Pulsatile human corticotropin-releasing hormone prevents dexamethasone-induced suppression of the plasma cortisol response to hypoglycemia in normal men.

0021-972x/92/7505-1358$03.00/0 Journal uf Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

Vol. 75, No. 5 Printed in USA

Pulsatile Human Corticotropin-Releasing Hormone Prevents Dexamethasone-Induced Suppression of the Plasma Cortisol Response to Hypoglycemia in Normal Men PETER C. AVGERINOS, CHRISTOS K. KIAMOURIS, IOANNIS IOANNIS K. KLEANTHOUS, PETROS A. ZORZOS, THEOLOGOS SOTOS A. RAPTIS, AND GORDON B. CUTLER, JR.

P. PETRAKOS, N. DIMITRIADIS,

Second Department of Internal Medicine (P.C.A., C.K.K., I.P.P., I.K.K., P.A.Z., T.N.D., S.A.R.), Propaedeutic, University of Athens Evangelismos Hospital, Athens 10676, Greece; and Developmental Endocrinology Branch (P.C.A., G.B.C.), National Institute of Child Health and Human Development, Bethesda, Mar.yland 20892 ABSTRACT Insulin-induced hypoglycemia causes a sequential stimulation of all three components of the hypothalamic-pituitary-adrenal axis. States of acute glucocorticoid excess, such as the overnight (1 mg) dexamethasone suppression test (DST), inhibit both the basal cortisol level and the response to an insulin tolerance test (ITT). However, whether this negative feedback effect is exerted primarily at the hypothalamic or the pituitary level is not clear. To explore this question further we have examined the cortisol response to insulin-induced hypoglycemia in three experimental settings, in the following order: 1) a control ITT performed at 0900 h after an overnight hospital stay (cITT); 2) an ITT at 0900 h after oral dexamethasone, 1 mg, at 2300 h on the previous evening (DST + ITT); and 3) an ITT at 0900 h after dexamethasone, 1 mg, at 2300 h and hCRH, 1 pg/kg iv, at 90 min intervals from 0100-0700 h (DST + hCRH + ITT). The response to ITT was defined as the peak cortisol increment (peak minus baseline). Since the study objective was to test whether overnight pulsatile hCRH could prevent dexamethasone-in-

duced suppression of the response to a morning ITT, only subjects that demonstrated a greater than 25% decrease in the cortisol response to DST + ITT vs. cITT received the full protocol (five of nine normal men). Basal ACTH and cortisol secretion remained suppressed throughout the night during both the Dex + ITT and Dex + hCRH + ITT studies when compared to the control study (cITT, P < 0.05). However, the cortisol response to hypoglycemia during DST + hCRH + ITT was significantly greater than during DST + ITT (P < 0.05) and was similar to the cITT response. Thus, pulsatile hCRH, administered during the 10 h between dexamethasone and the subsequent hypoglycemic stimulus, prevented acute suppression by dexamethasone of the cortisol response to hypoglycemia. We conclude that the dexamethasone-induced inhibition of the cortisol response to hypoglycemia results primarily from suppression by dexamethasone of basal hypothalamic corticotropin-releasing factor and the consequent impairment of corticotroph responsiveness to exogenous and endogenous corticotropin-releasing factor. (J Clin Endocrinol Metab 75: 1358-1361, 1992)

I

NSLJLIN -induced hypoglycemia is employed commonly to evaluate the hypothalamic-pituitary-adrenal (HPA) axis because it tests the integrity of all three components of the axis (l-6). States of acute glucocorticoid excess, such as the overnight (1 mg) dexamethasone suppression test (DST), inhibit both the basal cortisol level and the response to an insulin tolerance test (ITT, 7-9). However, whether this negative feedback effect is exerted primarily at the hypothalamic or the pituitary level is not clear (10-13). To gain further insight into this question we have examined whether overnight, exogenous, pulsatile hCRH administration, at a dose that produces physiological ACTH and cortisol levels in hypothalamic adrenal insufficiency (14), can prevent the DST-induced acute suppression of the plasma cortisol re-

sponse to ITT. We found that pulsatile hCRH did prevent the DST-induced suppression of the cortisol response to hypoglycemia, which is consistent with the hypothesis that dexamethasone acts at the hypothalamus to suppress endogenous corticotropin-releasing factor.

Received January 21, 1992. Address requests for reprints to: Gordon B. Cutler, Jr., M.D., National Institutes of Health, Building 10, Room lON262, Bethesda, Maryland 20892. Address correspondence to: Peter C. Avgerinos, M.D., National Institutes of Health, Building 13, Room lON262, Bethesda, Maryland 20892.

Subjects were admitted overnight on three occasions, separated by at least 1 week, to the Research Unit of the Second Department of Internal Medicine (Propaedeutic), Evangelismos Hospital. Admissions lasted from 2100-1030 h the following morning. A physician was present in the unit throughout the ITT. The sequence of the admissions and the procedures performed are outlined below:

Subjects and Methods Subjects Nine normal men (ages 24-39 yr) were studied after giving informed consent. The protocol was approved by the Evangelismos Hospital Scientific Committee and by the Institutional Review Board of the National Institute of Child Health and Human Development.

Protocol

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PULSATILE

hCRH

DURING

1) During the first admission, after an overnight stay in the hospital, a control insulin tolerance test (cITT) was performed at 0900 h (HumulinR 0.15 U/kg). 2) During the second admission 1 mg dexamethasone was administered orally at 2300 h, and an ITT was performed at 0900 h on the following morning (DST + ITT). Since the study objective was to evaluate whether hCRH could block dexamethasone suppression of the cortisol response to an ITT, only subjects who had more than a 25% decrease in the cortisol response to ITT during the second admission were selected to receive hCRH during the third admission (five of nine normal men). The other four men were not studied further, and their data are not included in the results, 3) During the third admission dexamethasone (1 mg orally) was administered at 2300 h, and five injections of hCRH were administered iv between 0100-0700 h as described in Pulsatile KRH administrntion. An ITT was then performed at 0900 h (DST + hCRH + ITT). After admission the subjects were asked to remain in bed throughout the study. No food or drink, apart from water, was allowed during the study. An indwelling catheter connected to a three-way stopcock was placed in a peripheral vein at 2200 h and was kept patent by a continuus infusion of normal saline. Starting at 2300 h, we obtained blood samples (5 mL) at 30.min intervals throughout each admission and at 15.min intervals during the ITT. The samples were placed on ice in EDTA tubes, and the plasma was separated within 6 h in a refrigerated centrifuge. The plasma was stored at -20 C until assayed for ACTH and cortisol. Additional blood samples (2 mL) were drawn at 15-min intervals during the ITT for measurement of blood sugar.

Pulsatile

hCRH administration

Synthetic hCRH was purchased from Bachem, Inc. (Torrance, CA) and prepared as previously described (15). A total of five injections of hCRH (1 kg/kg) were administered iv at 90-min intervals during the third admission. The first hCRH pulse was administered at 0100 h and the last at 0700 h. We showed in an earlier study that this regimen of nighttime pulsatile hCRH administration normalizes the early morning secretory pattern of plasma ACTH and cortisol in patients with hypothalamic adrenal insufficiency (14).

Hormone

and glucose assays

All samples from each individual subject were measured in duplicate in the same assay. Plasma ACTH (16) and cortisol (17) were measured by RIA as previously described (18). The assay detection limit was 1 pmol/L for ACTH and ranged from lo-20 nmol/L for cortisol. The intra- and interassay coefficients of variation were 9% and 20% for ACTH and 6% and 8% for cortisol, respectively. Blood sugar was measured by the glucose oxidase method (calorimetric end point).

Statistical

analysis

Since baseline ACTH and cortisol levels before the ITT varied according to whether dexamethasone was administered or not, the cortisol and ACTH responses to the ITT were expressed as the increments from baseline (mean of -30. and 0-min sample to peak levels). Overnight basal ACTH and cortisol levels were calculated as the mean of the values from 2300-0900 h the following morning preceding insulin administration. Overnight basal and hypoglycemia-stimulated responses of ACTH and cortisol between different admissions were compared by the paired one-tailed Student’s t test with the Bonferroni adjustment,

Results Blood sugar response during

ITT

Blood sugar dropped below 2.2 mmol/L, and subjects became symptomatic (hunger, drowsiness, and perspiration) during all insulin tolerance tests.

DEX

SUPPRESSION

Overnight

basal ACTH

1359 and cortisol levels

Both basal ACTH and cortisol levels throughout the night (means f SD of all values from 2300-0900 h the following morning) were significantly suppressed during the DST + ITT (2 + 1 pmol/L, range l-3 for ACTH; 20 + 10 nmol/L, range 20-30 for cortisol) and DST + hCRH + ITT (2 f 1 pmol/L, range 1-4 for ACTH; 30 + 20 nmol/L, range 20-60 for cortisol) when compared to the cITT study (4 + 1 pmol/ L, range 2-5 for ACTH, P < 0.05; 150 f 30 nmol/L, range 110-180 for cortisol, P < 0.05). ACTH

response to ITT

The mean + SD ACTH response hCRH + ITT (20 + 28 pmol/L, range between the control response (46 + and the response during DST + ITT l-27) and did not differ significantly

to ITT during DST + 3-70) was intermediate 38 pmol/L, range 8-99) (7 + 12 pmol/L, range from either (Fig. 1).

Cortisol response to ITT The mean + SD cortisol response to ITT during DST + hCRH + ITT (340 f 130 nmol/L, range 160-460) was significantly greater than during DST + ITT (130 f 150 nmol/L, range 30-400, P < 0.05) and was similar to the control ITT response (390 + 90 nmol/L, range 340-530, Fig. 1). Discussion We observed that pulsatile hCRH prevented acute suppression by dexamethasone of the cortisol response to hypoglycemia. These observations are consistent with preliminary studies in the rat in which CRH administered twice daily iv prevented dexamethasone-induced suppression of adrenal weight and plasma corticosterone (19). Four of the nine subjects in this study had less than a 25% suppression of the cortisol response to hypoglycemia during the DST, despite suppression of overnight basal cortisol levels to the assay detection limit in all subjects. Thus, suppressibility of basal HPA activity during the DST does not necessarily imply suppressibility of the HPA response to hypoglycemia. Factors likely to explain differences in the degree of suppression of the ITT response include differences in weight-adjusted dose of dexamethasone, in the absorption or metabolism of dexamethasone, in the level of hypoglycemia achieved, and in the subjects’ basal level of HPA activity. The available data do not permit us to distinguish among these possible explanations. Our observations may also help explain the resistance to dexamethasone suppression in depression and other pseudoCushing’s states (20, 21). In these disorders excessive endogenous CRH secretion is postulated to prevent inhibition of the pituitary adrenal axis during the dexamethasone suppression test (22, 23). Our finding that pulsatile hCRH administration will prevent dexamethasone-induced inhibition of the cortisol response to ITT is consistent with this hypothesis. Our observation that subjects in the DST + hCRH + ITT study had essentially normal cortisol responses to hypogly-


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AVGERINOS

ET

JCE & M. 1992 Vol75.No5

AL.

Sot 9 DST T DST

+ I-IT + hCRH

+ ITT

FIG. 1. Plasma

ACTH and cortisol responses to insulin-induced hypoglycemia (ITT) at baseline (cITT), after the 1-mg overnight dexamethasone suppression test (DST + ITT), and after the overnight dexamethasone suppression test followed by pulsatile hCRH administration (DST + hCRH + ITT). The left panels indicate the plasma ACTH (upper) and plasma cortisol (lower) levels for the three experimental settings. The control study (cITT) is shown as mean ? SD by the shaded region. The experimental studies (DST + ITT and DST + hCRH + ITT) are shown as mean ? SEM by the error bars. The open arrows indicate the time of hCRH pulses. The closed arrow indicates the time of insulin administration (ITT). The right panels indicate the increment of plasma ACTH (upper) and cortisol (lower) above baseline (A plasma ACTH or A cortisol during ITT). The control study is shown as mean + SD, the experimental studies (DST + ITT and DST + hCRH + ITT) as mean f SEM. Note break in scale for the ACTH results. See Subjects and Methods for additional details.

6-

2-

2

400-

E 1 i

300-

F 8 2

200-

% ii too-

24:00

02:oo

04:oo

06:OO

06:OO

lo:oo

HOUR

cemia, whereas those in the DST + ITT study do not, indicates: 1) that the hypothalamic response to hypoglycemia is not inhibited on the morning after acute administration of 1 mg dexamethasone (DST + hCRH + ITT); 2) that the corticotroph response to hypoglycemia-induced endogenous corticotropin-releasing factor (and/or other factors such as vasopressin; 5, 6) is inhibited on the morning after dexamethasone administration (DST + ITT); 3) that this diminished corticotroph cell response during hypoglycemia can be prevented by pulsatile hCRH administration, suggesting that

the diminished responsiveness to hypoglycemia results from dexamethasone-induced suppression of overnight endogenous hypothalamic corticotropin-releasing factor (acting directly or through the mediation of vasopressin; 24, 25); and 4) that corticotroph responsiveness during dexamethasone plus pulsatile hCRH depends upon the intensity of the stimulus, since it was normal to hypoglycemia but blunted to exogenous hCRH. The experimental design employed in this study, however, did not assess the reproducibility of dexamethasone-induced


PULSATILE

hCRH

DURING

suppression of the cortisol response to ITT. Thus, these studies do not exclude the possibility that some of the improved response to hypoglycemia during DST + ITT + CRH was due to intrasubject variability rather than an effect of pulsatile CRH. We conclude that dexamethasone-induced suppression of the cortisol response to hypoglycemia results primarily from inhibition by dexamethasone of basal overnight hypothalamic secretion of corticotropin-releasing factor, which in turn impairs corticotroph responsiveness to exogenous and endogenous corticotropin-releasing factor.

DEX

10.

11.

12.

13.

ACKNOWLEDGMENTS We wish to thank Miss Army T. Triandafyllopoulou and Mrs. Martina C. Tsatsika for their invaluable technical assistance. We are indebted to Drs. Kostas Markoglou, Panayotis Dunas, Vasilis Katsaros, and Nikos Stathopoulos for their contribution in carrying out the study.

14.

15.

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