
respectively) (11, 23, 27) and hydroxylation at C-6
(16) by a hepatic microsomal cytochrome P-450
3A4 (CYP3A4). Many drugs, diseases, and aging affect
corticosteroid metabolism. Glycyrrhizic acid, which is hydrolyzed to
much more active glycyrrhetinic acid in vivo and which affects
predominantly the 11-HSD2 in kidney, inhibits oxidation
(11
-dehydrogenation) of cortisol and corticosterone to the inactive
11-keto-steroids cortisone and 11-dehydrocorticosterone, respectively,
prolonging the half-life of cortisol and corticosterone. This results
in a condition resembling the syndrome of apparent mineralocorticoid
excess (23, 27). Furthermore, recent studies with 11-HSD1
knockout mice indicated that 11-HSD1 plays an important role in
regulating sensitivity to glucocorticoid negative feedback (4,
7). In contrast, abnormal overnight dexamethasone suppression tests in subjects receiving rifampicin (9) and
rifampicin-induced adrenal crises in patients with Addison's disease
receiving corticosteroid replacement (8) suggest that
corticosteroid metabolism is accelerated by rifampicin. Rifampicin
induces CYP3A4, increases urinary 6
-hydroxycortisol excretion, and thereby accelerates cortisol clearance (16,
30).
The present study was undertaken to evaluate the effects of altered corticosteroid metabolism on the hypothalamic-pituitary-adrenal axis in the absence of changes in circulating corticosteroid levels. In rats treated with glycyrrhizic acid or rifampicin, we studied the responses of plasma ACTH and corticosterone to the injection of human (h) corticosteroid-releasing factor (CRF) or insulin-induced hypoglycemia. Furthermore, to clarify the primary locus of these effects, we assayed CRF receptor binding in the pituitary, CRF content in the hypothalamus, and mRNA levels of CRF, CRF receptor, and 11-HSD1 in the pituitary and the hypothalamus.
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MATERIALS AND METHODS |
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Reagents and animals. Hydrocortisone sodium succinate, glycyrrhizic acid, hCRF, 125I-labeled-Tyr-CRF, and [1,2,6,7-3H]corticosterone (sp act 74 Ci/mmol) were purchased from Sumitomo-Upjohn (Tokyo, Japan), Sigma Chemical (St. Louis, MO), Peptides Institute (Osaka, Japan), Du Pont (Wilmington, DE), and Amersham Pharmacia Biotech (Buckinghamshire, UK), respectively. Rifampicin was kindly provided by Daiichi Pharmaceutical (Tokyo, Japan). All of the following experiments were conducted according to the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
Male Wistar rats weighing 200-250 g were housed individually in a controlled temperature (22°C) and light environment (12:12-h light-dark cycle; lights on from 0630 to 1830) and allowed to consume standard chow ad libitum. The animals were divided into three groups. The first group received 0.5 ml of distilled water two times per day by gavage and distilled water ad libitum for 7 days (control group). The second group received 30 mg/kg glycyrrhizic acid in 0.5 ml distilled water two times per day by gavage and 600 mg/l glycyrrhizic acid in distilled water ad libitum for 7 days (G group). The third group received 50 mg/kg rifampicin in 1.0 ml of distilled water daily by gavage and distilled water ad libitum for 7 days (R group). For evaluation of CRF content and mRNA levels, rats were decapitated, and the anterior lobe of the pituitary and the hypothalamus were removed immediately. They were kept frozen atTests. Twenty-four hours before the acute experiments (half-life of cortisol, hCRF test, and insulin-induced hypoglycemia), the animals were cannulated and implanted with polyethylene tubes (PE-50; Becton-Dickinson, Sparks, MD) of 0.58 mm inside diameter. Under aseptic conditions, a catheter filled with a 1,500 U/ml heparin solution was placed in the right atrium through a jugular vein under pentobarbital sodium anesthesia and was fitted with a 1,500 U/ml heparin lock with a metal tip plug. After cannulation, the animals were allowed to move freely. Each cannula was connected to a 30-cm length of PE-50 tubing 1 h before the acute experiments, and glycyrrhizic acid (10 mg/kg) was injected as a bolus through the cannula in the G group, whereas physiological saline was injected in the animals in the R and control groups.
To estimate the metabolism of corticosterone, 0.5 mg/kg hydrocortisone sodium succinate was injected as a bolus, and the rate of disappearance of cortisol was measured; little cortisol is synthesized in the rat, and both cortisol and corticosterone are metabolized via similar pathways. A sample of 0.2 ml of blood for measurement of plasma immunoreactive cortisol was drawn before and at 3, 5, 10, 15, 20, 30, 45, 60, 80, 120, 180, and 240 min. Synthetic hCRF (5 µg/kg) was administered as a bolus injection of 100-125 µl volume through the cannula, and blood samples were drawn at 0, 5, 15, 30, 45, and 60 min for measurement of plasma ACTH and corticosterone. For insulin-induced hypoglycemia, regular insulin (2 U/kg) was injected through the cannulas, and blood samples (0.5 ml) for measurement of plasma ACTH, corticosterone, and blood glucose were drawn before and at 15, 30, 60, and 90 min after. These three experiments were done separately using different animals and were started between 1200 and 1400. Amounts of physiological saline equivalent to the volume of blood drawn were replaced. Blood samples for measurement of plasma concentrations of ACTH and corticosterone were immediately collected into prechilled tubes containing EDTA, centrifuged for 10 min at 4°C, and then stored atQuantitation of mRNA levels of CRF, CRF receptor, and type 1 11-HSD in pituitary and hypothalamus. Because only a small amount of RNA could be obtained from an anterior lobe of the pituitary or a piece of the hypothalamus, we employed the quantitative competitive RT-PCR method to measure mRNA levels of CRF, CRF receptor, and 11-HSD1. Furthermore, the mRNA values were normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, to minimize the variations in DNase digestion and reverse transcription between samples. Total RNA was prepared from frozen tissues by use of Isogen (Nippon Gene, Toyama, Japan), and 1.8 µg of total RNA was treated with DNase I (GIBCO-BRL) and reverse transcribed with 160 units of Superscript II reverse transcriptase (GIBCO-BRL) in a 20-µl reaction volume containing 2.5 mM random 9 mers, 1 mM each dNTP, 8 units of placental RNase inhibitor, and the manufacturer's buffer. Each reaction was allowed to proceed at room temperature for 10-15 min followed by incubation at 42°C for 1.5 h. Competitors for each cDNA were prepared following the PCR MIMIC KIT protocol (Clontech, Palo Alto, CA). Gene-specific primers for each cDNA were as follows: CRF (24), 5'-CCG CCT GGG GAA CCT CAA C-3' [sense, nuclear transcript (nt) 1393-1411] and 5'-CCC TGG CCA TTT CCA AGA C-3' (antisense, nt 1682-1700); CRF receptor (17), 5'-CTC CTG GTG GCC TTT GTC CTC-3' (sense, nt 478-498) and 5'-GGG GCC CTG GTA GAT GTA GTC-3' (antisense, nt 883-903); 11-HSD1 (1) 5'-GAC ATG CTC ATT CTC AA-3' (sense, nt 415-431) and 5'-GCT GTT TCT GTG TCT ATG A-3' (antisense, nt 725-743); GAPDH (26), 5'-GCC AAG GTC ATC CAT GAC AAC-3' (sense, nt 482-497) and 5'-AGT GTA GCC CAG GAT GCC CTT-3' (antisense, nt 812-832). Quantitative competitive PCR was performed by addition of 2.5 pmol of a sense and a antisense primer to 0.5 µl of reverse-transcribed samples with 0.5 µl of various ranges of 2.5× serially diluted competitor in 5 µl of 67 mM Tris · HCl (pH 8.5), 16 mmol/l (NH4)2SO4, 2 mmol/l MgCl2, 17 µg/ml BSA, 5% glycerol, and 0.25 units of Ex Taq DNA polymerase (TaKaRa, Osaka, Japan) for CRH, 11-HSD1, or GAPDH. For CRF receptor, MasterAmp Taq DNA polymerase (Epicentre Technologies, Madison, WI) was used with premixed buffer E supplied by the manufacturer. Samples were subjected to initial denaturation at 96°C for 2.5 min, followed by 36-52 cycles of 96°C denaturation for 20 s, 62°C annealing for 20 s, and 72°C extension for 30 s. PCR products were subjected to electrophoresis in 2.5% agarose gels, stained with ethidium bromide, scanned with a GT-9000 image scanner (EPSON, Nagano, Japan), and analyzed using NIH Image 1.60 (W. Rasband, NIH). All of the mRNA levels of CRF, CRF receptor, and 11-HSD1 are calculated by normalizing each quantitated mRNA values for those of GAPDH to control for inefficiency in the reverse transcription steps.
CRF receptor binding assay in the pituitary.
The CRF receptor assay was performed with a modification of the method
previously described by Wynn et al. (29). After rats were
decapitated, pituitary glands were immediately removed and separated
into the neural-intermediate and anterior lobes. The latter were placed
in ice-cold 50 mM Tris · HCl (pH 7.4) containing 5 mM
MgCl2, 2 mM EGTA, and 1 mM dithiothreitol (buffer
A). For the assay of CRF receptors, 10 anterior lobes per
experiment were mechanically homogenized in buffer A with a
glass homogenizer. Homogenates were centrifuged at 800 g for
10 min, and the supernatant was centrifuged further at 30,000 g for 30 min. The pellet (crude membrane fraction) was
resuspended in buffer A to give a protein concentration of
100-300 µg/100 µl. For the binding assay, 100-µl aliquots of
the membrane suspension were incubated with 70,000 cpm
[125I]Tyr-CRF (0.14 nM) and various amounts of unlabeled
CRF (0 to 106 M) in a total 300-µl volume of 50 mM
Tris · HCl buffer (pH 7.4) containing 5 mM MgCl2, 2 mM EGTA, 0.15% BSA, 150 KIU/ml aprotinin, and 1 mM dithiothreitol.
Tubes were covered with 1.0 ml of oil made of dibutylphtalate and
dinorylolate (ratio, 2:3). After incubation for 60 min at 22°C, the
tubes were centrifuged at 10,000 g for 3 min, and the
supernatants were removed. After separation of the membrane pellet,
residual-bound radioactivities were counted. The protein concentration
was measured by using a BCA protein assay kit (Pierce Chemical). Under
these conditions, the total bound radioligand was 8.4-24.4%, and
nonspecific binding, determined in the presence of 10
6 M
unlabeled CRF, was 52.1 ± 2.1% of total radioactivity. These results are consistent with a previous report by Wynn et al.
(29). Calculation of the receptor affinity and
concentration was performed by Scatchard analysis using linear regression.
CRF content in the hypothalamus, hormone, and other determinations. CRF content in the hypothalamus was determined by a modification of a previously reported RIA using anti-CRF serum (Mitsubishi Petrochemical, Tokyo, Japan) and [125I]Tyr-CRF. Frozen hypothalami were incubated in MeOH-0.1 N HCl (1:1) for 10 min at 70°C and homogenized. The homogenate was centrifuged at 10,000 g for 10 min, and the supernatant was lyophilized. The extracted samples were used for measurement of CRF.
Plasma ACTH and corticosterone were measured by previously described methods (12) using a commercial two-site immunoradiometric assay kit (Mitsubishi Petrochemical) and a commercial direct RIA kit (ICN Biomedicals), respectively. Plasma cortisol was measured by a commercial direct RIA kit (Incstar, Stillwater, MN). The intra- and interassay coefficients of variation in these kits are as follows: ACTH, 3.5% at 120 pg/ml and 5.0% at 119 pg/ml; corticosterone, 7.1% at 166 ng/ml and 6.5% at 158 ng/ml; cortisol, 4.5% at 19.5 µg/dl and 6.5% at 20 µg/dl, respectively. Plasma glucose was measured with a Beckman glucose analyzer-2 (Beckman Instruments, Brea, CA).Assay of type 1 11-HSD activity in pituitary.
11-HSD1 activities, 11-dehydrogenation, and 11-oxo-reduction were
determined separately using the intact pituitary in the control group
and the G group (n = 5 each).
11-[3H]dehydrocorticosterone was synthesized from
[3H]corticosterone using the bacterial expression system
of 11-HSD2 (13). Immediately after removal, the anterior
lobe of the pituitary was cut into halves, weighed, washed in 10 mM
HEPES-buffered RPMI medium (pH 7.2), and incubated for 12 h at
37°C in 2 ml of the medium containing 10 nM cold
[3H]corticosterone or
11-[3H]dehydrocorticosterone with appropriate tritiated
tracer. Steroids were extracted from the medium and separated by TLC
plates (Whatman) using dichloromethane-acetone (82:18). Bands
consistent with each steroid were scraped and counted, and the
fractional conversion of [3H]corticosterone to
11-[3H]dehydrocorticosterone (dehydrogenation) or
11-[3H]dehydrocorticosterone to
[3H]corticosterone (reduction) was determined. Enzyme
activities were expressed as percent conversion per milligram of wet
tissue per hour.
Statistical analysis.
All data are expressed as means ± SE. The incremental value
(peak) was calculated as the difference between the peak and basal
values. The net area under the curve (net AUC) of ACTH and the
corticosterone response after stimulation with hCRF or insulin administration was calculated as AUC above the basal value. The parametric Bonferroni/Dunn test was used for group comparisons of the
responses in plasma ACTH and corticosterone to hCRF or insulin-induced
hypoglycemia, and the nonparametric Mann-Whitney test was used for
comparison of the remaining data. P < 0.05 was considered significant.
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RESULTS |
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As shown in Table 1, there were no
differences in serum potassium concentration or adrenal weights among
the control, G, and R groups. The half-life of plasma cortisol, which
is a surrogate for that of plasma corticosterone, was prolonged in the
G group (P < 0.01) and shortened in the R group
(P < 0.05) compared with that in the control group,
suggesting that the G and R groups had decreased and increased
metabolism of corticosterone, respectively.
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Responses of plasma ACTH and corticosterone to hCRF.
In response to hCRF, plasma ACTH in all groups increased rapidly and
reached a peak between 5 and 15 min after the injection (Fig.
1A). Plasma corticosterone
also increased, but the time to reach the peak value was slower than
that for ACTH (data not shown). There were no significant differences
in the basal concentration of plasma ACTH and corticosterone among the
three groups (Table 2). The plasma ACTH
response to hCRF in the G group was significantly higher than that in
the control group (P < 0.05) or the R group (P < 0.01). The ACTH response in the R group tended to
be lower, but with no significance, compared with that in the control
group (Fig. 1A). The peak incremental response (peak) and
net AUC of plasma ACTH were increased in the G group. In the R group,
the net AUC of plasma ACTH was smaller than in the control group (Table 2). There were no significant differences in peak,
peak, or net AUC
of the plasma corticosterone response to hCRF between the control group
and the G or R groups (Table 2).
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Responses of plasma ACTH and corticosterone to insulin-induced
hypoglycemia.
Insulin injection induced a fall in plasma glucose. The basal value of
blood glucose (control, 5.2 ± 0.2; G group, 5.5 ± 0.3; R
group, 5.5 ± 0.2 mmol/l), the nadir of blood glucose (control, 1.9 ± 0.1; G group, 2.0 ± 0.2; R group, 2.2 ± 0.1 mmol/l), and the blood glucose responses to insulin injection were
similar among the three groups. The plasma ACTH level reached a peak
value between 15 and 60 min after the insulin injection (Fig.
1B). Corticosterone increased, and the peak value was
obtained between 30 and 60 min after the insulin injection. There were
no significant differences in basal concentrations of plasma ACTH or
corticosterone among the three groups (Table
3). The plasma ACTH response in the G group was significantly higher than that in the control group (P < 0.01) or the R group (P < 0.01).
The ACTH response in the R group tended to be lower, but with no
significance, compared with that in the control group (Fig.
1B). The net AUC of plasma ACTH in the G group was greater
than that in the control group, and the peak and peak tended to be
high compared with those in control group. Peak,
peak, and net AUC
of plasma ACTH in the R group tended to be low but not significantly
different from those in the control group. There were no significant
differences in peak,
peak, or net AUC of plasma corticosterone among
the three groups (Table 3).
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Changes in mRNA levels of CRF, CRF receptor, and type 1 11-HSD in
pituitary and hypothalamus.
As shown in Fig. 2, left, the
hypothalamic mRNA levels of CRF receptor (top), which was
about 1/10 of that in the pituitary, CRF (middle), and
11-HSD1 (bottom), did not differ among the three groups.
However, in the pituitary (Fig. 2, right), the mRNA level of
CRF receptor (top) was increased in the G group but not
changed in the R group (control, 0.41 ± 0.03; G group, 1.18 ± 0.19; R group, 0.69 ± 0.11 × 103 CRF
receptor/GAPDH, n = 4). The mRNA level of CRF in the
pituitary (Fig. 2, right), which was about 1/12,000 of that
in the hypothalamus, was increased fourfold in the R group compared
with the control group (control, 2.6 ± 0.5; G group, 3.4 ± 0.8; R group, 10.2 ± 0.9 × 10
8 CRF/GAPDH,
n = 4). Finally, the mRNA level of 11-HSD1 in the pituitary was increased in the G group (control, 4.9 ± 0.6; G group, 14.5 ± 1.0; R group, 6.8 ± 0.5 × 10
3 of 11-HSD1/GAPDH, n = 4).
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Hypothalamic CRF content and pituitary CRF receptor binding assay. CRF content in the hypothalamus did not differ among the three groups (control, 2.50 ± 0.19; G group, 2.17 ± 0.18; R group, 2.21 ± 0.17 pg/mg wt, n = 12).
Pituitary CRF receptor concentration in the G group was statistically greater (P < 0.01) than that in the control or R groups (control, 57.4 ± 1.3; G group, 83.7 ± 4.7; R group, 51.6 ± 5.1 fmol/mg protein, mean ± SE of 5 experiments; Fig. 3, top). Although there were no significant differences in the dissociation constant of binding (Kd) between the control group and the G or R groups, Kd in the G group was higher (P < 0.05) than Kd in the R group (control, 0.88 ± 0.09, G group, 1.16 ± 0.11; R group, 0.75 ± 0.07 nM, mean ± SE of 5 experiments; Fig. 3, bottom).
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Type 1 11-HSD activity in intact pituitary.
In the intact pituitary halves, 11-reduction clearly predominated
11-dehydrogenation. 11
-Dehydrogenase activities in the G group
(0.051 ± 0.004%
conversion · mg
1 · h
1) were
lower (P < 0.01) than those in the control group
(0.138 ± 0.003), whereas 11-oxo-reductase activities in the G
group (0.498 ± 0.026) were higher (P < 0.01)
than those in the control group (0.316 ± 0.033).
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DISCUSSION |
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We demonstrated that the half-life of exogenous cortisol (which is presumably correlated with that of corticosterone) was longer in glycyrrhizic acid-treated rats (G group) and shorter in rifampicin-treated rats (R group) than in control rats, whereas basal concentrations of plasma corticosterone were similar among the three groups. Using these models of decreased and increased metabolism of corticosteroids, we performed two provocative tests (insulin tolerance and CRF tests) to assess the action of altered corticosterone metabolism on the hypothalamic-pituitary-adrenal axis.
We found that plasma ACTH responses to CRF and insulin-induced hypoglycemia were greater in the G group than in the control group, with no differences in plasma corticosterone responses. When the hypothalamic-pituitary-adrenal axis is driven in Addison's disease or metyrapone treatment, the ACTH response to CRF is enhanced (25). Hence, the increased ACTH responses to CRF in delayed corticosterone metabolism in our glycyrrhizic acid-treated rats seems to be paradoxical. However, plasma cortisol levels in Addison's disease or during metyrapone treatment are presumably low, unlike in our study.
CRF synthesis in hypothalamic paraventricular nuclei and its release into hypophysial portal blood undergo negative feedback regulation by glucocorticoids and are activated under various stresses (15). CRF secretion from the hypothalamus is suspected to be modulated by altered peripheral corticosterone metabolism and/or direct effects of drugs on the hypothalamus. Seckl et al. (22) reported that acute administration of 25 mg/kg glycyrrhetinic acid, the active component of glycyrrhizic acid, decreased CRF release into hypophysial portal blood without changing circulating glucocorticoid levels, which is probably the result of negative feedback of corticosterone that accumulates as a consequence of decreased conversion to its inactive 11-dehydro product at hypothalamic paraventricular nuclei. In our chronic study, however, neither hypothalamic content and mRNA expression of CRF nor mRNA expression of 11-HSD1 differed among the groups with increased or decreased metabolism of corticosteroids.
Chronic treatment with glycyrrhizic acid in our study revealed
upregulation of pituitary CRF receptor by both binding assays and mRNA
levels, consistent with the increased ACTH response to CRF and
insulin-induced hypoglycemia. The CRF receptor in the anterior
pituitary is known to be downregulated acutely by CRF and
glucocorticoids (5, 18, 29), but recent studies
demonstrated that acute stress causes biphasic changes in pituitary CRF
receptor mRNA expression with an early decrease followed by an increase and that chronic stress causes a sustained increase in pituitary CRF
receptor mRNA with a permissive role of glucocorticoids (14, 19). Interestingly, 11-HSD1 mRNA levels were increased by three times in the pituitary glands of our glycyrrhizic acid-treated rats. A
previous report (28) described that 75 mg/kg of
glycyrrhizic acid administration for 5 days inhibited
11-dehydrogenase activity in kidney and liver and reduced 11-HSD1
mRNA levels in kidney, liver, and pituitary. Our glycyrrhizic
acid-treated rats did show the decrease in 11
-dehydrogenase activity
but an increase in oxoreductase activity in the intact pituitary. The
mRNA expression of 11-HSD1 in the kidney of our glycyrrhizic
acid-treated rats was also increased (T. Mune, unpublished
observation). Because the dose or term of glycyrrhizic acid treatment
seems to be similar, the discrepancy about changes in pituitary or
kidney 11-HSD1 expression is not fully explained. There might be
differences in rat strain or in peripheral corticosterone concentrations.
Type 1 11-HSD has bidirectional (dehydrogenase and oxoreductase) activities in vitro and mainly dehydrogenase activity in tissue homogenates (10), but this isozyme mainly has oxoreductase activity in many tissues in vivo (6, 20). Indeed, our conversion assays showed an oxoreductase predominance in the intact pituitary. These observations indicate the role of 11-HSD1 as a potential enhancer of glucocorticoid action, whereas the role of 11-HSD2 is a protective mechanism for the mineralocorticoid receptor, because 11-HSD2 has only dehydrogenase activity and is expressed in mineralocorticoid target tissues. Recently described 11-HSD1 knockout mice have elevated basal levels and greater stress responses of plasma ACTH and corticosterone, together with diminished glucocorticoid feedback (4, 7). CRF mRNA expression in the hypothalamic paraventricular nuclei is similar in wild-type and 11-HSD1-deficient mice, but glucocorticoid receptor mRNA expression is reduced in 11-HSD1-deficient mice (4). Although glucocorticoid receptor expression was not examined in the present study, no changes in hypothalamic CRF, CRF receptor, and 11-HSD1 suggested that the pituitary gland was the primary lesion in our glycyrrhitic acid-treated rats, whereas 11-HSD1 knockout mice possibly have abnormalities also in higher central organs such as hypothalamus and hippocampus. In any case, the increased oxoreductase activity resulting from increased 11-HSD1 mRNA expression in the pituitary of our glycyrrhizic acid-treated rats should lead to intracellular accumulation of corticosterone within the cells, even under unchanged circulating corticosterone levels. Considering the sustained increase in pituitary CRF receptor mRNA under chronic stress (14, 19), it is plausible that chronically increased intrapituitary corticosterone with no detectable changes in hypothalamic CRF and 11-HSD1 upregulates pituitary CRF receptor expression.
The lack of differences in corticosterone responses compared with the differences in ACTH responses may indicate a kind of adrenal insensitivity, especially in glycyrrhitic acid-treated rats. This is in contrast to the increased adrenal sensitivity to ACTH reported in 11-HSD1 knockout mice (4) that have elevated plasma levels of ACTH and corticosterone as well as adrenal hypertrophy (7), none of which was seen in our glycyrrhitic acid-treated rats. These discrepancies might be because of the difference in affected sites of the central nervous system or the difference between supraphysiological changes, as in 11-HSD1 knockout mice, and physiological changes in our study. In our preliminary examination, the adrenal mRNA levels of 11-HSD1 and 11-HSD2 in our glycyrrhitic acid-treated rats tended to be increased and decreased, respectively. Further elucidation, including the changes in ACTH receptor or steroidogenic enzymes, will be necessary to clarify the role of adrenal 11-HSD isozymes in rats treated with glycyrrhitic acid.
Finally, our rifampicin-treated rats showed diminished plasma ACTH responses to CRF assessed by net AUC. Other parameters of ACTH responses showed similar tendencies but with no significance. Considering the milder decreases in cortisol half-life (compared with the greater increases by glycyrrhitic acid), the doses of rifampicin we used might not be enough to induce significant changes. Under these conditions, however, CRF expression in the pituitary was increased fourfold compared with the control group, although its level was only 1/12,000 that in the hypothalamus. Giraldi and Cavagnini (2) recently reported the production of CRF in rat corticotropes as in our study and suggested that intrapituitary CRH acts to maintain basal ACTH secretion. Increased CRF expression in our rifampicin-treated rats should cause a relative insensitivity to exogenous CRF. Because rifampicin is a prokaryotic RNA polymerase inhibitor and was recently shown to induce CYP3A4 via the orphan pregnane X receptor (3), one can speculate that rifampicin might directly affect CRF transcription, but CRF transcription was not examined in the present study.
In conclusion, our results suggest that chronic treatment with glycyrrhizic acid or the consequent decrease in metabolism of corticosterone upregulates CRF receptors in the pituitary concomitantly with induction of 11-HSD1 expression, resulting in an increased plasma ACTH response to stimuli. In contrast, rifampicin or the resulting increased metabolism of corticosterone might upregulate pituitary CRF expression. Our study suggests a possible novel mechanism of corticosteroid metabolic modulation and the involvement of pituitary 11-HSD1 and CRF in glucocorticoid feedback physiology.
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ACKNOWLEDGEMENTS |
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This work was supported in part by research grants for Disorders of the Adrenal Gland (1995-1998, 1999-2002) from the Ministry of Health, Labor, and Welfare, Japan.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Mune, Third Dept. of Internal Medicine, Gifu Univ. School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan (E-mail: mune{at}cc.gifu-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00065.2001
Received 20 February 2001; accepted in final form 27 September 2001.
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