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0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society

Vol. 83, No. 3 Printed in U.S.A.

Pulsatile Luteinizing Hormone Secretion in Patients with Addison’s Disease. Impact of Glucocorticoid Substitution* J. HANGAARD, M. ANDERSEN, E. GRODUM, O. KOLDKJÆR,

AND

C. HAGEN

Department of Endocrinology, Odense University Hospital (J.H., M.A., E.G., C.H.), DK-5000 Odense C; and the Department of Clinical Chemistry, Sønderborg Hospital (O.K.), DK-6400 Sønderborg, Denmark during hypocortisolism compared to that during eucortisolism (3.26 6 0.68 vs. 4.49 6 0.83 U/L; P , 0.05) and was associated with a clear decrease in LH pulse amplitude (1.09 6 0.33 vs. 1.96 6 0.53 U/L; P , 0.05). During high doses of glucocorticoids, the mean LH level in men was significantly lower than that during eucortisolism (3.81 6 0.88 vs. 4.49 6 0.83 U/L; P , 0.05). In both men and women, the mean PRL levels increased significantly (P , 0.05) during hypocortisolism, whereas high glucocorticoid doses suppressed the mean PRL level (P , 0.05). The LH and PRL responses to LH-releasing hormone and TRH were, however, similar during low, medium, and high cortisol levels in both men and women. In conclusion, our data suggest that the attenuation of pulsatile LH secretion in men during hypo- and hypercortisolism is due to variations in the hypothalamic opioid activity secondary to alterations in serum cortisol levels. A higher level of opioid receptor activity in men than in low estrogen women may explain the gender differences. (J Clin Endocrinol Metab 83: 736 –743, 1998)

ABSTRACT The physiological and pathophysiological role of cortisol in pulsatile LH release was investigated in 14 patients (5 men, 6 premenopausal women, and 3 postmenopausal women) with Addison’s disease. The explicit effect of cortisol in relation to the effect of corticotropin-releasing factor (CRF), ACTH, and opioids was ensured by hypo-, normo-, and hypercortisolism. Hypocortisolism was obtained by 24-h discontinuation of hydrocortisone (HC) followed by 23-h saline infusion. Eucortisolism was secured by infusion of HC (0.5 mg/kg) over 23 h. Stress-appropriate hypercortisolism was obtained by infusion of HC (2.0 mg/kg) over 23 h, preceded by treatment for 5 days with dexamethasone (1.5 mg/day). To imitate the normal diurnal rhythm for serum cortisol, HC was infused in graduated doses. Blood sampling was performed every 10 min during the last 10 h of the study period, followed by a LH-releasing hormone test (5 mg, iv) and a TRH test (10 mg, iv). In pre- and postmenopausal women, the mean LH level and the LH pulsatility pattern were similar on the 3 occasions. In contrast, the mean LH level in men was significantly reduced

T

HE NEUROENDOCRINE control of episodic LH secretion is initiated by the hypothalamic GnRH pulse generator (1), but a wide range of neuropeptides are known to have a modulating influence on pulsatile LH-releasing hormone (LHRH) and LH release (2–7). CRF and cortisol have been shown to suppress the reproductive axis (4, 8 –13), but the opioid and dopaminergic systems have also been implicated as potential mediators of stress-induced hypogonadism (4 – 6, 12, 14 –21). An important component of the hormonal response to stress is an activation of the hypothalamic-pituitary-adrenal axis (8, 20, 22–25). The almost invariable association of sustained hypercortisolism with hypothalamic hypogonadism may suggest a pathophysiological role of cortisol on the attenuated pulsatile LH release (8, 25). In vitro animal and human studies have demonstrated that glucocorticoids suppress basal and LHRH-stimulated LH release (25–27). Moreover, glucocorticoid receptors (GR) have been demonstrated in both LHRH neurons and the gonadotrophs (28 –31), and the activation of GR by endog-

enous glucocorticoids suppresses LH secretion (13). In accordance with these observations, the GR antagonist RU486 attenuates acute as well as chronic stress-induced inhibition of LH release under physiological conditions (13). However, in human studies of the interaction between stress and reproductive function, all three levels of the hypothalamicpituitary-adrenal axis are activated, and the implication of cortisol in the neuroendocrine derangements is controversial (32, 33). Both central and peripheral administration of CRF decreases LHRH and LH secretion, and central administration of CRF antagonists blocks the stress-induced inhibition of LH pulsatility (9, 10). Endogenous opioid peptides seem to be involved in the CRF-induced suppression of LH secretion (4, 19, 34 –36), and the existence of a CRF-opioid interaction is supported by the ability of naloxone to reverse the CRFinduced decrease in serum LH levels. Some investigators have claimed that increased opioid activity is the major pathophysiological factor during stress (19, 20, 34, 35), and that cortisol is unlikely to be responsible for the decrease in LH levels (33). The acute effect of physiological and pathophysiological variations in serum cortisol on pituitary LH secretion, either directly or through a modulation of hypothalamic secretagogues, cannot be fully assessed on the basis of the available data. This study was designed to investigate the effects of short term hypo- and hypercortisolism on pulsatile LH se-

Received April 30, 1997. Revision received November 25, 1997. Accepted December 4, 1997. Address all correspondence and requests for reprints to: Jørgen Hangaard, M.D., Department of Endocrinology, Odense University Hospital, DK-5000 Odense C, Denmark. * This work was supported by grants from the Clinical Research Institute, Odense University Hospital, and the Research Foundation for the Counties of Ribe, Ringkøbing and South Jutland.

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CORTISOL AND LH SECRETION

737

cretion. For that purpose, Addison patients can be used as an outstanding in vivo model. High glucocorticoid levels will ensure suppressed CRF/ACTH/opioid activity, and hypocortisolism will result in elevated CRF/ACTH/opioid activity. By investigating low estrogen women as well as men at three different levels of cortisol, the explicit effect of cortisol in relation to the effect of CRF and/or opioids on the hypothalamic-pituitary-gonadal axis is further emphasized. Subjects and Methods Subjects Fourteen patients with primary adrenocortical insufficiency were studied; five men (mean age, 46.4 6 1.8; range, 43–53 yr), six regularly menstruating and fertile women (mean age, 37.7 6 2.5; range, 30 – 43 yr), and three postmenopausal women (mean age, 64.7 6 3.0; range, 59 – 69 yr). The premenopausal women were studied on comparable days during the early follicular phase (days 3–5 from the onset of menses) of three menstrual cycles. The patients were carefully selected, i.e. all patients with other endocrine diseases, fertility problems, or medications besides substitution therapy had been excluded. All patients were well substituted for several years before inclusion in the study, and the mean duration of disease was 10.5 6 2.4 yr. Twelve patients had autoimmune Addison’s disease, one had adrenocortical insufficiency due to sarcoidosis, and one had previous tuberculosis. All patients were clinically and biochemically euthyroid, and baseline measures of hematological, hepatic, renal, and metabolic functions were normal. Their mean body mass index was 22.3 6 2.1 kg/m2. None of the patients was taking oral contraceptives, and they received no medication besides their usual substitution therapy of hydrocortisone (HC; median dose, 30 mg/day; range, 20 – 40 mg/day) and fludrocortisone (median dose, 0.01 mg/day; range, 0 – 0.2 mg/day). The Declaration of Helsinki II was observed, and the study was approved by the local committee on medical ethics. All subjects were volunteers and gave their written informed consent.

Protocol All 14 patients were investigated during low, medium, and high serum levels of cortisol, with an interval of 2–3 months between the three investigations (Fig. 1). The mineralocorticoid therapy was unchanged on all three occasions. They were admitted to our stationary clinic the evening before blood sampling, which was carried out through an iv catheter placed in a forearm vein. HC or saline was infused from 2000 – 1900 h the following day through an iv catheter in the other arm. The infusion rate of HC was varied during the study period to imitate the normal diurnal rhythm for serum cortisol. The low, medium, and high cortisol procedures were as follows: low, after 24-h withdrawal of HC, the patients had saline infusion for an additional 23 h; medium, in continuation of the conventional HC substitution, a medium dose of HC (0.5 mg/kg) was infused for 23 h; and high, dexamethasone was administered at a dose of 1.5 mg/day for 5 days before admission, followed by the infusion of a high dose of HC (2.0 mg/kg) over 23 h. The suppressibility of CRF and ACTH secretion in Addison patients is very heterogeneous, but we have previously shown that this dose and duration of dexamethasone pretreatment will secure a suppressed ACTH level (37). On all three occasions, low doses of LHRH (5 mg) and TRH (10 mg) were administered as an iv bolus at 1800 h, and LH and PRL secretion were followed for an additional 60 min. These doses were chosen to test the sensitivity and not the secretory reserve of the gonadotrophs and lactotrophs. The patients were not fasting, but received standard meals at 0800, 1200, and 1900 h. They slept from 2300 h until 0700 h and remained in a bed or chair during sampling.

Blood sampling Blood was drawn every 10 min from 0800 –1900 h. LH was determined every 10 min, PRL was determined every 20 min, and ACTH and cortisol were determined every hour. Serum estradiol and serum testosterone were determined at 0800, 1200, and 1800 h.

FIG. 1. Study design and time schedule for the investigations during low, medium, and high serum cortisol levels.

Assays Serum LH was determined by an immunofluorometric assay [Delfia, Wallac OY, Turku, Finland; sensitivity, 0.05 U/L; intraassay coefficient of variation (CV), ,3.0% for LH values 1.0 –27.4 U/L]. Serum cortisol was determined by competitive RIA (Orion Diagnostics, Espoo, Finland; sensitivity, 5 nmol/L; intraassay CV, ,3%). Plasma ACTH was determined by immunoradiometric assay (Allegro-IRMA, Nichols Institute Diagnostics, San Juan Capistrano, CA; sensitivity, 0.6 pmol/L; intraassay CV, ,4%). Serum PRL was determined by a Delfia immunofluorometric assay (Wallac; sensitivity, 0.02 mg/L; intraassay CV, ,4.0% for PRL values 2.0 –24 mg/L). Serum testosterone was measured by RIA (own method by use of antibodies from Orion Diagnostics; sensitivity, 0.5 nmol/L; intraassay CV, ,8%). Serum estradiol was measured by RIA (Orion Diagnostics; sensitivity, 0.03 nmol/L; intraassay CV, 20% for estradiol values ,0.27 nmol/L and ,5% for values .1.14 nmol/L). Cortisol-binding globulin (transcortin) was analyzed by an immunochemical assay (antitranscortin antibody from Dako, Copenhagen, Denmark; sensitivity, 0.10 mmol/L; intraassay CV, ,7%). To avoid interassay variation, all samples from an individual subject were analyzed in the same assay.

Peak detection The patterns of episodic LH secretion were characterized using the computerized peak detection scheme for serial hormone data reported by Clayton et al. (38). A LH value was defined as a peak if the minimum amplitude on either side of the potential peak was more than 2 sd and there was a minimum mean amplitude of 3 sd, achieving a specified type I error (false positive rate) of 5%. The pulse amplitude was defined as VP 2 1⁄2 3 (NL 1 NR), where VP is the peak value, and NL and NR are the left and right nadir values. All identified pulses during the 10-h

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High Low

32.60 6 9.71b 4.69 6 1.27b 13.0 6 1.0b 45.4 6 3.8b 46.3 6 4.1b 83.1 6 16.9b 52.2 6 9.5b 4056 6 924b – ,0.05b 34.30 6 10.17 6.00 6 1.5 11.3 6 0.3 50.6 6 1.3 49.7 6 0.9 87.7 6 20.2 51.1 6 8.3 4312 6 1140 – ,0.05 3.90 6 0.49b 1.31 6 0.19b 10.7 6 0.7b 54.5 6 2.7b 55.0 6 3.5b 14.2 6 4.5b 10.4 6 4.0b 576 6 96b – 0.34 6 0.03b 4.10 6 0.35b 1.12 6 0.25b 11.8 6 1.1b 48.3 6 4.6b 50.2 6 5.2b 10.7 6 1.7b 6.1 6 1.4b 536 6 65b – 0.25 6 0.05b 4.29 6 0.30 1.24 6 0.23 11.5 6 1.1 53.4 6 6.8 53.8 6 7.9 11.4 6 1.0 7.1 6 1.0 576 6 48 – 0.30 6 0.05 3.81 6 0.88a 1.86 6 0.24b 7.2 6 1.1b 87.8 6 21.4b 83.2 6 21.7b 15.7 6 3.2b 12.7 6 2.4b 757 6 151b 14.8 6 0.8a –

Postmenopausal women (n 5 3)

Medium High Low

Premenopausal women (n 5 6)

Medium High

3.26 6 0.68a 1.09 6 0.15a 8.6 6 1.1b 66.8 6 8.3a 67.2 6 7.6b 13.9 6 3.1b 10.6 6 2.5b 695 6 164b 20.6 6 2.2b –

Value are the mean 6 SE. a P , 0.05, low and high vs. medium cortisol levels. b P 5 NS, low and high vs. medium cortisol levels.

The PRL mean levels for all patients during low, medium, and high serum levels of cortisol are shown in Table 2. In men and pre- and postmenopausal women, the mean PRL levels were significantly increased (P , 0.05) during low cortisol levels and suppressed (P , 0.05) during high cortisol levels compared to those during eucortisolism. Comparing the results from all 3 days, a significant inverse correlation between the serum levels of cortisol and PRL was found in both men and women (r 5 20.6; P , 0.001), and the glucocorticoidmediated decrease in PRL diminished during the day in parallel with the steadily declining serum cortisol levels (data not shown).

4.49 6 0.83 1.96 6 0.24 7.2 6 0.7 81.9 6 6.7 80.8 6 6.9 16.8 6 4.4 12.5 6 3.5 835 6 220 18.8 6 1.7 –

PRL levels

Low

The mean LH levels and pulsation data for the 10-h period during low, medium, and high cortisol levels are shown in Table 1 and Figs. 2– 4. In men (Fig. 2), the LH mean level was significantly reduced during low cortisol levels compared to that during medium cortisol levels (3.26 6 0.68 vs. 4.49 6 0.83 U/L, respectively; P , 0.05). Also, the mean LH pulse amplitude and the wave length of LH pulses were significantly decreased during low cortisol level compared to medium cortisol level (1.09 6 0.33 vs. 1.96 6 0.53 U/L and 66.8 6 8.3 vs. 81.9 6 6.7 min, respectively; P , 0.05). During the administration of high doses of glucocorticoids, the LH mean level was significantly lower compared to medium cortisol levels (3.81 6 0.88 and 4.49 6 0.83 U/L, respectively; P , 0.05), with no significant changes in pulse amplitude or wave length. The pulse frequency was similar during low, medium, and high levels of cortisol. In premenopausal (Fig. 3) and postmenopausal (Fig. 4) women, the LH mean levels, pulse amplitude, frequency, and wave length were similar during low, medium, and high levels of cortisol. A ratio plot of serum LH concentrations during medium cortisol levels compared to low (Fig. 5a) and high (Fig. 5b) cortisol levels shows the differences (Table 1; P , 0.05) in the glucocorticoid-mediated alterations in LH in men and low estrogen women.

Men (n 5 5)

Results LH levels and pulsatility pattern

Serum measurements

The mean LH and PRL concentrations were calculated using all sampling values in the individual patient obtained during the 10-h period. The LH and PRL values during low, medium, and high cortisol levels were compared using one-way ANOVA for repeated measures, and the LH pulse frequencies and amplitudes were compared using Friedman’s two-way ANOVA. If significant differences were found, the difference between paired values was tested by Wilcoxon signed rank test. During LHRH and TRH stimulation (1800 –1900 h), the incremental area under the curve (AUC) for LH and PRL was calculated according to Tai’s model (39). Data are given as the mean 6 se. P , 0.05 was considered statistically significant.

TABLE 1. LH pulsation data and gonadal steroids in 14 patients with Addison’s disease during the infusion of low, medium, and high doses of cortisol

Statistical analysis

LH mean level (U/L) LH pulse amplitude (U/L) LH pulse frequency (burst/10 h) LH pulse wavelength (min) LH pulse interval (min) Peak LH during LHRH (U/L) Dmax LH during LHRH (U/L) AUC LH during LHRH (U/L min) Total testosterone (nmol/L) Total estradiol (nmol/L)

period (0800 –1800 h) were used to calculate the mean pulse amplitude for each subject. The wave length of the LH pulses was defined as the time interval from prepeak nadir to postpeak nadir, and the pulse interval was the time interval from postpeak nadir to prepeak nadir.

31.91 6 9.42b 6.87 6 1.58b 11.3 6 1.5b 53.3 6 6.2b 56.0 6 7.0b 98.6 6 25.6b 57.5 6 12.1b 5079 6 1464b – ,0.05b

HANGAARD ET AL.

Medium

738


739

FIG. 2. a, Serum LH levels (mean 6 SE) in five men with Addison’s disease during low (*), normal (l), and high (f) serum cortisol levels. b, Serum LH levels and pulsatility pattern for a typical man with Addison’s disease during low (*), normal (l), and high (f) serum cortisol levels.

FIG. 3. a, Serum LH levels (mean 6 SE) in six premenopausal women with Addison’s disease during low (*), normal (l), and high (f) serum cortisol levels. b, Serum LH levels and pulsatility pattern for a typical premenopausal woman with Addison’s disease during low (*), normal (l), and high (f) serum cortisol levels.

LHRH and TRH test

Cortisol and ACTH levels

The AUC and the peak and the maximum increments (Dmax) of LH responses to LHRH were highest in postmenopausal women, but similar (P . 0.05) during low, medium, and high cortisol levels in men and pre- and postmenopausal women. The AUC and the peak and the maximum increments (Dmax) of PRL responses to TRH were significantly higher in premenopausal women than in men and postmenopausal women, but were similar (P . 0.05) during the three glucocorticoid levels in men and women.

The serum levels of cortisol and ACTH during the adjusted infusion of medium and high doses of HC and during saline infusion are shown in our previous paper (40). During saline infusion (AUC for cortisol, 327 6 102 nmol/Lzh) the mean cortisol level was 34 6 12 nmol/L at 0800 h and 25 6 6 nmol/L at 1900 h. During the infusion of a medium dose of HC (AUC for cortisol, 2996 6 166 nmol/Lzh), the serum concentration of cortisol was 312 6 27 nmol/L at 0800 h, with a decline during the day to 199 6 10 nmol/L at 1900 h. During the infusion of high doses of HC (AUC for cortisol, 7429 6 234 nmol/Lzh), the serum cortisol level declined from 891 6 28 nmol/L at 0800 h to 548 6 20 nmol/L at 1900 h. The corresponding mean ACTH values at 0800 h were 174 6 38, 36 6 13, and 0.5 6 0.2 pmol/L during low, medium, and high cortisol levels (P , 0.001). During the infusion of medium doses of HC, 10 patients had normal plasma ACTH levels (4.7 6 1.5 pmol/L), and 4 had elevated plasma ACTH levels (74 6 56 pmol/L). All had suppressed plasma ACTH levels during high doses of cortisol. During HC withdrawal, the mean ACTH level was significantly elevated above the

Gonadal steroids

In men, the mean serum level of testosterone was significantly reduced during high doses of glucocorticoids compared to the level during medium doses of HC (14.8 6 0.8 vs. 18.8 6 1.66 nmol/L, respectively; P , 0.05), whereas the serum levels were similar during low and medium cortisol levels. In premenopausal women, serum estradiol levels were unchanged (P . 0.05) during the three levels of cortisol.

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HANGAARD ET AL.

FIG. 4. a, Serum LH levels (mean 6 SE) in three postmenopausal women with Addison’s disease during low (*), normal (l), and high (f) serum cortisol levels. b, Serum LH levels and pulsatility pattern for a typical postmenopausal woman with Addison’s disease during low (*), normal (l), and high (f) serum cortisol levels.

normal range (2–14 pmol/L) and fell gradually during the day. Cortisol-binding globulin

The mean serum levels of transcortin in the 14 patients were 0.65 6 0.02, 0.71 6 0.02, and 0.67 6 0.02 mmol/L during low, medium, and high serum cortisol levels, respectively (normal range, 0.60 –1.00 mmol/L). The transcortin levels were similar in men and pre- and postmenopausal women (P . 0.05), and no significant difference was found on the three occasions (P . 0.05). Discussion

In the present in vivo model, well substituted Addison patients have basal LH levels, and frequency and amplitude of spontaneous pulsatile LH secretion comparable to those previously reported in normal healthy adults (3, 7, 18, 41, 42). Forty-seven-hour discontinuation of cortisol provoked a clearly reduced LH mean level in men, but not in pre- and postmenopausal women. The decreased LH level in men during hypocortisolism was associated with a decline in pulse amplitude and wave length. The changes in spontaneous LH pulsatility pattern were not associated with any

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FIG. 5. a, Ratio plot of the serum LH concentrations during medium cortisol levels divided by the LH concentrations during low cortisol levels during the 10-h study period in men (f), premenopausal (f) and postmenopausal women (*). b, Ratio plot of the serum LH concentrations during medium cortisol levels divided by the LH concentrations during high cortisol levels during the 10-h study period in men (f), premenopausal women (f), and postmenopausal women (*).

decrease in pituitary responsiveness to a submaximal dose of exogenous LHRH. Our data suggest that the amplitude suppression of pulsatile LH release in men is mediated by a decreased LHRH burst mass and duration (42, 43). During the withdrawal of HC, the plasma ACTH levels were significantly elevated, which implies increased POMC transcription and, hence, increased b-endorphin production (44 – 46). Substantial evidence has accrued suggesting that endogenous opioids play a key role in the regulation of LH secretion, accomplished by a tonic inhibition of hypothalamic LHRH release (14, 19, 47– 49). These observations suggest that our results obtained in men during hypocortisolism may be explained by an increased opioidergic inhibition of LHRH and LH release. Our data agree with those obtained in adrenalectomized male animals (50), but contrast with the results of Vierhapper et al. (51), who found that the basal LH concentrations in five men with Addison’s disease were similar during regular substitution therapy and after 84-h withdrawal of glucocorticoids. This contradiction may be explained by the improved sensitivity of our LH assay.

3.98 6 0.76a 29.1 6 8.2b 24.7 6 7.6b 1588 6 425b

High

4.85 6 0.82 32.0 6 13.1 26.2 6 11.6 2033 6 843

8.32 6 0.71 28.5 6 8.8b 21.4 6 8.7b 1840 6 620b

a

Low

4.05 6 0.69 43.7 6 12.9b 37.9 6 12.4b 2272 6 667b 5.49 6 1.23 48.1 6 13.2 42.7 6 13.2 2464 6 673 Values are the mean 6 SE. a P , 0.05, low and high vs. medium cortisol levels by ANOVA, P , 0.05. b P 5 NS, low and high vs. medium cortisol levels.

PRL mean level (mg/L) Peak PRL during TRH (mg/L) Dmax PRL during TRH (mg/L) AUC PRL during TRH (mg/L min)

3.46 6 0.19 26.4 6 3.8 21.9 6 4.1 1316 6 136

4.92 6 0.21 24.5 6 4.5b 18.3 6 3.5b 1300 6 181b

a

a

2.95 6 0.53 19.1 6 2.0b 16.0 6 2.6b 999 6 83b

7.29 6 1.53 42.6 6 9.9b 33.5 6 8.7b 2137 6 473b

a

High Low

Premenopausal women (n 5 6)

Medium High Low

Men (n 5 5)

Medium Serum measurements

TABLE 2. PRL levels in 14 patients with Addison’s disease during the infusion of low, medium, and high doses of cortisol

a

Medium

Postmenopausal women (n 5 3)


741

Although only three postmenopausal women were studied, the elevated levels of LH failed to respond to the proposed increased b-endorphin level during hypocortisolism. Also in the premenopausal women, no change in the mean LH level or LH pulsatility pattern was found. These findings agree with several studies that have confirmed the involvement of endogenous opioids in the regulation of pulsatile LH secretion only during the high estrogen phases of the menstrual cycle, but not in the early follicular phase (52–54). Gonadal steroids may influence the binding characteristics of opioid receptors (55, 56) and seem to regulate POMC gene expression in a complex and time-dependent manner (54, 57, 58). Although hypothalamic and pituitary POMC transcription are also regulated by CRF and glucocorticoids (12, 59 – 61), the CRF-induced suppression of LH release seems to be mediated primarily by endogenous opioids (4, 19, 20, 34 –36). The gender differences in the LH response to hypocortisolism in this study support the hypothesis that LHRH neurons are regulated indirectly by endogenous opiates. The augmentation of CRF and opioid activity during hypocortisolism, in terms of elevated ACTH concentrations (61, 62), was equal in men and women, which suggests a reduced expression of opioid receptor activity in women during low circulating levels of gonadal steroids (53). This assumption is certified by the results in animal studies, which showed that treatment with estrogen induced an increased number of m-opioid receptors (56, 63). Our results could not be explained by different cortisol or ACTH levels, as the ACTH, cortisol, and transcortin levels were equal in men and women. The unchanged LH response to LHRH during hypocortisolism is also consistent with a change in the suprapituitary regulation of LH secretion. Our data contrast with some earlier observations of enhanced LH responses to LHRH in unsubstituted Addison patients (51) and after pretreatment with metyrapone in normal men and women (26). This contradiction may be explained by our evaluation of the pituitary sensitivity by the use of 5 mg LHRH, whereas other studies have assessed the pituitary response to 100 mg LHRH. Several studies have established an inhibitory effect of pharmacological doses of glucocorticoids on basal and LHRH-induced LH release (26). In addition, chronic glucocorticoid excess of endogenous origin is characterized by hypogonadotropic hypogonadism in both men and women (21, 64, 65). Some studies have presented data that suggest a regulatory function of endogenous cortisol on basal and stress-induced secretion of LH (8, 13, 27, 51, 66). Other experiments have argued against any role of cortisol on the variations in LH secretion occurring during acute stress (32). Studying the impact of glucocorticoids on LH secretion, not only the dose, pharmacokinetics, and receptor affinity, but also the time course of the glucocorticoid actions are important (44). In the present study, stress-appropriate doses of glucocorticoids significantly lowered the LH mean level and pulse amplitude in men. In pre- and postmenopausal women, however, the mean basal LH levels and the pulsatility pattern were not significantly different from the values during eucortisolism. The LH responses to LHRH were similar during the three different serum cortisol levels in both

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HANGAARD ET AL.

men and women, suggesting that the inhibitory action of short term, moderate hypercortisolism on LH release in men is exerted at a suprapituitary level. GR have been localized in LHRH neurons (31) as well as in the pituitary gonadotrophs (28). The modulation of LHRH gene expression by GR is influenced by the central opiate receptor function, which has been shown to be crucial for the expression of the inhibitory action of glucocorticoids on LH release (30). A higher opioid receptor activation in men than in women may in part explain the gender differences in glucocorticoid-mediated suppression of LH release during hypercortisolism. High doses of glucocorticoids suppressed serum testosterone in men, but did not alter the estradiol level in premenopausal women. The inappropriate low LH secretion in men suggests that the impairment of gonadal steroid synthesis is secondary to the suppressive effect on LHRH/LH release (20), but a supplementary direct effect on the testicular function of glucocorticoids may occur (67, 68). The mean PRL levels were significantly increased during hypocortisolism and significantly reduced during high glucocorticoid levels in both men and women. The PRL responses to TRH, however, were similar during the three different glucocorticoid levels. Glucocorticoid excess is a known inhibitor of PRL gene transcription, but the unchanged PRL responses to TRH suggest that the variations in basal PRL secretion may in part be due to alterations in hypothalamic regulatory mechanisms. Exogenous opioid peptides consistently induce a prompt release of PRL in normal human males and females (69, 70), whereas the implication of endogenous opioids under physiological and pathophysiological conditions is unclear (5, 15, 71, 72). Our data imply that the glucocorticoid modulation of PRL secretion may involve an influence on the opioid-dopaminergic system (72), although other hypothalamic secretagogues may be involved. We infer that the gender differences in the attenuation of pulsatile LH secretion during hypo- and hypercortisolism are due to a higher opioid receptor activation in men than in low estrogen women. The direct effect of short term, pathophysiological changes in serum cortisol on pituitary LH and PRL release were minor, but physiological cortisol levels were crucial for the preservation of normal pulsatile LH secretion in patients with adrenocortical insufficiency.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27.

28.

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