Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles

Previous studies have shown that hypoglycemia may reduce counterregulatory responses to subsequent hypoglycemia in healthy subjects and in patients with diabetes. The effect of hypoglycemia on the hormonal response to a nonhypoglycemic stimulus is uncertain. To test the hypothesis that the cortisol response to corticotropin (ACTH) infusion is independent of antecedent hypoglycemia, 10 healthy subjects received a standard ACTH infusion (0.25 mg Cosyntropin [Organon, West Orange, NJ] intravenously over 240 minutes) at 8:00 AM on day 1 and day 3 and a hypoglycemic insulin clamp study (1 mU/kg/min) at 8:00 AM on day 2. During the hypoglycemic clamp, plasma glucose decreased from 5.0 mmol/L to 2.8 mmol/L for two periods of 120 minutes (mean glucose, 2.9 +/- 0.03 and 2.8 +/- 0.02 mmol/L, respectively) separated by a 60-minute interval of euglycemia (mean glucose, 4.7 +/- 0.01 mmol/L). Seven subjects also had paired control studies in random order during which a 330-minute euglycemic clamp (mean glucose, 5.0 +/- 0.11 mmol/L) instead of a hypoglycemic clamp was performed on day 2. Basal ACTH (4.6 +/- 0.7 v 2.6 +/- 0.4 pmol/L, P < .02) and basal cortisol (435 +/- 46 v 317 +/- 40 nmol/L, P < .02) both decreased from day 1 to day 3 following intervening hypoglycemia. In contrast, with intervening euglycemia, neither basal ACTH (5.9 +/- 1.5 v 4.5 +/- 1.0 pmol/L) nor basal cortisol (340 +/- 38 v 318 +/- 60 nmol/L) were reduced significantly on day 3 compared with day 1. Following interval hypoglycemia, the area under the curve (AUC) for the cortisol response to successive ACTH infusions was increased (4,734 +/- 428 nmol/L over 240 minutes [day 3] v 3,526 +/- 434 nmol/L over 240 minutes [day 1], P < .01). The maximum incremental cortisol response was also significantly increased (805 +/- 63 nmol/L (day 3) v 583 +/- 58 nmol/L (day 1), P < .05). In contrast, the AUC for the cortisol response to successive ACTH infusions with interval euglycemia (3,402 +/- 345 nmol/L over 240 minutes [day 3] v 3,709 +/- 391 nmol/L over 240 minutes [day 1] and the incremental cortisol response (702 +/- 62 nmol/L [day 3] v 592 +/- 85 nmol/L [day 1] were unchanged. Following exposure to intermittent hypoglycemia in healthy humans, fasting morning ACTH and cortisol levels are reduced and the incremental cortisol response to an infusion of ACTH is enhanced. The enhanced cortisol response to exogenous ACTH infusion after intervening hypoglycemia (but not intervening euglycemia) may reflect priming of the adrenal gland by endogenous ACTH produced during the hypoglycemia. These data suggest that adrenal function testing by exogenous ACTH administration is not impaired by prior exposure to hypoglycemia. Moreover, the reduced cortisol response to recurrent hypoglycemia in patients with well-controlled diabetes is not likely the result of impaired adrenal responsiveness.     

The frequency of late-onset 21-hydroxylase and 11 beta-hydroxylase deficiency in women with polycystic ovary syndrome

OBJECTIVE: To determine the frequency of late-onset adrenal hyperplasia (LOCAH) due to 21-hydroxylase (21-OH) and 11 beta-hydroxylase (11 beta-OH) deficiency in women with clinical and biochemical features of polycystic ovary syndrome (PCOS). DESIGN: Eighty-three consecutively selected women with PCOS and eighteen normal women were included in the study. METHODS: Ultrasound, clinical and hormonal parameters were used to define PCOS. Basal FSH, LH, testosterone, free testosterone, androstenedione, dehydroepiandrosterone sulfate (DHEA-S), sex hormone-binding globulin (SHBG) and cortisol levels were measured. Serum 17-hydroxyprogesterone (17-OHP) and 11-deoxycortisol (11-DOC) levels were also measured before, 30 and 60 min after a single bolus injection of 0.25 mg ACTH (1-24) at 0900 h during the mid-follicular phase of the cycle. ACTH-stimulated 17-OHP levels > 30 nmol/l were considered as the criteria of 21-OH deficiency. The diagnosis 11 beta-OH deficiency was made if the adrenal 11-DOC response to ACTH stimulation exceeded threefold the 95th percentile of controls. RESULTS: Basal serum testosterone, free testosterone, androstenedione, DHEA-S, cortisol and 11-DOC levels were significantly higher in PCOS than in control subjects. ACTH-stimulated 17-OHP (P < 0.05) and 11-DOC (P < 0.0005) levels were found to be significantly higher in patients with PCOS than in controls. Seven (8.4%) patients had an 11-DOC response to ACTH higher than threefold the 95th percentile of controls, while no patients showed evidence of 21-OH deficiency. CONCLUSIONS: We have found that 8.4% of the women with clinical and biochemical features of PCOS could be presumed to have 11 beta-OH deficiency. No patients among the women with PCOS showed evidence of 21-OH deficiency. 11 beta-OH deficiency is unexpectedly more common than 21-OH deficiency in women with PCOS.     

Adrenal androgen excess in the polycystic ovary syndrome: sensitivity and responsivity of the hypothalamic-pituitary-adrenal axis

Over 50% of patients with the polycystic ovary syndrome (PCOS) demonstrate excess levels of adrenal androgens (AAs), particularly dehydroepiandrosterone sulfate (DHS). Nonetheless, the mechanism for the AA excess remains unclear. It has been noted that in PCOS the pituitary and ovarian responses to their respective trophic factors (i.e. GnRH and LH, respectively) are exaggerated. Similarly, we have postulated that excess AAs in PCOS arises from dysfunction of the hypothalamic-pituitary-adrenal axis, due to 1) exaggerated pituitary secretion of ACTH in response to hypothalamic CRH, 2) excess sensitivity/responsivity of AAs to ACTH stimulation, or 3) both. To test this hypothesis we studied 12 PCOS patients with AA excess (HI-DHS; DHS, > 8.1 mumol/L or 3000 ng/mL), 12 PCOS patients without AA excess (LO-DHS; DHS, < 7.5 mumol/L or 2750 ng/mL), and 11 controls (normal subjects). Each subject underwent an acute 90-min ovine CRH stimulation test (1 microgram/kg) and an 8-h incremental i.v. stimulation with ACTH-(1-24) at doses ranging from 20-2880 ng/1.5 m2.h) with a final bolus of 0.25 mg. All patient groups had similar mean body mass indexes and ages, and both tests were performed in the morning during the follicular phase (days 3-10) of the same menstrual cycle, separated by 48-96 h. During the acute ovine CRH stimulation test, no significant differences in the net maximal response (i.e. change from baseline to peak level) for ACTH, dehydroepiandrosterone (DHA), androstenedione (A4), or cortisol (F) or for the DHA/ACTH, A4/ACTH, or F/ACTH ratios was observed. Nonetheless, the net response of DHA/F and the areas under the curve (AUCs) for DHA and DHA/F indicated a greater response for HI-DHS vs. LO-DHS or normal subjects. The AUC for A4 and A4/F and the delta A4/delta F ratio (delta = net maximum change) indicated that HI-DHS and LO-DHS had similar responses, which were greater than that of the normal subjects, although the difference between LO-DHS patients and normal subjects reached significance only for the AUC of the A4 response. No difference in the sensitivity (i.e. threshold or minimal stimulatory dose) to ACTH was noted between the groups for any of the steroids measured. Nonetheless, the average dose of ACTH-(1-24) required for a threshold response was higher for DHA than for F and A4 in all groups. No difference in mean responsivity (slope of response to incremental ACTH stimulation) was observed for DHA and F between study groups, whereas the responsivity of A4 was higher in HI-DHS patients than in normal or LO-DHS women. The net maximal and the overall (i.e. AUC) responses of DHA were greater for HI-DHS than for normal or LO-DHS women. The response of A4 and the delta A4/delta F ratio were greater for HI-DHS patients than for LO-DHS patients or normal subjects. Alternatively, HI-DHS and LO-DHS patients had similar overall responses (i.e. AUC) for A4 or A4/F, although both were greater than those of normal subjects. The relative differences in response to incremental ACTH stimulation between steroids was consistent for all subject groups studied, i.e. A4 > F or DHA. In conclusion, our data suggest that AA excess in PCOS patients is related to an exaggerated secretory response of the adrenal cortex for DHA and A4, but not to an altered pituitary responsivity to CRH or to increased sensitivity of these AAs to ACTH stimulation. Whether the increased responsivity to ACTH for these steroids is secondary to increased zonae reticularis mass or to differences in P450c17 alpha activity, particularly of the delta 4 pathway, remains to be determined.     

Variation of dorsal horn cell dendritic spread with map scale

OBJECTIVE: To examine the hypothesis that, in polycystic ovary syndrome (PCOS), ovarian steroids induce adrenal enzyme dysfunction or adrenal androgen hyperresponsiveness to ACTH. DESIGN: Prospective controlled clinical study. SETTING: Reproductive endocrinology unit of an academic medical center. PATIENTS: Twelve women with PCOS who had adrenal androgen excess were compared with five weight-matched ovulatory women. In half of the women with PCOS, prestudy screening was suggestive of mild 3 beta-hydroxysteroid dehydrogenase (HSD) deficiency. INTERVENTIONS: Basal and adrenal dynamic blood sampling before and after GnRH agonist (GnRH-a) administration for 6 months. MAIN OUTCOME MEASURES: Basal E2 and androgen levels as well as dexamethasone-suppressed, ACTH-stimulated 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, and androgen levels before and after ovarian suppression. RESULTS: Although none of the subjects with PCOS proved to have mild 3 beta-HSD deficiency, the majority of them (58%) met the criteria for 17,20 lyase hyperactivity before and after GnRH-a therapy. As a group, the remaining subjects with PCOS exhibited an elevated DHEAS response to ACTH before GnRH-a treatment, which may have normalized after GnRH-a treatment. CONCLUSION: Adrenal androgen excess in PCOS may be heterogeneous in etiology, whereas 17,20 lyase hyperactivity appears to be an intrinsic adrenal disorder, adrenal androgen hyperresponsiveness to ACTH may be ovarian induced. Reliance on historical controls may lead to overdiagnosis of mild 3 beta-HSD deficiency.     

A critical review of the origin and control of adrenal androgens

The reticularis and fasciculata zones of the adrenal cortex are the predominant sources of dehydroepiandrosterone (DHEA) and DHEA-sulphate and contribute directly or indirectly 60-75% of androstenedione and testosterone in women. The specific control of adrenal androgens remains unclear. While ACTH stimulates adrenal androgen secretion, the dissociation of cortisol and androgens occurring during adrenarche and under pathological conditions suggests other factors are involved. Recent studies using human adrenal cells in vitro have demonstrated that the ratio of androgen to cortisol produced is substantially independent of the age and gender of the adrenal, indicating that extra-adrenal factors are of greater importance. beta-Endorphin and joining peptide have been shown to stimulate androgen production in human adrenal cells and to influence ACTH-stimulated steroidogenesis in a manner that promotes adrenal androgen production. The activity of these pro-opiomelanocortin-derived peptides may explain the physiological and pathological dissociations of androgens and cortisol.     

Tuft-to-capsule adhesions and their precursors: differences between the vascular and tubular poles of the human glomerulus

The hypothalamo-pituitary-adrenal (HPA) axis is modulated by sex hormones. Few data exist on the relation between acute estrogen deficit and HPA axis response to corticotropin-releasing hormone (CRH). The effects of a sudden drop in estradiol levels on basal and CRH-stimulated levels of ACTH, cortisol, testosterone, androstenedione and 17-hydroxyprogesterone (17-OHP) were assessed in nine premenopausal women (44-48 years of age), before and after ovariectomy. The CRH test was performed before and 8 days after ovariectomy. A significant reduction in ACTH and adrenal steroids but not in cortisol response to CRH was observed after ovariectomy. The ratio of deltamax androstenedione/17-OHP after CRH stimulation was substantially the same before and after ovariectomy, whereas deltamax 17-OHP/cortisol was significantly lower in ovariectomized women showing increased 21- and 11beta-hydroxylase activity. The results show that the acute estrogen deficit induces changes in the HPA axis characterized by reduced stimulated secretion of ACTH and steroids but normal stimulated cortisol production.     

17 alpha-Hydroxyprogesterone responses to leuprolide and serum androgens in obese women with and without polycystic ovary syndrome offer dietary weight loss

Insulin resistance and increased ovarian cytochrome P450c17 alpha activity (i.e. increased 17 alpha-hydroxylase and, to a lesser extent, increased 17,20-lyase) are both features of the polycystic ovary syndrome (PCOS). Evidence suggests that hyperinsulinemia may stimulate ovarian P450c17 alpha activity in obese women with PCOS. We hypothesized that weight loss would decrease serum insulin and P450c17 alpha activity in PCOS. Therefore, we measured serum steroid concentrations and 17 alpha-hydroxyprogesterone responses to leuprolide administration and performed oral glucose tolerance tests before and after 8 weeks of a hypocaloric diet in 12 obese women with PCOS (PCOS group) and 11 obese women with normal menses (control group). Serum insulin decreased in both groups. In the PCOS group, basal serum 17 alpha-hydroxyprogesterone decreased from 4.2 +/- 0.6 to 3.0 +/- 0.5 nmol/L (P < 0.05), and leuprolide-stimulated peak serum 17 alpha-hydroxyprogesterone decreased from 14.9 +/- 2.6 to 8.9 +/- 0.8 nmol/L (P < 0.025). Serum testosterone decreased from 2.47 +/- 0.52 to 1.56 +/- 0.33 nmol/L (P < 0.05), and free testosterone decreased from 9.03 +/- 1.39 to 5.95 +/- 0.50 pmol/L (P < 0.02). None of these values changed in the control group. Serum sex hormone-binding globulin increased by 4.5- and 3-fold in the PCOS (P < 0.003) and control (P < 0.007) groups, respectively. We conclude that dietary weight loss decreases ovarian P450c17 alpha activity and reduces serum free testosterone concentrations in obese women with PCOS, but not in obese ovulatory women. The changes in women with PCOS may be related to a reduction in serum insulin.     

Hypofunction of the stress axis in Sj?gren's syndrome

OBJECTIVE: To examine the functional integrity of the hypothalamic-pituitary-adrenal (HPA) and thyroid axes in Sj?gren's syndrome (SS) via the assessment of basal and stimulated adrenocorticotropin (ACTH), cortisol, thyroid stimulating hormone (TSH), and prolactin levels. METHODS: Pituitary function of the HPA axis was assessed by determining the basal plasma levels of ACTH in the late afternoon, as well as the ACTH released to ovine corticotropin releasing hormone (oCRH) stimulation; adrenal function was assessed by measuring plasma cortisol levels in the late afternoon at baseline and after release of the endogenous ACTH during oCRH stimulation. Basal and thyrotropin releasing hormone (TRH) stimulated levels of TSH and prolactin were also assessed. Healthy volunteers were used as controls. RESULTS: Patients with SS, compared to controls, were characterized by significantly lower ACTH levels (pg/ml), (5.1 +/- 0.5 vs 11.4 +/- 1.5, respectively; p < 0.05) and cortisol levels (microg/ml), (2.4 +/- 0.6 vs 5.9 +/- 1.2, respectively; p < 0.05). Furthermore, a blunted pituitary and adrenal response to oCRH compared to controls was observed: peak plasma ACTH and cortisol levels for patients with SS were 46.2 +/- 5.4 pg/ml and 15.7 +/- 1.6 microg/ml, respectively, and for controls 61.5 +/- 3.8 and 19.6 +/- 0.7, respectively (p < 0.05). Basal TSH levels were significantly elevated in patients (1.3 +/- 0.3 microIU/ml vs 0.9 +/- 0.05 microIU/ml; p < 0.05). CONCLUSION: The above findings indicate hypoactivity of the HPA axis in patients with SS. Further studies are needed to definitively identify the locus of the defects and assess the significance of the pattern of the perturbations to the pathogenesis and expression of SS.     

Medication use patterns among demented, cognitively impaired and cognitively intact community-dwelling elderly people

OBJECTIVE: The present study was conducted in order to describe human hypothalamo-pituitary adrenal (HPA) axis adaptation in a model of repeated physical stress (endurance training) that causes a moderate increase in cortisol levels. SUBJECTS: We performed the same stimulation tests (adrenal stimulation with ACTH or pituitary stimulation with combined CRH/LVP) in a population of 8 endurance-trained athletes in two distinct situations: resting (baseline cortisol values) and 2 h after the end of strenuous exercise (increased cortisol values) to evaluate the HPA axis sensitivity to endogenous sustained increases in cortisol concentrations. MEASUREMENTS: During these tests, saliva and plasma cortisol (Fs and Fp, respectively) were assessed and compared. RESULT: Cortisol values in both plasma and saliva at the end of 2 h of exercise were significantly higher than in rested controls: Fs 11.5 +/- 1.3 vs 6.5 +/- 0.8 nmol.l-1 and Fp 428 +/- 36 vs 279 +/- 27 nmol.l-1 (post exercise vs post rest sessions, respectively, P < 0.001 for both). After either hormone test (CRH/LVP or ACTH), cortisol levels in plasma and saliva increased similarly when rest was compared to post exercise. Saliva variations (delta %) under exogenous hormone stimulation were dramatically greater than plasma variations. For example, under ACTH stimulation, the relative increments in cortisol were on control day: delta Fs 980 +/- 139 vs delta Fp 218 +/- 43% (saliva vs plasma, respectively, P < 0.05) and on exercise day: delta Fs 605 +/- 89 vs delta Fp 102 +/- 14% (saliva vs plasma, respectively, P < 0.05). CONCLUSIONS: In endurance-trained athletes, displaying a moderate but sustained endogenous cortisol increase: (1) ACTH responses following pituitary stimulation are not blunted, (2) cortisol responses following maximal adrenal stimulation are not blunted. Our results favour the hypothesis of a decreased pituitary sensitivity to cortisol negative feedback whereas the hypothesis of a major decreased adrenal sensitivity to ACTH was discarded. The greater ability of saliva assays to detect a cortisol increase strongly supports its use in the study of HPA physiology, whether under basal or dynamic conditions.     

Treatment system for drug-dependence syndrome

OBJECTIVE: There is emerging evidence that women with visceral obesity may have hyper-responsiveness of the hypothalamic-pituitary-adrenal axis. There are no studies on basal daily secretory pattern of ACTH and cortisol in subjects with different obesity phenotypes. DESIGN AND PATIENTS: In this study we examined daytime pulsatile secretion of ACTH and cortisol in two groups of premenopausal obese women with visceral (V-BFD) (BMI 37.1 +/- 1.7) and subcutaneous (S-BFD) (BMI 38.8 +/- 1.5) body fat distribution (measured by CT scan) and in a group of normal weight healthy controls (BMI 21.1 +/- 0.5). After an overnight fast, blood samples were taken at 15-minute intervals for 12 h (49 samples, from 0800 h until 2000 h). All women avoided breakfast but had a normal lunch and dinner, both containing similar food, energy and nutrient composition. ACTH and cortisol responses to mixed meals at noon and in the evening were also investigated. RESULTS: Mean values of ACTH and cortisol did not differ between the groups. However, ACTH pulse frequency was significantly higher in V-BFD (P < 0.06) and S-BFD (P < 0.02) obese women than in controls, without any significant differences between the two obese subgroups. Mean ACTH pulse amplitude was lower in the V-BFD than in S-BFD obese (P < 0.02) and control (P < 0.05) groups. Cortisol episodic characteristics did not differ between V-BFD and S-BFD obese and controls. All differences in ACTH pulsatile parameters between obese and controls and between the two obese subgroups were evident only in the morning, with no further significant differences during the early and late afternoon. There were no significant differences in cortisol parameters during the three periods of the day between the various groups, apart from late afternoon cortisol pulse frequencies, which were significantly lower in V-BFD than in controls. After lunch, ACTH and cortisol levels significantly increased in all groups, but the cortisol increase tended to be more rapid in V-BFD than in the other two groups. After dinner, ACTH significantly increased in V-BFD and controls but not in the S-BFD group, whereas cortisol rose significantly in all groups, but significantly less in S-BFD than in V-BFD and controls. CortisolAUC (but not ACTHAUC) after lunch was significantly higher than after dinner in all groups. ACTH response after each meal was similar in all groups, but cortisolAUC after dinner was significantly lower in S-BFD than in V-BFD women. CONCLUSION: This study demonstrates that in premenopausal women, obesity, particularly the visceral phenotype, is associated with several abnormalities of ACTH pulsatile secretion, particularly in the morning. On the contrary, no major differences were present in either blood concentrations, diurnal rhythm or secretory pattern of cortisol between obese and controls. The responses to meals seem to indicate a much more rapid cortisol response after lunch in women with visceral obesity and a reduced activation of the hypothalamic-pituitary-adrenal axis after dinner in women with subcutaneous obesity.     

Effect of glycemic control on glucose counterregulation during hypoglycemia in NIDDM

OBJECTIVE: We examined the effect of glycemic control of NIDDM on counterregulatory hormone responses to hypoglycemia and compared the effect with that seen in patients with IDDM. RESEARCH DESIGN AND METHODS: Eleven subjects with NIDDM and eight age- and weight-matched control subjects and ten subjects with IDDM and ten age- and weight-matched control subjects were studied. All subjects underwent a stepped hypoglycemic-hyper-insulinemic clamp study during which plasma glucose levels were lowered in a stepwise manner from 5.0 to 2.2 mmol/l in steps of 0.6 mmol/l every 30 min. Counterregulatory hormones (epinephrine, norepinephrine, glucagon, ACTH, cortisol, and growth hormone [GH]) were measured, and a symptom survey was administered during the last 10 min of each 30-min interval. RESULTS: The threshold for release of epinephrine, norepinephrine, ACTH, and cortisol occurred at higher plasma glucose levels in NIDDM than in IDDM patients (P < 0.05-0.01). The glucose threshold for release of epinephrine and norepinephrine correlated with glycemic control as measured by glycosylated hemoglobin (P < 0.05-0.01). However, for a given level of glycemic control, the threshold for release of epinephrine and norepinephrine occurred at a higher glucose level in NIDDM versus IDDM patients (P < 0.05-0.01). At the nadir level of hypoglycemia, glucagon, ACTH, and cortisol levels were all higher in NIDDM compared with IDDM subjects, whereas GH levels were lower. CONCLUSIONS: Glycemic control alters counterregulatory responses to hypoglycemia in NIDDM as has been previously reported in IDDM. However, at similar levels of glycemic control, NIDDM patients release counterregulatory hormones at a higher plasma glucose level than patients with IDDM. In addition, subjects with NIDDM maintain their glucagon response to hypoglycemia. These data suggest that patients with NIDDM may be at reduced risk of severe hypoglycemia when compared with a group of IDDM patients in similar glycemic control, thus providing a more favorable risk-benefit ratio for intensive diabetes therapy in NIDDM.     

Leptin down-regulates the steroid producing system in the adrenal

OB protein leptin inhibits the secretion of cortisol in primary cultures of bovine adrenocortical cells and down-regulates 17alpha-hydroxylase cytochrome P450 mRNA expression. To analyze if leptin regulates other major enzymes involved in adrenal steroidogenesis we tested its effect on mRNA expression for two further key enzymes, C21-hydroxylase (P450C21) and side-chain cleavage enzyme (P450SCC). Cultured bovine cortical cells were stimulated for 24 hours with 10 nM ACTH, with 10 nM ACTH plus 100 ng/ml leptin or left unstimulated as controls. Stimulation with ACTH led to a 1.75-fold increase of P450C21 mRNA and a 3.31-fold increase of P450SCC mRNA compared to unstimulated controls. Addition of leptin led to a reduction of ACTH-stimulated mRNA accumulation of 73% for P450C21 and of 45% for P450SCC. We therefore suggest that leptin reduces cortisol synthesis in the adrenal by down-regulating the steroid producing enzyme cascade in the cortical cell.     

Immunolocalisation of P450(C17) in the fetal sheep adrenal gland during gestation and in response to ACTH and glucocorticoid administration

In sheep, increased output of cortisol from the fetal adrenal gland is critical to organ maturation and parturition. Cortisol synthesis is determined in part by the activity of P450(C17) enzyme. We have used immunohistochemistry and Western immunoblotting to examine the distribution of P450(C17) in the ovine fetal adrenal during gestation, and after ACTH or dexamethasone administration to fetuses between Days 125 and 130. The patterns were compared with changes in 3beta-hydroxysteroid dehydrogenase (3beta-HSD) localisation and levels. Adrenal tissue was obtained from four fetuses at each of Days 63-65, 100, 125-130 and term (>140 days). Further animals were chronically catheterised and infused with ACTH, dexamethasone or saline for 96 h beginning on Day 125. Immunohistochemistry for P450(C17), 3beta-HSD, and phenylethanolamine-N-methyl transferase (PNMT) was conducted using standard techniques. At Day 63-65 of pregnancy immunoreactive (ir-)P450(C17) was present in cords of cells throughout the adrenal gland. Ir-P450(C17) was reduced or was undetectable at Day 100, but had increased by Day 125-130, and was present throughout the zona fasciculata of the adrenal cortex of term animals. An increase in P450(C17) protein was also seen between Day 100 and 125 by Western blotting, and after ACTH treatment. Dexamethasone administration led to a marked reduction in ir-P450(C17) levels. In contrast, ir-3beta-HSD was present in the fetal adrenal cortex between Day 100 and term, and was less affected by ACTH or dexamethasone treatment. We conclude that P450(C17) in the fetal sheep adrenal is responsive to regulation by ACTH, and that changes in its levels correlate with previously reported alterations in patterns of cortisol output by the fetal adrenal gland.     

Functioning of the porcine pituitary-adrenocortical axis during neonatal development

A study was conducted with neonatal boars to measure age-related changes in functioning of the pituitary-adrenocortical axis. Pigs were randomly assigned to control (n = 7-10/age) or treated (1-min restraint, n = 9-11/age) groups to be sampled at either 12, 19, or 26 days of age. Blood samples were taken via catheter 10 min before and 3, 10, and 20 min after restraint or at similar time intervals in controls. One day later, pigs were killed and adrenal glands obtained for ACTH receptor measurements. Basal plasma ACTH concentrations were greatest (p = 0.035) on day 12 when compared with later ages, but basal plasma cortisol concentrations were comparable at the three ages. Compared with controls, restraint elevated incremental plasma ACTH and cortisol responses at each age (p < 0.004). On day 12, maximal plasma ACTH (p = 0.0006) and incremental cortisol (p < 0.006) responses to restraint were greater than at later ages. Binding to adrenal ACTH receptors was greatest (p < 0.05) at day 13, which may help explain the apparently increased in vivo response of the adrenal gland to ACTH at this time. Restrained pigs had increased growth rates with increasing age (p = 0.016) whereas growth rates for control pigs did not differ with age. At day 27, 24 h after the 1-min restraint, body weights of restrained pigs exceeded those of control pigs (p = 0.045). At day 20, adrenal DNA and protein in pigs restrained 24 h previously were greater than in control pigs (p < 0.05). These data suggest age-related changes in functioning of the pituitary-adrenal axis in neonatal boars, and an absence of period during neonatal life when the porcine pituitary adrenocortical axis cannot respond to a stressor. The data also indicate both rapid and long-term responses of the adrenal to a very modest stressor and suggest an extreme sensitivity of neonatal pigs to environmental perturbations.     

Regulation of corticotropin and steroidogenic enzyme mRNAs in human fetal adrenal cells by corticotropin, angiotensin-II and transforming growth factor beta 1

Using cultured human fetal adrenal cells, we have investigated the basal secretion of cortisol and dehydroepiandrosterone sulfate (DHAS) and the effect of corticotropin (ACTH), angiotensin-II (A-II) and transforming growth factor beta 1 (TGF beta 1) on the secretion of these steroids and on the mRNA levels of ACTH receptor (ACTHR), cytochrome P-450scc (cholesterol side-chain cleavage), P450 17 alpha (17 alpha-hydroxylase/17-20 lyase) and 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). The basal DHAS/cortisol ratio declined progressively between 12.5 and 21 weeks. ACTH treatment enhanced the secretion of cortisol and to a lesser extent that of DHAS, and increased the steroidogenic response to an acute stimulation with ACTH. These changes were associated with increased mRNA levels of ACTHR and of the steroidogenic enzymes. A-II treatment also increased the secretion of both DHAS and cortisol, but less than ACTH, enhanced the responsiveness to ACTH and increased ACTHR, P450scc and P450 17 alpha mRNA levels. In contrast, TGF beta 1 alone or together with ACTH decreased DHAS secretion, but not cortisol secretion. Moreover, TGF beta 1 had no effect on ACTHR and P450scc mRNA levels, decreased by about 50% the mRNA levels of P450 17 alpha both in the absence or presence of ACTH, but enhanced the stimulatory effects of ACTH on 3 beta-HSD mRNA. These results, along with those previously reported, suggest that both A-II and TGF beta may play a role in fetal adrenal function. In addition, they show that the effects of both peptides are qualitatively different from, even sometimes opposite to, those previously reported in bovine and ovine adrenal cells.     

Hormonal responses during various phases of autoimmune adrenal failure: no evidence for 21-hydroxylase enzyme activity inhibition in vivo

Adrenal autoantibodies (ACA) are markers of adrenal cortex involvement in idiopathic Addison's disease. Recently the 21-hydroxylase (21-OH) enzyme has been discovered to be the major autoantigen of the ACA. A potential role of these antibodies in determining adrenal failure by inhibition of the 21-OH has been recently postulated. To test this hypothesis, cortisol and aldosterone (final products of adrenal steroid synthesis) and 17-hydroxyprogesterone (17-OH-progesterone) (as a marker of 21-OH impairment) have been investigated in baseline conditions and after ACTH (1-24) stimulation test in a group of 42 patients positive for both ACA and 21-OH autoantibodies. Patients were divided into five groups according to the stages (0-4) of adrenal failure. With progression toward overt Addison's disease, baseline 17-OH-progesterone, cortisol, and aldosterone remained almost unchanged but with impairment of their responses to ACTH (1-24) stimulation. The 17-OH-progesterone/cortisol ration remained normal both in basal conditions and after stimulation at stages 0-3. At stage 4 (overt Addison's disease), this ratio increased in baseline condition with no changes after ACTH (1-24), probably because of persistent 17-OH-progesterone gonadal production. In conclusion, there was a progressive and concomitant impairment of the synthesis of all steroids tested over various phases of adrenal failure. The pattern of response of the 17-OH-progesterone/cortisol ratio to ACTH stimulation in patients with 21-OH autoantibodies was not consistent with the autoantibodies inhibiting the 21-OH activity. This suggests that the inhibiting effect of 21-OH autoantibodies on 21-OH activity is not usually evident in vivo.     

11 beta-Hydroxysteroid dehydrogenase activity in Cushing's syndrome: explaining the mineralocorticoid excess state of the ectopic adrenocorticotropin syndrome

A characteristic feature of the ectopic ACTH syndrome is a state of mineralocorticoid excess, although the etiology remains obscure. Some forms of endocrine hypertension, such as licorice ingestion, have been explained by cortisol acting as a mineralocorticoid in the setting of inhibition or deficiency of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). This enzyme is responsible for the conversion of cortisol (F) to hormonally inactive cortisone, and its activity in vivo can be inferred from the ratio of the urinary excretion of tetrahydrocortisol (THF) and its isomer (5 alpha THF) to tetrahydrocortisone. Twenty-two patients with Cushing's syndrome (11 pituitary dependent, 9 ectopic, and 2 adrenal adenomas) and 13 controls were studied. Compared to controls. Cushing's patients had a significant increase (P < 0.001) in the excretion of all principal metabolites of F, secondary to a 5- to 6-fold increase in the cortisol secretion rate [median, 34.0 (range, 13.3-327) mg/day in Cushing's vs. 6.1 (range, 2.5-10.3) mg/day in controls]. The THF plus 5 alpha THF/tetrahydrocortisone ratio was significantly increased in Cushing's syndrome regardless of etiology [mean, 1.81 (range, 1.09-9.99) in Cushing's vs. 0.81 (range, 0.51-1.47) in controls; P < 0.001), indicative of defective 11 beta HSD activity. Furthermore, compared to patients with pituitary-dependent Cushing's, this ratio was significantly higher in patients with the ectopic ACTH syndrome (4.12 vs. 1.49; P < 0.01) and was inversely correlated with serum potassium levels (r = -0.57; P = 0.01; n = 22). One explanation for the mineralocorticoid excess state of the ectopic ACTH syndrome appears to be that cortisol gains inappropriate access to the mineralocorticoid receptor through failure of its normal metabolism by 11 beta HSD. The reason for the defective 11 beta HSD activity is unclear, but it may be secondary to substrate saturation, inhibition by other adrenal steroids, or product inhibition.     

Differences in corticotropin-releasing hormone-stimulated adrenocorticotropin and cortisol before and after weight loss

Little is known about the effects of intentional weight loss on the function of the hypothalamic-pituitary-adrenal (HPA) axis of obese individuals. We studied the HPA axis of 34 healthy obese women (body mass index, 40.2 +/- 7.9 kg/m2) before and after a 21.0 +/- 7.9-kg weight loss induced by a 26-week weight loss program that included 12 weeks of a 3350 kJ/day (800 Cal/day) liquid formula diet, 6 weeks of gradual refeeding, and 6 weeks of caloric stabilization at 5020-6280 kJ/day (1200-1500 Cal/day). Obese subjects were evaluated twice: before caloric restriction and during the last 3 weeks of caloric stabilization with a 3-h evening 1 microg/kg ovine CRH (oCRH) stimulation test. CRH-stimulated ACTH and cortisol values were compared to those of a control group of 12 normal weight women. Before caloric restriction, both ACTH and cortisol responses to oCRH were similar in obese women and normal weight controls. Weight loss did not significantly alter the ACTH response to oCRH; however, the total plasma cortisol response to oCRH decreased significantly with weight loss (area under the curve, 96,320 +/- 21,040 nmol/L x min before weight loss; 82,450 +/- 22,460 nmol/L x min after weight loss; P < 0.001). Cortisol-binding globulin also decreased significantly after weight loss (2,270 +/- 1,050 nmol/L) compared either to values obtained before weight loss (3,590 +/- 1,360 nmol/L; P < 0.001) or to those of normal weight controls (3,910 +/- 1,400 nmol/L; P < 0.001). Assay for plasma free cortisol, either before or 180 min after oCRH treatment, showed no significant changes in cortisol responses resulting from weight loss. As plasma free cortisol was not altered by weight reduction, the decrease in the total cortisol response to oCRH after weight loss appears to be secondary to significant decreases in cortisol-binding globulin. We conclude that when obese women lose large amounts of weight with a 3350 kJ/day, very low energy diet, such weight reduction does not significantly affect the HPA axis.     

Menstrual abnormalities in women with Cushing's disease are correlated with hypercortisolemia rather than raised circulating androgen levels

Menstrual irregularity is a common complaint at presentation in women with Cushing's syndrome, although the etiology has been little studied. We have assessed 45 female patients (median age, 32 yr; range, 16-41 yr) with newly diagnosed pituitary-dependent Cushing's syndrome. Patients were subdivided into 4 groups according to the duration of their menstrual cycle: normal cycles (NC; 26-30 days), oligomenorrhea (OL; 31-120 days), amenorrhea (AM; > 120 days), and polymenorrhea (PM; < 26 days). Blood was taken at 0900 h for measurement of LH, FSH, PRL, testosterone, androstenedione, dehydroepiandrosterone sulfate, estradiol (E2), sex hormone-binding globulin (SHBG), and ACTH; cortisol was sampled at 0900, 1800, and 2400 h. The LH and FSH responses to 100 micrograms GnRH were analyzed in 23 patients. Statistical analysis was performed using the nonparametric Mann-Whitney U and Spearman tests. Only 9 patients had NC (20%), 14 had OL (31.1%), 15 had AM (33.3%), and 4 had PM (8.8%), whereas 3 had variable cycles (6.7%). By group, AM patients had lower serum E2 levels (median, 110 pmol/L) than OL patients (225 pmol/L; P < 0.05) or NC patients (279 pmol/L; P < 0.05), and higher serum cortisol levels at 0900 h (800 vs. 602 and 580 nmol/L, respectively; P < 0.05) and 1800 h (816 vs. 557 and 523 nmol/L, respectively; P < 0.05) and higher mean values from 6 samples obtained through the day (753 vs. 491 and 459 nmol/L, respectively; P < 0.05). For the whole group of patients there was a negative correlation between serum E2 and cortisol at 0900 h (r = -0.50; P < 0.01) and 1800 h (r = -0.56; P < 0.01) and with mean cortisol (r = -0.46; P < 0.05). No significant correlation was found between any serum androgen and E2 or cortisol. The LH response to GnRH was normal in 43.5% of the patients, exaggerated in 52.1%, and decreased in 4.4%, but there were no significant differences among the menstrual groups. No differences were found in any other parameter. In summary, in our study 80% of patients with Cushing's syndrome had menstrual irregularity, and this was most closely related to serum cortisol rather than to circulating androgens. Patients with AM had higher levels of cortisol and lower levels of E2, while the GnRH response was either normal or exaggerated. Our data suggest that the menstrual irregularity in Cushing's disease appears to be the result of hypercortisolemic inhibition of gonadotropin release acting at a hypothalamic level, rather than raised circulating androgen levels.     

Effect of maternal betamethasone administration at midgestation on baboon fetal adrenal gland development and adrenocorticotropin receptor messenger ribonucleic acid expression

Although fetal pituitary ACTH is important to fetal adrenal growth and steroidogenesis in the second half of primate pregnancy, its role in adrenal development and function has not been established in vivo in the first half of gestation. In the present study, therefore, baboons were treated at midgestation with betamethasone to determine the effect of fetal pituitary ACTH on fetal adrenal growth, development, and ACTH receptor and P-450 enzyme messenger ribonucleic acid (mRNA) levels. The administration of betamethasone to baboon mothers on days 60-99 of gestation (term = 184 days) decreased fetal pituitary POMC mRNA levels by 54% (P < 0.01) and fetal serum ACTH levels to undetectable values (P < 0.05). The decline in ACTH was associated with decreases in fetal adrenal weight (P < 0.001), cortical cell size (P < 0.05), appearance of apoptosis and cellular disorganization, and a loss of immunocytochemically demonstrable definitive zone-specific delta5-3beta-hydroxysteroid dehydrogenase expression. The concomitant administration of ACTH and betamethasone restored these aspects of adrenal integrity to normal. Moreover, there was approximately a 95% decrease (P < 0.01) in fetal adrenal expression of ACTH receptor, P-450 cholesterol side-chain cleavage, and P-450 17alpha-hydroxylase 17/20-lyase mRNA levels after betamethasone administration. We conclude that fetal pituitary ACTH is necessary for the growth and development of fetal and definitive cortical zones and the marked coordinated increase in ACTH receptor and maintenance of P-450 cholesterol side-chain cleavage/P-450 17alpha-hydroxylase 17/20-lyase expression in the baboon fetal adrenal gland during the first half of gestation.