Episodic secretion of ACTH in rats

While the circadian rhythm of pituitary adrenocorticotropin (ACTH) secretion has been well characterized, the ultradian rhythm has been less thoroughly investigated. To study the episodic nature of ACTH secretion, unrestrained, unanesthetized rats were bled continuously through indwelling jugular venous cannulae and blood sampled for up to 75 minutes at one-minute intervals beginning at 1100 hr (n=6) or 1730 hr (n=4). Sporadic low-amplitude micropulses were observed at both times of day. In addition, infrequent "superpulses" were observed in the evening. Analysis of pulse parameters revealed a significant (p less than 0.001) difference in pulse amplitude but no difference in pulse frequency of interpeak interval between morning and evening. As with other episodically secreted hormones, the threshold for pulse identification and the sampling interval were found to influence the observed pulse parameters.     

Seasonal, circadian and episodic variations of human immunoreactive beta-MSH, ACTH and cortisol

The 24-hour profile of plasma levels of immunoreactive beta-MSH (IR-beta MSH), ACTH and cortisol was obtained at 15-min intervals in six normal males in summer and winter. In the radioimmunoassay used, dilution curves of human beta-MSH and human beta-LPH were not parallel. A seasonal variation in basal pituitary-adrenal secretion, with higher levels in winter than in summer, was demonstrated. A circadian rhythm was found to be significant for ACTH, IR-beta MSH and cortisol in all investigations. Whereas ACTH and cortisol patterns were largely concordant in all studies, there was a significant desynchronization of the circadian rhythm of IR-beta MSH as compared to ACTH in five cases. Eighty-three percent of the secretory spikes of cortisol but only 68% of the IR-beta MSH spikes were concomitant with an ACTH spike. Correlations between maximal levels of concomitant spikes were higher during the quiescent period of pituitary-adrenal secretion (22.00--04.00) for ACTH-cortisol whereas for ACTH-IR-beta MSH, highest correlations were found during the active early morning secretory phase (04.00--10.00). For the three plasma constituents studied, longer apparent half-lives were found to occur when the basal level before spiking was already elevated, suggesting that ACTH, beta-LPH and cortisol are secreted in bursts superimposed on a continuous basal secretion. Absolute increments of concentration appeared to be relatively independent of the level before spiking. It is suggested that the dissociations between ACTH and IR-beta MSH fluctuations in plasma observed here result from in vitro proteolysis of beta-LPH.     

Response to low-dose pulsatile cortisol in Addison's disease with suspected corticotropinoma

A 65 year old woman with long-standing Addison's disease treated with oral glucocorticoid and mineralocorticoid replacement had persistently high ACTH levels, inadequate suppression of ACTH on low-dose dexamethasone, sellar enlargement, and pigmentation, and thus resembled patients alleged to develop corticotropinomas while on oral replacement for adrenal insufficiency. Since animal studies suggested that rapid rises of corticosteroids within the physiologic range can inhibit ACTH release, we administered brief infusions of cortisol every three hours with total daily dose equal to her chronic dose. Prompt suppression of ACTH and immunoreactive beta-endorphin occurred during each cortisol dose profiled, suggesting a role for ultradian cortisol fluctuations in tonic inhibition of ACTH secretion in humans, and a possible therapeutic benefit of mimicking ultradian cortisol rhythms during replacement therapy.     

Circadian cortisol secretion and circadian adrenal responses to ACTH are maintained in dexamethasone suppressed capuchin monkeys (Cebus apella)

We tested the hypothesis that the capuchin monkey adrenal (Cebus apella) gland has oscillatory properties that are independent of adrenocorticotropic hormone (ACTH) by exploring under ACTH suppression by dexamethasone: (i) maintenance of a circadian rhythm of plasma cortisol and (ii) clock time dependency of plasma cortisol response to exogenous ACTH. The capuchin monkey had a clear ACTH and plasma cortisol rhythm. Dexamethasone treatment resulted in low non-rhythmic ACTH levels and decreased cortisol to 1/10 of control values; nevertheless, the circadian rhythm of plasma cortisol persisted. We found that cortisol response to exogenous ACTH was clock time-dependent. The maximal response to ACTH occurred at the acrophase of the cortisol rhythm (0800 h). These results suggest that the capuchin monkey adrenal cortex may possess intrinsic oscillatory properties that participate in the circadian rhythm of adrenal cortisol secretion and in the circadian cortisol response to ACTH.     

Neuro-endocrine correlations of hypothalamic-pituitary-thyroid axis in healthy humans

Neuro-endocrine hormone secretion is characterized by circadian rhythmicity. Melatonin, GRH and GH are secreted during the night, CRH and ACTH secretion peak in the morning, determining the circadian rhythm of cortisol secretion, TRH and TSH show circadian variations with higher levels at night. Thyroxine levels do not change with clear circadian rhythmicity. In this paper we have considered a possible influence of cortisol and melatonin on hypothalamic-pituitary-thyroid axis function in humans. Melatonin, cortisol, TRH, TSH and FT4 serum levels were determined in blood samples obtained every four hours for 24 hours from ten healthy males, aged 36-51 years. We correlated hormone serum levels at each sampling time and evaluated the presence of circadian rhythmicity of hormone secretion. In the activity phase (06:00 h-10:00 h-14:00 h) cortisol correlated negatively with FT4, TSH correlated positively with TRH, TRH correlated positively with FT4 and melatonin correlated positively with TSH. In the resting phase (18:00 h-22:00 h-02:00 h) TRH correlated positively with FT4, melatonin correlated negatively with FT4, TSH correlated negatively with FT4, cortisol correlated positively with FT4 and TSH correlated positively with TRH. A clear circadian rhythm was validated for the time-qualified changes of melatonin and TSH secretion (with acrophase during the night), for cortisol serum levels (with acrophase in the morning), but not for TRH and FT4 serum level changes. In conclusion, the hypothalamic-pituitary-thyroid axis function may be modulated by cortisol and melatonin serum levels and by their circadian rhythmicity of variation.     

Temporal characteristics of cortisol metabolism in adrenalectomized primates

The rhythmicities observed in the plasmatic levels of cortisol are generally attributed to rhythms of production and release of the hormone. Since the plasmatic concentration of any given substance is a function of its production and its removal from the circulation, it is conceivable that the metabolism of cortisol also occurs in an oscillating fashion. To test this hypothesis Rhesus monkeys were submitted to bilateral adrenalectomy; cortisol was replaced at a constant infusion rate while blood was sampled at hourly intervals for the measurement of cortisol plasma levels. Rhythmic oscillations in the cortisol levels were observed. These rhythms exhibited two major components: a circadian and an ultradian component. The authors suggest that these rhythms be considered whenever normal or pathologic hormone rhythmicities are analyzed.     

Age-related changes in diurnal rhythms and levels of gonadotropins, testosterone, and inhibin B in male rhesus monkeys (Macaca mulatta)

Testosterone shows circadian rhythms in monkeys with low serum levels in the morning hours. The decline relies on a diminished frequency of LH pulses. Inhibin B shows no diurnal patterns. In elderly men, the diurnal rhythm of testosterone is blunted and inhibin levels fall. Here we explore whether aging exerts similar effects in the rhesus monkey. We collected blood samples from groups of young (6-9 yr) and old (12-16 yr) male rhesus monkeys at 20-min intervals for a period of 24 h under remote sampling via a venous catheter. We determined moment-to-moment changes in plasma levels of testosterone, FSH, and LH by RIA, and of inhibin B by ELISA. We found significant diurnal patterns of testosterone in both groups. The circadian rhythm in testosterone was enhanced in older monkeys. Testosterone levels and pulse frequencies dropped significantly below those of young monkeys during midday hours. Diminished pulse frequency of LH appeared to be responsible for the midday testosterone decrease in old monkeys, while LH and testosterone pulse frequency did not change in young monkeys at corresponding time points. Old monkeys showed extended periods of LH-pulse quiescence in the morning and midday hours. Inhibin B and FSH levels were generally lower in old monkeys compared with the young group, but neither inhibin B nor FSH showed circadian rhythms. We conclude from these data that old rhesus monkeys have a more prominent circadian rhythm of LH and testosterone resulting from an extended midday period of quiescence in the hypothalamus-pituitary-gonadal axis.     

A stochastic differential equation model of diurnal cortisol patterns

Circadian modulation of episodic bursts is recognized as the normal physiological pattern of diurnal variation in plasma cortisol levels. The primary physiological factors underlying these diurnal patterns are the ultradian timing of secretory events, circadian modulation of the amplitude of secretory events, infusion of the hormone from the adrenal gland into the plasma, and clearance of the hormone from the plasma by the liver. Each measured plasma cortisol level has an error arising from the cortisol immunoassay. We demonstrate that all of these three physiological principles can be succinctly summarized in a single stochastic differential equation plus measurement error model and show that physiologically consistent ranges of the model parameters can be determined from published reports. We summarize the model parameters in terms of the multivariate Gaussian probability density and establish the plausibility of the model with a series of simulation studies. Our framework makes possible a sensitivity analysis in which all model parameters are allowed to vary simultaneously. The model offers an approach for simultaneously representing cortisol's ultradian, circadian, and kinetic properties. Our modeling paradigm provides a framework for simulation studies and data analysis that should be readily adaptable to the analysis of other endocrine hormone systems.     

Hypothalamic pituitary adrenal function in patients with anorexia nervosa.

Hypothalamic pituitary adrenal function was studied in 14 patients with anorexia nervosa. Although basal plasma cortisol levels in the morning were elevated in most cases, basal plasma ACTH levels were not suppressed. Oral administration of 1 mg dexamethasone 10 hr before blood sampling failed to suppress plasma ACTH and cortisol levels in most patients with anorexia nervosa. Apparent biological half-life of exogenous cortisol was prolonged in all 4 patients with anorexia nervosa tested. The cortisol response to insulin-induced hypoglycemia and exogenous ACTH appeared to be blunted in these patients. It is concluded that anorexia nervosa has dysfunctions of hypothalamic pituitary adrenal axis, especially an abnormal feedback mechanism on ACTH secretion.     

Patterns of serum cortisol levels in ovariectomized females with and without androgen administration.

Serum cortisol levels in humans and primates display a circadian rhythm. A study in monkeys showed that orchiectomy abolishes this circadian rhythm. The present study compared the patterns of serum cortisol levels between 9 a. m. and 3 p. m. in two groups of ovariectomized females, one treated with testosterone (T) and one without sex steroid administration. Over the first 80 minutes of the sampling period cortisol levels declined similarly in both groups, probably due to waning of stress of the experiment. Thereafter, levels fell further in the T-administered group, but not in the group without sex steroid administration. From this pilot study it is tentatively concluded that ovariectomy, in analogy with orchiectomy in monkeys, produces a loss of a circadian pattern of cortisol levels, which is reversed by administration of T. Furthermore, upon comparison of mean serum cortisol levels in the two groups, T appeared to have a suppressive effect on values of serum cortisol levels.     

Suppression of basal plasma cortisol and adrenal androgen concentrations, but not of ACTH and cortisol responses to hCRH by low-dose dexamethasone in rhesus monkeys

Glucocorticoid treatment at replacement doses does not result in a suppression of ACTH and cortisol responses to corticotropin-releasing hormone (CRH), while basal plasma concentrations of cortisol and adrenal androgens are efficiently suppressed 34 h after starting treatment. This finding could be demonstrated in rhesus monkeys receiving a continuous infusion of dexamethasone (1 microgram/kg per h) for 48 h and confirms our observations in patients on alternate-day prednisone therapy and in patients with congenital adrenal hyperplasia on glucocorticoid replacement therapy. We conclude that the decrease of basal adrenal steroid secretion resulting from glucocorticoid replacement therapy represents an effect on hypothalamic rather than on pituitary function.     

Steroid secretion by ACTH-stimulated human fetal adrenal tissue during the first week in organ culture

Fetal adrenal tissue has been reported to lose its in vivo secretory pattern by virtue of a loss of fetal zone cells after the first week in culture. Consequently, we studied the steroidogenic capacity and the responsiveness to ACTH of human fetal adrenal tissue during the first week in organ culture. The culture medium was removed daily and assayed for cortisol and dehydroisoandrosterone sulfate (DS). First, as the concentration of ACTH in the medium was increased from 0 to 1 micrograms/ml steroid secretion increased. When tissue fragments were maintained in the absence of ACTH for 3 to 4 days, there was a striking increase in steroid secretion upon addition of ACTH to the medium, with larger rates of secretion of cortisol than DS being observed. Second, the steroidogenic capacity of the separate zones of the fetal adrenal gland was assessed. Tissue from the fetal zone secreted large amounts of DS and small amounts of cortisol, whereas neocortex tissue secreted similar quantities of DS and cortisol. Third, fetal zone tissue was maintained the absence of ACTH for 4 days and thereafter ACTH was added to the media for an additional 6 days. In this experiment, there was a marked increase in DS secretion rate after the addition of ACTH and a smaller increase in cortisol secretion.     

Influence of the hypothalamic-pituitary-adrenal axis on the menstrual cycle and the pituitary responsiveness to estradiol in the female rhesus monkey (Macaca mulatta)

In order to examine the effect of glucocorticoids on the menstrual cycle of rhesus monkeys, cortisol was injected twice daily during the follicular phase. This cortisol treatment did not alter basal gonadotropin secretion but blocked the normal follicular rise of estrogens, the gonadotropin surge and the luteal rise of progesterone, and delayed the onset of the next cycle. In a second study, estradiol benzoate (E2B) was injected on the sixth day following the start of menstrual bleeding either with or without concurrent adrenocorticotropic hormone (ACTH) treatment. E2B injection was able to stimulate surges of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) whether or not the animals had been treated with ACTH. These data suggest that, the action of cortisol, the final mediating step in the hypothalamic-pituitary-adrenal axis, occurs at the level of the gonads versus the pituitary in the rhesus monkey. While the pituitary response to endogenous gonadotropin-releasing hormone or exogenous E2B stimulation appears to remain unaffected, normal folliculogenesis is disrupted, preventing the follicular secretion of estrogens and the subsequent gonadotropin surges. The effects of corticosteroids are temporary, with normal cycling returning when plasma corticosteroids return to basal concentrations, albeit after a delay.     

Noninvasive technique for the repeated sampling of salivary free cortisol in awake,unrestrained squirrel monkeys

The use of noninvasive measures of hypothalamic-pituitary-adrenal (HPA) axis function is of growing interest among preclinical and clinical investigators. This report describes a method for the repeated assessment of salivary free cortisol in awake, unrestrained squirrel monkeys (Saimiri sciureus) based on a saliva sampling technique previously developed for rhesus monkeys. Individually housed adult male squirrel monkeys were trained to chew on dental rope attached to a pole, from which saliva was extracted by centrifugation and analyzed for cortisol by radioimmunoassay (RIA). Eight of nine monkeys readily acquired the task, reliably providing adequate saliva samples for the assay. Salivary free cortisol levels were examined in these subjects under basal conditions and in response to two types of neuroendocrine challenge. Levels of salivary free cortisol showed relatively low intra- and interindividual variability, with mean individual morning levels ranging between 17.1 and 37.9 microg/dl. Squirrel monkeys demonstrated a consistent daily rhythm in salivary free cortisol ranging from a high of 27.4 +/- 5.2 microg/dl (mean +/- SEM) at 12 P.M. to a low of 7.5 +/- 1.6 microg/dl at 6 P.M. Intravenous (IV) challenges with 1 microg/kg ACTH, or 10 and 50 microg/kg CRF resulted in significant increases in salivary free cortisol. The described sampling technique provides a reliable and sensitive means for repeated measurement of HPA activity in unrestrained, awake squirrel monkeys. In addition, our findings illustrate several features of HPA system rhythmicity and reactivity using salivary cortisol instead of blood plasma or serum.     

Effect of aminoglutethimide on ACTH plasma levels in Cushing's disease and Nelson syndrome]

Out of all steroidogenesis inhibitors aminoglutethimide is most frequently used agent for so-called chemical adrenalectomy, especially in oncological cases. The present studies aimed at assessing an effect of the inhibition of cortisol synthesis on plasma ACTH in patients treated with aminoglutethimide. According to the rules of negative feedback, an increase in plasma ACTH should be expected. Aminoglutethimide has been administered to 24 patients with Cushing's disease for 1-6 months. Plasma ACTH did not increase but statistically significantly decreased despite a decrease in blood cortisol. It indicates that aminoglutethimide directly inhibits ACTH secretion. No return of the normal circadian rhythm of cortisol and ACTH release suggests that the drug exerts an effect on ACTH release regulating mechanisms. No definite results were achieved in patients with Nelson syndrome treated with aminoglutethimide for a short period of time. Plasma ACTH levels tend to decrease but no statistical significance was observed in comparison with placebo. It may depend on markedly increased corticotrophin secretion in Nelson tumors.     

Detecting regulatory mechanisms in endocrine time series measurements

The regulatory mechanisms underlying pulsatile secretion are complex, especially as it is partly controlled by other hormones and the combined action of multiple agents. Regulatory relations between hormones are not directly observable but may be deduced from time series measurements of plasma hormone concentrations. Variation in plasma hormone levels are the resultant of secretion and clearance from the circulation. A strategy is proposed to extract inhibition, activation, thresholds and circadian synchronicity from concentration data, using particular association methods. Time delayed associations between hormone concentrations and/or extracted secretion pulse profiles reveal the information on regulatory mechanisms. The above mentioned regulatory mechanisms are illustrated with simulated data. Additionally, data from a lean cohort of healthy control subjects is used to illustrate activation (ACTH and cortisol) and circadian synchronicity (ACTH and TSH) in real data. The simulation and the real data both consist of 145 equidistant samples per individual, matching a 24-hr time span with 10 minute intervals. The results of the simulation and the real data are in concordance.     

The inhibitory action of corticotropin-releasing hormone on gonadotropin secretion in the ovariectomized rhesus monkey is not mediated by adrenocorticotropic hormone

Earlier observations in our laboratory indicated that i.v. infusion of human/rat corticotropin-releasing hormone (hCRH) suppresses pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in ovariectomized rhesus monkeys. Since cortisol secretion increased significantly as well, it was not possible to exclude the possibility that this inhibitory effect of hCRH on gonadotropins was related to the activation of the pituitary/adrenal axis. The purpose of the present study was to determine the role of pituitary/adrenal activation in the effect of hCRH on LH and FSH secretion. We compared the effects of 5-h i.v. infusions of hCRH (100 micrograms/h, n = 7) and of human adrenocorticotropic hormone (ACTH) (1-24) (5 micrograms/h, n = 3; 10 micrograms/h, n = 3, 20 micrograms/h, n = 3) to ovariectomized monkeys on LH, FSH, and cortisol secretion. As expected, during the 5-h ACTH infusions, cortisol levels increased by 176-215% of baseline control, an increase similar to that observed after CRH infusion (184%). However, in contrast to the inhibitory effect observed during the CRH infusion, LH and FSH continued to be released in a pulsatile fashion during the ACTH infusions, and no decreases in gonadotropin secretion were observed. The results indicated that increases in ACTH and cortisol did not affect LH and FSH secretion and allowed us to conclude that the rapid inhibitory effect of CRH on LH and FSH pulsatile release was not mediated by activation of the pituitary/adrenal axis.     

Immunoneutralization of corticotropin-releasing hormone prevents the diurnal surge of ACTH

To determine whether CRH is required for the evening rise in plasma ACTH, rats were injected at 0800 hr with CRH antiserum (anti-CRH) or normal rabbit serum (NRS). Blood samples were taken through venous catheters at 0800 hr before treatment and at 1300, 1700, and 2100 hr. Plasma was assayed for immunoreactive ACTH and corticosterone. There was no significant difference in pretreatment values between the two groups. Immunoneutralization of CRH abolished the rise in plasma ACTH seen at 1700 hr in the NRS group but had little effect on earlier levels. The diurnal elevation in plasma corticosterone continued after anti-CRH treatment, but peak levels occurred earlier. Plasma ACTH and corticosterone were significantly correlated at the time of the diurnal surge, but not at 0800 hr or 1300 hr in the NRS controls or at any time point in the anti-CRH group. These results suggest that CRH is required for the diurnal surge of plasma ACTH. They also confirm previous observations by others that the adrenal cortex does not require active CRH or a diurnal surge of ACTH in order to exhibit a significant diurnal increase in secretion of corticosterone, and that factors other than CRH may be relatively more active than CRH in regulation of ACTH secretion during the time of circadian inactivity.     

Differences in cortisol, aldosterone and testosterone responses to ACTH 1-17 administered at two different times of day

The effects of 100 micrograms, i.m. of the analog ACTH 1-17 administered at 0800 and 1800 on the secretion of cortisol, aldosterone and testosterone have been studied in normal subjects: 8 male and 8 female. The group as a whole and the males had significantly greater absolute and percent increments in plasma cortisol after administration at 1800. In the females, there was only a greater percent increment in cortisol after the evening administration. The heptadecapeptide always significantly stimulated serum aldosterone, with no difference between the two times of administration. In the females, ACTH 1-17 significantly stimulated testosterone, with a more protracted secretion after the evening administration. In the males, there was always a significant testosterone decrease after the administration of the drug, with no difference between morning and evening. In conclusion, 100 micrograms i.m. of the analog ACTH 1-17 stimulates cortisol secretion more when given during the circadian nadir of plasma cortisol, but only in men. ACTH 1-17 increases testosterone in women and decreases it in men, whereas it seems to increase aldosterone secretion in both sexes.     

Circadian and circannual rhythms of hormonal variables in elderly men and women

A group of fourteen men (73 ± 5 yr of age), and eighteen women (77 ± 7 yr of age) institutionalized at the Berceni Clinical Hospital, Bucharest, Romania, were studied over a 24-hr span once during each season (winter, spring, summer and fall). All subjects followed a diurnal activity pattern with rest at night and ate three meals per day with breakfast at about 0830, lunch at about 1300 and dinner at about 1830. The meals were similar, although not identical for all subjects during all seasons. On each day of sampling blood was collected at 4-hr intervals over a 24-hr span. Seventeen hormonal variables were determined by radioimmunoassay. Statistically significant circadian rhythms were detected and quantitated by population mean cosinor analysis in pooled data from all four seasons in both sexes for ACTH, aldosterone, Cortisol, C-peptide, dehydroepiandrosterone-sulfate (DHEA-S), immunoreactive insulin, prolactin, 17-OH progesterone, testosterone, total T₄ and TSH. In women, estradiol and progesterone also were determined and showed a circadian rhythm during all seasons. Total T, and FSH showed circadian rhythm detection by cosinor analysis in the men only; LH showed no consistent circadian rhythm as group phenomenon in men or women.

A circannual rhythm was detected using the circadian means of each subject at each season as input for the population mean cosinor in the women for ACTH, C-peptide, DHEA-S, FSH, LH, progesterone, 17-OH progesterone and TSH. In the men, a circannual rhythm was detected for ACTH, FSH, insulin, LH, testosterone and T₃. There were phase differences between men and women in ACTH, FSH and LH. In those functions in which both the circadian and circannual rhythms were statistically significant, a comparison of the amplitudes showed in the women a higher circannual rather than circadian amplitude for DHEA-S. In 17-OH progesterone, TSH and C-peptide, the circadian amplitude in women was larger. In men, the circannual amplitude of T₃ was larger than the circadian amplitude and in insulin the circadian amplitude was larger than the circannual amplitude. There was no statistically significant difference between the circadian and circannual amplitudes in the women in ACTH and progesterone and in the men in ACTH and testosterone.