Effect of glucocorticoid blockade on inflammatory responses to acute sleep fragmentation in male mice.

The association between sleep and the immune-endocrine system is well recognized, but the nature of that relationship is not well understood. Sleep fragmentation induces a pro-inflammatory response in peripheral tissues and brain, but it also activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing glucocorticoids (GCs) (cortisol in humans and corticosterone in mice). It is unclear whether this rapid release of glucocorticoids acts to potentiate or dampen the inflammatory response in the short term. The purpose of this study was to determine whether blocking or suppressing glucocorticoid activity will affect the inflammatory response from acute sleep fragmentation (ASF). Male C57BL/6J mice were injected i.p. with either 0.9% NaCl (vehicle 1), metyrapone (a glucocorticoid synthesis inhibitor, dissolved in vehicle 1), 2% ethanol in polyethylene glycol (vehicle 2), or mifepristone (a glucocorticoid receptor antagonist, dissolved in vehicle 2) 10 min before the start of ASF or no sleep fragmentation (NSF). After 24 h, samples were collected from brain (prefrontal cortex, hypothalamus, hippocampus) and periphery (liver, spleen, heart, and epididymal white adipose tissue (EWAT)). Proinflammatory gene expression (TNF-α and IL-1ß) was measured, followed by gene expression analysis. Metyrapone treatment affected pro-inflammatory cytokine gene expression during ASF in some peripheral tissues, but not in the brain. More specifically, metyrapone treatment suppressed IL-1ß expression in EWAT during ASF, which implies a pro-inflammatory effect of GCs. However, in cardiac tissue, metyrapone treatment increased TNF-α expression in ASF mice, suggesting an anti-inflammatory effect of GCs. Mifepristone treatment yielded more significant results than metyrapone, reducing TNF-α expression in liver (only NSF mice) and cardiac tissue during ASF, indicating a pro-inflammatory role. Conversely, in the spleen of ASF-mice, mifepristone increased pro-inflammatory cytokines (TNF-α and IL-1ß), demonstrating an anti-inflammatory role. Furthermore, irrespective of sleep fragmentation, mifepristone increased pro-inflammatory cytokine gene expression in heart (IL-1ß), pre-frontal cortex (IL-1ß), and hypothalamus (IL-1ß). The results provide mixed evidence for pro- and anti-inflammatory functions of corticosterone to regulate inflammatory responses to acute sleep loss.

Effects of the combination of chronic unpredictable stress and environmental enrichment on anxiety-like behavior assessed using the elevated plus maze in Swiss male mice: Hypothalamus-Pituitary-Adrenal Axis-mediated mechanisms.

Environmental enrichment (EE) is a paradigm that offers the animal a plethora of stimuli, including physical, cognitive, sensory, and social enrichment. Exposure to EE can modulate both anxiety responses and plasma corticosterone. In this study, our objective was to explore how chronic unpredictable stress (CUS) impacts anxiety-related behaviors in male Swiss mice raised in EE conditions. Additionally, we investigated corticosterone and adrenocorticotropic hormone (ACTH) levels to assess the involvement of the hypothalamic-pituitary-adrenal (HPA) axis in mediating these responses. Mice were housed under either EE or standard housing conditions for 21 days. Afterward, they were exposed to 11 days of CUS while still reared in their distinct housing conditions, with half of the mice receiving daily pretreatment with the vehicle and the other half receiving daily metyrapone (MET) injections, an inhibitor of steroid synthesis, 30 mins before CUS exposure. Blood samples were obtained to assess plasma corticosterone and ACTH levels. The 11-day CUS protocol induced anxiety-like phenotype and elevated ACTH levels in EE mice. Chronic MET pretreatment prevented anxiety-like behavior in the EE-CUS groups, by mechanisms involving increased plasma corticosterone levels and decreased ACTH. These results suggest a role of the HPA axis in the mechanism underlying the anxiogenic phenotype induced by CUS in EE mice and shed light on the complex interplay between environmental factors, stress, and the HPA axis in anxiety regulation.

Prolonged Hypokalemia Following Metyrapone Treatment for Primary Bilateral Macronodular Adrenal Cortical Disease.

Surgical treatment is generally the standard therapeutic regimen used for primary bilateral macronodular adrenal cortical disease (PBMACD). However, in cases for which surgery is difficult or in which there is mild cortisol hypersecretion, metyrapone treatment can be selected. Although hypokalemia has been occasionally noted following metyrapone administration for Cushing syndrome associated with an adrenal adenoma, all such cases have been reported to be transient. Hypokalemia induced by metyrapone treatment is thought to occur due to excessive suppression of cortisol secretion, resulting in overproduction of adrenocorticotropic hormone from the pituitary gland, ultimately leading to excessive production of 11-deoxycorticosterone (DOC) in the adrenal cortex. A 52-year-old man diagnosed with PBMACD and started on metyrapone treatment subsequently presented with persistent hypokalemia. Interestingly, following initiation of metyrapone, blood test findings indicated marginal changes in serum cortisol, adrenocorticotropic hormone, and dehydroepiandrosterone sulfate levels, even when DOC levels were already markedly elevated. In addition to the effects of metyrapone, the present findings suggest a unique DOC synthesis regulatory mechanism involved in the pathogenesis of PBMACD.

Transcriptomic data of human adrenocortical NCI-H295R cells treated with cortisol biosynthesis inhibitors.

Adrenal corticosteroid biosynthesis dysregulation can give rise to various pathological conditions, such as Cushing's syndrome, a disorder characterized by the sustained and excessive production of cortisol. Despite the development of several classes of steroidogenesis inhibitors to treat human diseases associated with cortisol overproduction, their use is limited by insufficient efficacy, adverse effects, and/or tolerability. Recently, we identified a series of benzimidazolylurea derivatives, including the representative compound CJ28, as novel cortisol biosynthesis inhibitors [1]. They significantly inhibited both basal and stimulated production of cortisol in NCI-H295R cells, a human adrenocarcinoma cell line. The inhibitory effects were attributed to both attenuated steroidogenesis and de novo cholesterol biosynthesis. Here, we provide transcriptomic (RNA-seq) data from adrenal cell cultures in response to treatment with either CJ28 or metyrapone (MET), an inhibitor of 11ß-hydroxylase). Total RNA was extracted from the cells treated with vehicle (0.1% DMSO), CJ28 (30 µM), or MET (30 µM) for 24 h. Primary sequence data were acquired using paired-end sequencing on an Illumina NovaSeq 6000 platform. The raw RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database (GSE236435). This dataset is a useful resource for providing valuable information on the gene expression networks underlying adrenocortical steroidogenesis.

Discontinuation of Drug Treatment in Cushing's Disease Not Cured by Pituitary Surgery.

OBJECTIVE: When transsphenoidal surgery (TSS) does not cure Cushing's disease (CD), 4 treatments are available: drug treatment (DT), second TSS (2nd TSS), bilateral adrenalectomy (BA), and pituitary radiotherapy (PR). DT is attractive but supposes long-term continuation, which we aimed to evaluate. DESIGN AND METHODS: Retrospective study, in a center prioritizing 2nd TSS, of 36 patients, including 19 with TSS failure and 17 with recurrence, out of 119 patients with CD treated by a first TSS, average follow-up 6.1 years (95% confidence interval 5.27-6.91). Control was defined as normalization of urinary free cortisol (UFC) and final treatment (FT) as the treatment allowing control at last follow-up. We also analyzed discontinuation rates of DT in published CD prospective clinical trials. RESULTS: Control was achieved in 33/36 patients (92%). DT was initiated in 29/36 patients (81%), allowing at least 1 normal UFC in 23/29 patients (79%) but was discontinued before last follow-up in 18/29 patients (62%). DT was FT in 11/29 patients (38%), all treated with cortisol synthesis inhibitors. Second TSS was FT in 8/16 (50%), BA in 14/14 (100%), and PR in 0/5. In published trials, discontinuation of DT was 11% to 51% at 1 year and 32% to 74% before 5 years. CONCLUSION: DT allowed at least 1 normal UFC in 23/29 patients (79%) but obtained long-term control in only 11/29 (38%), as discontinuation rate was high, although similar to published data. Interestingly, a successful 2nd TSS was the cause for discontinuing efficient and well-tolerated DT in 5 patients. Further studies will show whether different strategies with cortisol synthesis inhibitors may allow for a lower discontinuation rate in patients not candidates for a 2nd TSS so that BA may be avoided in these patients.

Cortisol enhances aerobic metabolism and locomotor performance during the transition to land in an amphibious fish.

Amphibious fishes on land encounter higher oxygen (O2) availability and novel energetic demands, which impacts metabolism. Previous work on the amphibious mangrove killifish (Kryptolebias marmoratus) has shown that cortisol becomes elevated in response to air exposure, suggesting a possible role in regulating metabolism as fish move into terrestrial environments. We tested the hypothesis that cortisol is the mechanism by which oxidative processes are upregulated during the transition to land in amphibious fishes. We used two groups of fish, treated fish (+metyrapone, a cortisol synthesis inhibitor) and control (-metyrapone), to determine the impact of cortisol during air exposure (0 and 1 h, 7 days) on O2 consumption, terrestrial locomotion, the phenotype of red skeletal muscle, and muscle lipid concentration. Metyrapone-treated fish had an attenuated elevation in O2 consumption rate during the water to air transition and an immediate reduction in terrestrial exercise performance relative to control fish. In contrast, we found no short- (0 h) or long-term (7 days) differences between treatments in the oxidative phenotype of red muscles, nor in muscle lipid concentrations. Our results suggest that cortisol stimulates the necessary increase in aerobic metabolism needed to fuel the physiological changes that amphibious fishes undergo during the acclimation to air, although further studies are required to determine specific mechanisms of cortisol regulation.

Long-term Treatment With Metyrapone in a Man With Ectopic Cushing Syndrome.

This is a unique case of ectopic adrenocorticotropic hormone (ACTH)-secreting mediastinal neuroendocrine tumor, presumably thymic in origin, with suspected lung metastasis in a 61-year-old man who was successfully managed with long-term metyrapone alone. He presented with severe hypokalemia and hypertension, complicated with psychosis and vertebral collapse. He survived through a complicated course of pulseless ventricular tachycardia arrest and a severe Cushing storm due to drug nonadherence. For 3 years since diagnosis, he remained stable on metyrapone, and was able to achieve biochemical eucortisolism, with normalization of ACTH and cortisol levels. In addition, his tumor was reduced in size and the suspicious lung metastasis regressed.

Pharmacological Treatment of Cushing's Syndrome.

The 1st line treatment of Cushing's syndrome is surgery, whatever the aetiology. The role of pharmacological treatment is clear in cases where surgery fails or is impossible, in cases of metastases, or while awaiting the delayed effects of radiotherapy. However, certain situations remain controversial, in particular the possible role of pharmacological treatment as a preparation for surgery. This situation must be divided into 2 parts, severe hypercortisolism with immediate vital risk and non-severe hypercortisolism with diagnostic delay. The initiation and adjustment of treatment doses is also controversial, with the possibility of titration by gradual dose increase based on biological markers, or a more radical "block and replace" approach in which the ultimate goal is to achieve hypocortisolism, which can then be supplemented. Each of these approaches has its advantages and drawbacks and should probably be reserved for different patient profiles depending on the severity of hypercortisolism. In this review, we will focus specifically on these 2 points, namely the potential role of preoperative pharmacological treatment and, more generally, the optimal way to initiate and monitor drug treatment to ensure that eucortisolism or hypocortisolism is achieved. We will define for each part which profiles of patients should be the most adapted to try to give advice on the optimal management of patients with hypercortisolism.

Controlling intrusive thoughts of future fears under stress.

Negative outlooks of our future may foster unwanted and intrusive thoughts. To some extent, individuals have control over their ability to suppress intrusions and downregulate their frequency. Acute stress impairs intentional suppression, leading to an increased frequency of intrusions. The aim of this study was to gain insight into the mechanism underlying stress-induced impairments in intentional suppression of intrusions by investigating the combined and independent roles of the two major stress hormones, noradrenaline and cortisol. Healthy participants (N = 181) were administered propranolol (to block the noradrenergic response), metyrapone (to block the cortisol response), or a placebo before being exposed to the Maastricht Acute Stress Test. Intrusive thoughts of autobiographical future fears were then measured via the Imagine/No-Imagine task. Results demonstrated that the stress response was successfully altered because of the drug and stress manipulations. In all groups, repeated suppression of future fears reduced intrusions. Across the sample, an enhanced decrease over time was associated with greater attenuation of anxiety towards the related fears. The groups did not differ in the total frequency of intrusions. Though, trait anxiety increased the total number of intrusions. Our findings show that stress hormones did not influence the ability to suppress intrusions. However, our results do add support to previous research linking anxiety to memory control deficits. When using autobiographical content, future research should focus on the quality and characteristics of the individual memories to explain more of the variation observed in intentional memory control.

Metyrapone Treatment Protects Low-Density Lipoprotein Receptor Knockout Mice against Hypercorticosteronemia Development without Changing Atherosclerosis Susceptibility.

The steroid 11beta-hydroxylase inhibitor metyrapone is able to effectively reverse the hypercortisolemia detected in human Cushing's Syndrome patients. In this current preclinical study, we investigated whether metyrapone monotherapy can also reverse the hypercortisolemia-associated increase in atherosclerotic cardiovascular disease risk. In this instance, female low-density lipoprotein receptor knockout mice fed a cholic acid-containing high cholesterol/high fat diet to induce the development of hypercorticosteronemia and atherosclerotic lesions were treated twice daily with 100 mg/kg metyrapone for 4 weeks. Metyrapone effectively protected against hypercorticosteronemia development with endpoint plasma corticosterone levels remaining 43% lower than in controls (p < 0.01). Gene expression analysis in livers and adrenals validated that glucocorticoid receptor signaling was also reduced. Importantly, metyrapone treatment did not impact plasma cholesterol levels or alter atherosclerotic plaque areas or lesional collagen contents. However, metyrapone induced significant systemic lymphocytopenia as evident from marked decreases in splenic white pulp contents and thymus weights (-48% and -41%, respectively; p < 0.001). In conclusion, we have shown that treatment with metyrapone diminishes hypercorticosteronemia without affecting atherosclerosis susceptibility in cholic acid-containing high cholesterol/high fat diet-fed low-density lipoprotein receptor knockout mice. These preclinical findings highlight that restoring plasma glucocorticoid levels to normal is not necessarily sufficient to overcome the cardiovascular co-morbidities associated with human Cushing's disease.

Case Report: Severe McCune-Albright syndrome presenting with neonatal Cushing syndrome: navigating through clinical obstacles.

Background: Café-au-lait skin macules, Cushing syndrome (CS), hyperthyroidism, and liver and cardiac dysfunction are presenting features of neonatal McCune-Albright syndrome (MAS), CS being the rarest endocrine feature. Although spontaneous resolution of hypercortisolism has been reported, outcome is usually unfavorable. While a unified approach to diagnosis, treatment, and follow-up is lacking, herein successful treatment and long-term follow-up of a rare case is presented. Clinical case: An 11-day-old girl born small for gestational age presented with deterioration of well-being and weight loss. Large hyperpigmented macules on the trunk, hypertension, hyponatremia, hyperglycemia, and elevated liver enzymes were noted. ACTH-independent CS due to MAS was diagnosed. Although metyrapone (300 mg/m2/day) was started on the 25th day, complete remission could not be achieved despite increasing the dose up to 1,850 mg/m2/day. At 9 months, right total and left three-quarters adrenalectomy was performed. Cortisol decreased substantially, ACTH remained suppressed, rapid tapering of hydrocortisone to physiological dose was not tolerated, and supraphysiological doses were required for 2 months. GNAS analysis from the adrenal tissue showed a pathogenic heterozygous mutation. During 34 months of follow-up, in addition to CS due to MAS, fibrous dysplasia, hypophosphatemic rickets, and peripheral precocious puberty were detected. She is still regularly screened for other endocrinopathies. Conclusion: Neonatal CS due to MAS is extremely rare. Although there is no specific guideline for diagnosis, treatment, or follow-up, addressing side effects and identifying treatment outcomes will improve quality of life and survival.

Optimal timing of oral metyrapone intake for the suppression of cold-pressor stress-induced cortisol release.

BACKGROUND: Pharmacological manipulation of cortisol levels is instrumental in elucidating mechanisms underlying acute stress effects and for distinguishing the physiological and behavioral effects of cortisol from those of the adrenergic system. Administration (oral or IV) of hydrocortisone is a direct and efficient method to elevate cortisol, and thus, frequently used in psychobiological stress research. However, lowering of cortisol (i.e. blockade of stress cortisol) requires a more sophisticated approach, such as the administration of the corticostatic compound metyrapone (MET). However, there is insufficient knowledge about the temporal dynamics of MET for the blocking of stress-induced cortisol reactivity. Thus, the present study aimed to build up an experimental protocol suitable to suppress acute behavioral stress-induced cortisol secretion by MET. METHODS: 50 healthy young men were randomly assigned to one of five treatment groups. They received 750 mg oral MET either 30 (n = 9), 45 (n = 11), or 60 (n = 10) minutes before exposure to a combined cold pressor and mental arithmetic test (stress induction), or were subjected to two different control treatments (placebo 60 min before stress (n = 10) or MET 30 min before non-stressful warm-water condition (n = 10)). Salivary cortisol concentration, hemodynamics, and subjective ratings were assessed. RESULTS: Suppression of cold stress-induced cortisol release was strongest when MET intake was scheduled 30 min prior to stress onset. Cardiovascular stress-responses and subjective ratings remained unaffected by MET. CONCLUSION: In healthy young males, 750 mg of MET efficiently block cold stress-induced cortisol release when oral administration is scheduled 30 min prior to stress onset. This finding may guide future research in improving timing of suppression of stress-induced cortisol secretion.

Morning salivary dehydroepiandrosterone (DHEA) qualifies as the only neuroendocrine biomarker separating depressed patients with and without prior history of depression: An HPA axis challenge study.

BACKGROUND: Hypothalamic-pituitary-adrenal (HPA) axis abnormalities in major depression (MDD) have been consistently reported in psychiatry and extend to several neurosteroids. However, recurrence and chronicity may heavily influence HPA axis dynamics in MDD along its course and also explain conflicting results in literature. Thus, the mechanistic understanding of HPA axis (re)activity changes over time could be of major importance for unravelling the dynamic pathophysiology of MDD. METHODS: This study simultaneously assessed several baseline and dynamic HPA-axis-related endocrine biomarkers in both saliva (dehydroepiandrosterone, DHEA; sulfated DHEA, DHEA-s; cortisol, CORT) and plasma (CORT; adrenocorticotropic hormone, ACTH; copeptin, CoP) over three consecutive days using overnight HPA axis stimulation (metyrapone) and suppression (dexamethasone) challenges in order to investigate differences between antidepressant-free MDD patients (n = 14) with and without history of prior depressive episodes (i.e., first vs. recurrent episode). RESULTS: Our results suggest group differences only with respect to saliva DHEA levels, with recurrent-episode MDD patients showing overall lower saliva DHEA levels across the three days, and statistically significant differences mainly at day 1 (baseline) across all three timepoints (awakening, +30 min, +60 min), even after adjustment for confounders. CONCLUSIONS: Our study supports that salivary DHEA levels could represent a significant biomarker of MDD progression and individual stress resilience. DHEA deserves additional attention in the research of pathophysiology, staging and individualized treatment of MDD. Prospective longitudinal studies are needed to evaluate HPA axis reactivity along MDD course and progression to better understand temporal effects on stress-system-related alterations, related phenotypes and appropriate treatment.

LC-MS/MS method for simultaneous quantification of osilodrostat and metyrapone in human plasma from patients treated for Cushing's Syndrome.

Steroidogenesis inhibitors such as metyrapone (MTP) and osilodrostat (ODT) have a key role in the medical treatment of endogenous Cushing's Syndrome (ECS). Both drugs are characterized by a high inter-individual variability of response and require a dose-titration period to achieve optimal control of cortisol excess. However, PK/PD data remain scarce for both molecules and a pharmacokinetically guided approach could help reaching eucortisolism more rapidly. We aimed to develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ODT and MTP in human plasma. After addition of isotopically labeled internal standard (IS), plasma pretreatment consisted in protein precipitation with acetonitrile including 1% formic acid (v/v). Chromatographic separation was performed on Kinetex® HILIC (4.6 × 50 mm; 2.6 µm) analytical column with an isocratic elution during the 2.0-min run time. The method was linear from 0.5 to 250 ng/mL for ODT and from 2.5 to 1250 ng/mL for MTP. Intra- and inter-assay precisions were < 7.2%, with an accuracy ranging from 95.9% to 114.9%. The IS-normalized matrix effect ranged from 106.0% to 123.0% (ODT) and from 107.0% to 123.0% (MTP) and the range of the IS-normalized extraction recovery was 84.0-101.0% for ODT and 87.0-101.0% for MTP. The LC-MS/MS method was successfully applied in patients' plasma samples (n = 36), trough concentration of ODT and MTP ranged from 2.7 ng/mL to 8.2 ng/mL and from 10.8 ng/mL to 27.8 ng/mL, respectively. Incurred sample reanalysis exhibits less than 14% difference between the first and the second analysis for both drugs. This accurate and precise method, meeting all validation criteria, can therefore be used for plasma drug monitoring of ODT and MTP within the dose-titration period.

An evaluation of pharmacological options for Cushing's disease: what are the state-of-the-art options?

INTRODUCTION: Untreated Cushing's syndrome (CS) is associated with significant morbidity and mortality. Cortisol normalization is a key goal to treatment. Pituitary surgery remains the first-line approach for Cushing's disease, but sometimes it is impracticable, unsuccessful, or complicated by recurrence. Medical therapy has been historically considered a palliative. However, in the latest years, interest on this topic has grown due to both the availability of new drugs and the reevaluation of the old, commonly used drugs in clinical practice. AREAS COVERED: In this article, we will discuss the current options and future directions of medical therapy for CS, aiming at fitting best patients' features. An extensive literature search regarding already approved and investigational principles was conducted (PubMed, ClinicalTrials.gov. Available drugs include inhibitors of ACTH secretion, steroidogenesis inhibitors, and glucocorticoid receptor antagonists; drugs acting at different levels can be also combined in uncontrolled patients. EXPERT OPINION: Since there is still no standardized pharmacological approach and the superiority of one drug over another has not been established yet in the absence of comparative studies, each time clinicians' choices should be patient-tailored. Age, gender, tumor features, severity of hypercortisolism, comorbidities/complications, rapidity of action, side effects, drug-drug interactions, contraindications, availability, patients' preferences, and costs should be all considered.

Metyrapone abolishes spike-wave discharge seizures in genetic absence epilepsy rats from Strasbourg by reducing stress hormones.

OBJECTIVE: Stress is one of the most commonly reported triggers for seizures in patients with epilepsy, although the mechanisms that mediate this effect are not established. The clinical evidence supporting this is derived from patients' subjective experience of stress, and how this influences their own seizures. Animal models can be used to explore this phenomenon in controlled environments, free from subjective bias. Here, we used genetic absence epilepsy rats from Strasbourg (GAERS), a genetic rat model of absence epilepsy, to explore the influence of stress and stress hormones on spontaneous seizures. METHODS: Adult male GAERS (n = 38) and nonepileptic control (NEC) rats (n = 4) were used. First, rats were subjected to 30-min restraint stress to assess hypothalamic-pituitary-adrenal axis function. Next, we assessed the effects of 30-min noise stress, and cage tilt stress, on spike-wave discharge seizures in GAERS. We then performed pharmacological experiments to assess the direct effects of stress hormones on seizures, including corticosterone, metyrapone, and deoxycorticosterone. RESULTS: GAERS exhibited elevated baseline corticosterone levels, compared to NEC rats. Noise stress and cage tilt stress significantly enhanced seizure incidence (p < .05), but only during stress periods. Exogenous corticosterone administration also significantly increased seizure occurrence (p < .05). Metyrapone, an inhibitor of corticosterone synthesis, completely abolished seizures in GAERS, and seizures remained suppressed for >2 h. However, deoxycorticosterone, the precursor of corticosterone, increased seizures. SIGNIFICANCE: These results suggest that GAERS exhibit elevations in stress hormones, and this may contribute to seizures. Inhibiting corticosterone synthesis with metyrapone prevents seizures in GAERS, and shows potential for repurposing this drug as a future antiseizure medication.

The resilience of adolescent male rats to acute stress-induced delayed anxiety is age-related and glucocorticoid release-dependent.

Studies investigated how stressful experiences modulate physiological and behavioral responses and the consequences of stress-induced corticosterone release in anxiety-like behavior. Adolescence is crucial to brain maturation, and several neurobiological changes in this period lead individuals to increased susceptibility or resilience to aversive situations. Despite the effects of stress in adults, information about adolescents' responses to acute stress is lacking. We aimed to understand how adolescence affects acute stress responses. Male adolescent rats (30 days old) were 2 h restrained, and anxiety-like behaviors were measured immediately or 10 days after stress in the elevated plus-maze (EPM) and the light-dark box (LDB) tests. To verify the importance of CORT modulation in stress-induced anxiety, another group of rats was treated, 30 min before restraint, with metyrapone to blunt the stress-induced CORT peak and tested immediately after stress. To show that stress effects on behavior were age-dependent, another set of rats was tested in two different periods - early adolescence (30 days old) and mid-adolescence (40 days old) and were treated or not with metyrapone before the stress session and tested immediately or ten days later in the LDB test. Only early adolescent male rats were resilient to delayed anxiety-like behavior in EPM and LDB tests. Metyrapone treatment increased the rats' exploration immediately and ten days after stress. These data suggest a specific age at which adolescent rats are resilient to the delayed effects of acute restraint stress and that the metyrapone treatment has long-term behavioral consequences.

How best to monitor the specific side effects of medical treatments of Cushing's disease.

The first-line treatment of Cushing's disease is transsphenoidal surgery. Medical treatment of Cushing's disease can be considered in several situations: as a presurgical treatment in patients with severe comorbidities, when surgery fails, or while waiting for the maximal efficacy of radiation techniques. Several modalities of medical treatment are possible, from adrenal-targeting drugs (steroidogenesis inhibitors) to pituitary-targeting drugs (somatostatin receptor ligand pasireotide or the dopamine agonist cabergoline), or even drugs that antagonize the glucocorticoid receptor (mifepristone). Given the morbidities associated with hypercortisolism, and the fact that medical treatment can be delivered on a long-term basis, it is important to obtain eucortisolism and to monitor the drug effectively. The efficacy of these drugs will not be detailed in this review, nor their roles in the therapeutic algorithm of Cushing's disease. This review will rather focus specifically on adverse events associated with these drugs (ketoconazole, levoketoconazole, metyrapone, osilodrostat, pasireotide, cabergoline and mifepristone), and the way in which to monitor and treat them, based on retrospective studies and the most recently published prospective studies.

Evaluation of plasma ACTH in the metyrapone test is insufficient for the diagnosis of secondary adrenal insufficiency.

Objective: To determine whether a single measurement of ACTH instead of less available in daily practice 11-deoxycortisol assay is sufficient to rule out or confirm secondary adrenal insufficiency (SAI) in the short Metyrapone test. Design: A retrospective analysis of diagnostic tests (Metyrapone and Synacthen tests) performed at our Center between 2016 and 2018 in patients with suspicion of secondary adrenal insufficiency. Material and methods: In 103 patients short metyrapone test was performed with assessment of 11-deoxycortisol and ACTH concentration after Metyrapone administered at midnight. In 89 of them short Synacthen (SST) test was also done (1 or/and 250 mcg 1-24ACTH). ROC curves have been performed to evaluate the diagnostic performance of ACTH level in metyrapone test as the predictor of secondary adrenal insufficiency (SAI) analysing sensitivity and specificity for various possible thresholds proposed in literature. Results: 40 (39%) of examined subjects were diagnosed as SAI, basing on post-Metyrapone 11-deoxycortisol concentration below 70 µg/l. In this group ACTH concentration was 128.1 ng/l (95% CI 96.8-159.4) versus 289.9 ng/l (95% CI 249.1-330.9) in patients with proper adrenal response. There was only a moderate positive correlation between ACTH and 11-deoxycortisol concentrations (r=0.5; p<0.05). The best cut off value of ACTH in relation to 11-deoxycortisol serum concentrations was 147 ng/l - with sensitivity of 73.2% and specificity 83.9%. However, plasma ACTH was>200ng/ml (the highest threshold proposed in literature) in 8 cases (20%) with positive diagnosis of SAI made on the basis of low 11-deoxycortisole and confirmed in short Synacthen test. Conclusion: Our results indicate that for a valuable evaluation of the results of the metyrapone test, the more readily available plasma ACTH assay cannot replace the measurement of 11-deoxycortisol concentrations.