Adrenal function in relation to cytokines and outcome in non-critically ill patients with COVID-19.

PURPOSE: We aimed to identify whether hypothalamic-pituitary-adrenal (HPA) axis dysfunction is related to deterioration in a percentage of patients who progress to severe COVID-19. METHODS: In this cohort observational study, we evaluated HPA axis activation by measuring cortisol, adrenocorticotropic hormone (ACTH), dehydroepiandrosterone sulfate (DHEA-S) levels, whole blood expression levels of the key glucocorticoid receptor, GCR-α, and the glucocorticoid-induced leucine zipper (GILZ), and cytokines, as markers of the inflammatory phase, in 149 patients with respiratory infection admitted in the ward, without known adrenal disease and/or confounding medications (glucocorticoids). One hundred and four (104) patients were SARS-CoV-2 positive (C +) and controls consisted of 45 SARS-CoV-2-negative patients (NC). RESULTS: No differences in cortisol levels were observed between the C + and the NC patients. Cortisol levels correlated with ACTH (r = 0.284, p = 0.001) and IL-6 (r = 0.289, p = 0.04). In C + patients, cortisol levels mainly correlated with IL-6 levels (r = 0.28; p = 0.017). GCR-α expression was significantly higher in C + patients compared to NC. Patients with higher cortisol levels were more likely to progress to respiratory function deterioration or die. Both GCR-α and GILZ expression were significantly higher in C + non-survivors. CONCLUSION: Our findings indicate that cortisol serves as an indicator of disease severity. GILZ expression appears to be a more effective marker of mortality prediction in moderate COVID-19 cases. However, routine measurement of GILZ levels is currently unavailable. Elevated levels of cortisol may be indicative of patients with moderate COVID-19 who are at a higher risk of deterioration. This information can aid in identifying individuals who require early medical attention.

The ongoing challenge of discrepant growth hormone and insulin-like growth factor I results in the evaluation of treated acromegalic patients: a systematic review and meta-analysis.

OBJECTIVE: Growth hormone (GH) and insulin-like growth factor I (IGF-I) are the principal biomarkers used to assess disease activity in acromegaly, and any discrepancy between them renders interpretation of results inconclusive. Purpose of this study was to assess the frequency of this discrepancy and identify parameters that might affect its occurrence. DESIGN: A systematic review of MEDLINE and Scopus was performed (1987-2013) followed by a meta-analysis to address the frequency of discrepant results between GH and IGF-I levels. Meta-regression and subgroup analyses were performed assessing the effects of the year of publication, the different types of GH testing and GH assays used, as well as the impact of treatment with somatostatin analogues (SSAs) on the occurrence of this discrepancy. RESULTS: The analysis retrieved 39 eligible studies totalling 7071 patients. The pooled discordance rate between GH and IGF-I was 25·7% (95% CI: 22·3-29·4), and the predominant format was that of elevated IGF-I with normal GH levels (15·3%, 95% CI: 12·5-18·7). No significant correlation between the discordance rate and the year of publication was shown; whereas, the use of ultrasensitive GH assays resulted in higher discordance rates (30·7%, 95% CI: 25·9-35·9 vs 19·8%, 95% CI: 14·1-27·2, P = 0·04) as did treatment with SSAs (32·5%, 95% CI: 27·8-37·4) vs (21·6%, 95% CI: 17·8-25·6, P = 0·001). CONCLUSIONS: Discrepancy between GH and IGF-I results is encountered in a quarter of treated patients with acromegaly, especially when using ultrasensitive GH assays or in patients receiving SSAs, a fact that the clinician should take into consideration when making clinical decisions.

Impact of gsp mutations in somatotroph pituitary adenomas on growth hormone response to somatostatin analogs: a meta-analysis.

OBJECTIVE: Somatic mutations in the GNAS1 gene, which encodes the alpha-subunit of G stimulatory proteins (gsp), are frequently detected in somatotroph pituitary tumors and have been associated to specific clinical and histopathological characteristics. However, the question whether the presence of a somatic gsp mutation affects the response to somatostatin analog treatment remains unresolved. DESIGN: Following a literature search, we performed a meta-analysis, including 8 eligible studies, in order to estimate the effect of gsp mutation on the percent reduction of growth hormone (GH) levels during an acute octreotide suppression test (OST). A total of 310 patients with acromegaly [126 gsp (+) and 184 gsp (-)] were included in the analysis. RESULTS: The presence of the gsp mutation was related with a greater reduction in GH levels on OST [Weighted Mean Difference (WMD): 9.08 % (95 % CI, 2.73, 15.42); p = 0.005; random effects model]. There was significant heterogeneity for this effect estimate (I(2) = 58 %, p value for heterogeneity = 0.02). A sensitivity analysis after exclusion of a study with different methodology of OST provided similar estimates [WMD: 6.93 % (95 % CI, 1.40, 12.46); p = 0.01], albeit with no significant heterogeneity (I(2) = 35 %, p value for heterogeneity = 0.16). CONCLUSIONS: The present meta-analysis suggests a role for gsp mutation as a prognostic factor of treatment response to somatostatin analogs.

Clinical and biochemical impact of BCL1 polymorphic genotype of the glucocorticoid receptor gene in patients with adrenal incidentalomas.

BACKGROUND: Adrenal incidentaloma (AI) is a common clinical problem. Subtle hormonal abnormalities are present in a substantial proportion of patients. BCL1 gene polymorphism of the glucocorticoid receptor (GR) is associated with increased sensitivity to glucocorticoid action. The genotype- phenotype associations of this polymomorphism in patients presenting with AI has not been extensively investigated. AIM: A cross-sectional study in secondary/tertiary care centers. SUBJECTS/METHODS: Ninety-five subjects with AI were genotyped for the BCL1 GR gene polymorphism. Patients underwent an oral glucose tolerance test and a dexamethasone suppression test (DST). The presence of subclinical hypercortisolism, features of metabolic syndrome, and osteoporosis/ osteopenia were also assessed. RESULTS: No significant differences in markers of adrenal function between BCL1 carriers and non-carriers were revealed. Also, no difference was found in the features of metabolic syndrome, as well as in bone metabolism and density between these 2 groups. However, DST suppressor patients belonged more frequently to the BCL1 carriers group (41 out of 69 patients, 59.4% vs 9 out of 26 patients, 34.6%, p=0.0039), had smaller total adenoma size (2.4±0.2 cm vs 3.5±0.4 cm, p=0.04), and lower incidence of bilateral adrenal masses (18.8% vs 46.2%, p=0.01). CONCLUSIONS: AI patients who also carry the polymorphic BCL1 variant exhibit smaller size adrenal nodules. Those AI patients with complete DST suppression had a higher incidence of the polymorphic BCL1 variant. However, this study failed to demonstrate any significant impact of BCL1 GR polymorphism on the frequency of cortisol-dependent co-morbidities in patients with AI.

Inferior petrosal sinus sampling in Cushing's syndrome: usefulness and pitfalls.

In patients with ACTH-dependent Cushing's syndrome (CS), the differentiation between Cushing's disease (CD) and ectopic ACTH secretion (EAS) can often be challenging. The traditionally used biochemical tests have limited diagnostic accuracy and imaging modalities may fail to detect the culprit lesion. Inferior petrosal sinus sampling (IPSS) was introduced more than 3 decades ago as a test with optimal diagnostic accuracy and has since become the gold standard in the differential diagnosis between CD and EAS. However, several, albeit rare, pitfalls may limit its diagnostic accuracy and awareness is needed to avoid incorrect interventions. Moreover, it is an invasive and demanding procedure, available in a limited number of centers worldwide. This review aims to critically present the usefulness and pitfalls of IPSS and define strategies for its optimal place in the contemporary management of CS.

Cushing's disease: the desmopressin test as a compelling predictive marker of long-term remission after transsphenoidal surgery.

Transsphenoidal surgery (TSS) is the treatment of choice in Cushing's disease. However, recurrence rates are substantial and currently there are no robust predictors of late prognosis. As accumulating evidence challenge the accuracy of the traditionally used early postoperative cortisol values, alternative tests are required. The study of Cambos et al., published in a recent issue of the European Journal of Endocrinology, adds to the existing data that support a role of the desmopressin test as an early and reliable predictive marker in successfully TSS-treated patients. However, despite these promising data, the use of this test is hampered by the fact that it can be applied only in patients with a documented preoperative positive test. Moreover, the lack of robust criteria to define positive postoperative responses represents another major limitation.

DKA cases over the last three years: has anything changed?

BACKGROUND: Diabetic ketoacidosis (DKA) has been related mainly to type 1 diabetes mellitus (T1DM). However, it is not solely related to T1DM. The purpose of this study was to assess the prevalence of DKA among type 1 and type 2 patients with diabetes mellitus, who were hospitalized in our Clinic due to DKA, as well as to determine the etiology beyond DKA. PATIENTS AND METHODS: A cohort of 109 patients with DKA, 17-86 years of age, who were hospitalized in the Department of Endocrinology, Diabetes and Metabolism of our hospital between 2015 and 2017, were included in the study. RESULTS: Among the 109 patients, 50 (45.9%) had mild DKA, 48 (44.1%) had moderate DKA, whereas 11 patients (10%) had severe DKA. Sixty-five patients (60%) developed DKA as the first manifestation of T1DM, 30 patients (27%) developed DKA in the context of type 2 diabetes (T2DM), mainly due to the co-existence of serious infections, 11 patients (10%) had T1DM, but had omitted their insulin dosages, and 3 patients (3%) developed DKA due to unknown reasons. CONCLUSIONS: Most patients with DKA presented with mild and moderate DKA and only a minority presented with the severe form of the disease. The etiology of DKA was mainly T1DM and less frequent uncontrolled T2DM, usually due to the co-existence of severe infections, while only in a tiny minority, the causes remained unidentifiable.

Monitoring of patients with type 2 diabetes and nephropathy in a specialized diabetic nephropathy clinic seems to be beneficial.

BACKGROUND: The purpose of this study was to evaluate the outcome of DM2 patients with nephropathy when they are under surveillance of a joined clinic run by endocrinologists & nephrologists. PATIENTS AND METHODS: A cohort of 106 patients with DM2, 42-83 years of age, and eGFR < 60 ml/min/m2 were included. Age, sex, duration of diabetes, duration of attending our clinic, smoking habits, BMI, data regarding ischemic heart disease and induction of hemodialysis, urine albumin excretion (UAE) levels, eGFR (MDRD equation) and values of various biochemical parameters were recorded too. Follow-up period ranged from one to 25 years. Paired samples t-test and non-parametrical Kruskal-Wallis test were used for the analyses of the data. RESULTS: Fifty percent of patients had no further progression, 25.9% improvement, while 24.1% had worsening of the UAE levels. During the follow-up in the joined clinic, there was a smaller than the expected from the medical literature decrease in median eGFR, i.e. 2,3 ml/min/m2 and a statistically significant improvement in glycosylated hemoglobin levels from 8.0% to 7.4% (p = 0.016). Time in years of follow-up in the joined clinic of our hospital appeared to be the most significant factor in the improvement or stabilization against deterioration of the UAE levels (p = 0.018). CONCLUSIONS: Close follow-up of DM2 patients with eGFR < 60 ml/min/m2 has resulted in a minor annual eGFR decrease. Monitoring of these patients in a specialized diabetic nephropathy clinic is beneficial for this group of patients for delaying the occurrence of end-stage renal disease.

The application of the combined corticotropin-releasing hormone plus desmopressin stimulation during petrosal sinus sampling is both sensitive and specific in differentiating patients with Cushing's disease from patients with the occult ectopic adrenocorticotropin syndrome.

CONTEXT: Although bilateral inferior petrosal sinus sampling (BIPSS) with CRH stimulation is the most accurate procedure for the differential diagnosis of ACTH-dependent Cushing's syndrome (CS), 4-15% of patients with Cushing's disease (CD) fail to demonstrate diagnostic gradients. Preliminary data suggest that a more potent stimulation by the combined administration of CRH plus desmopressin during BIPSS may provide some diagnostic advantage. A crucial issue, however, is whether such an amplified stimulation may affect the specificity of the procedure, and this was the main aim of the present study. OBJECTIVE: We investigated the diagnostic accuracy of BIPSS performed by CRH plus desmopressin stimulation. DESIGN AND SETTING: A retrospective analysis was conducted at a single tertiary care center. PARTICIPANTS: Fifty-four patients were admitted for the investigation of ACTH-dependent CS. CD was diagnosed in 47 patients; occult ectopic ACTH syndrome (oEAS) was histologically confirmed in seven patients. INTERVENTION(S): All patients underwent BIPSS with CRH plus desmopressin administration. Additional noninvasive tests included CRH test, high-dose dexamethasone suppression test, desmopressin test, and pituitary magnetic resonance imaging. MAIN OUTCOME MEASURES: Gradients of inferior petrosal sinus (IPS) to peripheral (IPS/P) ACTH were calculated before and after stimulation with CRH plus desmopressin. RESULTS: The sensitivity for a basal IPS/P gradient greater than 2 was 61.7%, with 100% specificity and a diagnostic accuracy of 66.7%. After stimulation with CRH plus desmopressin, receiver operating characteristic (ROC) curve analysis showed that a cutoff gradient of more than 2 offers the best test performance. In total, 46 of 47 patients with CD had an IPS/P gradient greater than 2, but none of the patients with oEAS, resulting in a sensitivity of 97.9%. The specificity was 100%, diagnostic accuracy was 98.2%, and the positive and negative predictive values were 100 and 87.5%, respectively. A subgroup of 18 patients (16 with CD and two with oEAS) had contradictory responses to routine tests with CRH and/or high-dose dexamethasone suppression test; sensitivity, specificity, and accuracy of BIPSS in this subgroup were 100%. CONCLUSIONS: The application of a combined stimulation with CRH plus desmopressin during BIPSS is associated with a high sensitivity but no loss of specificity.

The Desmopressin Test Predicts Better Than Basal Cortisol the Long-Term Surgical Outcome of Cushing's Disease.

CONTEXT: Cushing's disease (CD) has a significant relapse rate after successful transsphenoidal surgery (TSS). Many CD patients respond aberrantly to the desmopressin test (DT). Disappearance of this response after surgery may suggest complete removal of abnormal corticotrophs and a lower possibility of recurrence. OBJECTIVE: The utility of postoperative DT to predict long-term outcome compared to the widely used postoperative cortisol level. DESIGN: Retrospective analysis. SETTING: Tertiary hospital. PATIENTS: Seventy-three patients underwent TSS and postoperative DT; 51 had sustained remission, defined as normal dexamethasone suppression and urinary free cortisol at 6 months. After excluding 12 patients with short follow-up, negative or no preoperative DT, we analyzed 39 patients. INTERVENTION(S): Measurements of morning cortisol at 1-2 weeks and DT within 6 months after TSS. MAIN OUTCOME MEASURE(S): Recurrence or remission at latest follow-up. RESULTS: Mean follow-up was 63 ± 50 months. Recurrence occurred in seven patients. In logistic regression analysis, postoperative cortisol levels were not associated with remission. Apart from the percentage increment of cortisol, all other DT criteria (peak cortisol, peak ACTH, absolute cortisol increment [ΔCort], absolute ACTH change, and percentage absolute ACTH change) were significant predictors of outcome. In receiver operating characteristic analysis, the ΔCort had the best diagnostic performance. ΔCort <7.4 µg/dL had a sensitivity of 97% to detect remission. Comparison of Kaplan-Meier curves showed that ΔCort <7.4 µg/dL was associated with remission, whereas ΔCort ≥7.4 µg/dL had a hazard ratio of recurrence of 24.7 (95% confidence interval, 10.6-448.5) at 60 months (median). CONCLUSION: Loss of desmopressin response indicates favorable prognosis and, if used in addition to basal cortisol levels, improves the accuracy of the postoperative assessment of CD.

Biochemical and clinical benefits of unilateral adrenalectomy in patients with subclinical hypercortisolism and bilateral adrenal incidentalomas.

OBJECTIVE: The treatment of subclinical hypercortisolism in patients with bilateral adrenal incidentalomas (AI) is debatable. We aimed to compare the biochemical and clinical outcome of unilateral adrenalectomy vs a conservative approach in these patients. DESIGN: Retrospective study. METHODS: The study included 33 patients with bilateral AI; 14 patients underwent unilateral adrenalectomy of the largest lesion (surgical group), whereas 19 patients were followed up (follow-up group). At baseline and at each follow-up visit, we measured 0800 h plasma ACTH, midnight serum cortisol (MSF), 24-h urinary-free cortisol (UFC) and serum cortisol following a standard 2-day low-dose-dexamethasone-suppression test (LDDST). We evaluated the following comorbidities: arterial hypertension, impaired glucose tolerance or diabetes mellitus, dyslipidemia and osteoporosis. RESULTS: Baseline demographic, clinical characteristics and the duration of follow-up (53.9±21.3 vs 51.8±20.1 months, for the surgical vs the follow-up group) were similar between groups. At the last follow-up visit the surgical group had a significant reduction in post-LDDST cortisol (2.4±1.6 vs 6.7±3.9 µg/dl, P=0.002), MSF (4.3±2 vs 8.8±4.6 µg/dl, P=0.006) and 24-h UFC (50.1±21.1 vs 117.9±42.4 µg/24 h, P=0.0007) and a significant rise in mean±s.d. morning plasma ACTH levels (22.2±9.6 vs 6.9±4.8 pg/ml, P=0.002). Improvement in co-morbidities was seen only in the surgical group, whereas no changes were noted in the follow-up group. CONCLUSIONS: Our early results show that removal of the largest lesion offers significant improvement both to cortisol excess and its metabolic consequences, without the debilitating effects of bilateral adrenalectomy. A larger number of patients, as well as a longer follow-up, are required before drawing solid conclusions.

Abnormal Responsiveness to Dexamethasone-Suppressed CRH Test in Patients With Bilateral Adrenal Incidentalomas.

CONTEXT: The bilateral formation of nodules indicates that the pathogenesis of bilateral adrenal incidentalomas (AI) may differ from that of unilateral AI. A possible role of hypothalamo-pituitary-adrenal (HPA) axis dysregulation in their formation has not been investigated. OBJECTIVE: The objective of the study was to evaluate the presence of altered feedback regulation of HPA axis in patients with bilateral AI. DESIGN: The dexamethasone (DEX) suppression-CRH test was used to assess ACTH and cortisol responses in controls and patients with unilateral and bilateral AI. SETTING: The study was conducted at endocrine departments of two tertiary centers. PATIENTS: We studied 24 controls and 39 patients with unilateral and 46 with bilateral AI. INTERVENTIONS: All subjects underwent standard low-dose dexamethasone suppression followed by iv bolus administration of human CRH (100 µg). RESULTS: Bilateral AI had higher levels of ACTH and cortisol after the DEX-CRH challenge compared with both controls (P < .01 for ACTH and P < .001 for cortisol) and unilateral AI (P < .01 for ACTH and cortisol). A positive response, defined as peak ACTH greater than 10 pg/mL at 15 and/or 30 minutes followed by a significant rise in cortisol levels, was noted in 41.3% of bilateral vs 2.6% in unilateral AI (P < .001). Bilateral responders did not differ from nonresponders in demographic or hormonal characteristics, but they had larger total adrenal size compared with nonresponders. CONCLUSIONS: A significant proportion of patients with bilateral AI demonstrate positive responses to the DEX-CRH test compared with unilateral AI, providing ground for potential involvement of HPA axis dysregulation in the pathogenesis, in at least a subgroup, of bilateral AI patients.

Acetylcholine and norepinephrine stimulate the release of corticotropin-releasing factor-41 from the rat hypothalamus in vitro.

The effects of the two putative neurotransmitters acetylcholine and norepinephrine on immunoreactive CRF-41 release from incubated rat hypothalami were studied. Acetylcholine at concentrations of 10(-11) to 10(-7) M stimulated CRF-41 release. This effect was blocked in a dose-dependent manner by the muscarinic antagonist atropine (10(-9) to (-7) M). The nicotinic antagonist hexamethonium was ineffective at a dose of 10(-7) M, but produced slight inhibition of this response at 10(-5) M. Norepinephrine at concentrations of 10(-10) to 10(-6) M also produced a dose-dependent stimulation of CRF-41 release. The beta-adrenoceptor antagonists propranolol (10(-5) M) and timolol (10(-6) M) blocked norepinephrine-induced CRF-41 release. The alpha 1-adrenoceptor antagonists thymoxamine (10(-5) M), prazosin (10(-5) M), and corynanthine (10(-4) M), and the alpha 2-antagonist idazoxan (10(-5) M), were ineffective. Potassium depolarization (56 mM) caused stimulation of CRF-41 release which was dependent on the presence of calcium in the incubation medium. Authenticity of immunoreactive CRF-41 released was demonstrated by chromatographic criteria using gel filtration and reversed phase HPLC. These results provide evidence for a stimulatory role of acetylcholine and norepinephrine on CRF-41 release, and consequently on hypothalamo-pituitary-adrenal axis in the rat, through actions at a hypothalamic level. The stimulatory effect of acetylcholine is mediated principally through muscarinic receptors and that of norepinephrine through beta-adrenoceptors.

Morphine directly modulates the release of stimulated corticotrophin-releasing factor-41 from rat hypothalamus in vitro.

The actions of opioids and opiates on the hypothalamo-pituitary-adrenal axis are currently controversial. In the rat, morphine is reported to both stimulate and inhibit ACTH and corticosterone secretion, but the precise sites and mechanisms of these effects have remained unclear. To analyze further the hypothalamic actions of morphine, we have investigated its effect on hypothalamic fragments in vitro and measured the major CRF, CRF-41, by a specific RIA. The acute effects of morphine on both basal and stimulated ACTH release from dispersed pituitary cells were also investigated. Morphine (10(-8)-10(-6) M) did not significantly alter the basal secretion of CRF-41. However, similar concentrations of morphine inhibited CRF-41 release stimulated by norepinephrine in a dose-dependent manner. Similarly, morphine (10(-6) M) inhibited acetylcholine (10(-9) M)- and serotonin (10(-7) M)-stimulated CRF-41 release. The stimulatory effect on CRF-41 release induced by veratridine (10(-6) M) was inhibited by approximately 50% in the presence of morphine. KCl (28 nM)-mediated CRF-41 release was also significantly inhibited by morphine. Naloxone (10(-7)-10(-5) M) had no significant effect on either basal or norepinephrine-induced CRF-41 release, but reversed the inhibitory effect of morphine on norepinephrine-induced CRF-41 secretion in a dose-dependent manner. Morphine (10(-6)-10(-5) M) had no effect on either basal or CRF-41-stimulated ACTH release from dispersed pituitary cells. These data suggest that the predominant effect of morphine on hypothalamic CRF-41 release in vitro is suppression of the release induced by a variety of putative neurotransmitters and depolarizing agents. This inhibitory effect is reversed by naloxone, suggesting that it is mediated by opiate receptors, presumably situated directly on CRF-41 neurons.

Interleukins-1 and -6 stimulate the release of corticotropin-releasing hormone-41 from rat hypothalamus in vitro via the eicosanoid cyclooxygenase pathway.

It has previously been shown that interleukin-1 (IL-1) directly stimulates the release of CRH-41 from rat hypothalamus in vitro, suggesting that cytokines may mediate the effects of changes in immune state on the hypothalamo-pituitary adrenal axis (HPA). However, it is likely that several cytokines can cause changes in neuroendocrine function, and we have now investigated a series of others for central activity on the HPA: IL-2, IL-6, IL-8, tumor necrosis factor (cachectin), interferon-alpha 2, and interferon-gamma. The static rat hypothalamic incubation system used involves fresh hypothalamic explants with consecutive 20-min incubation, and estimation of CRH-41 concentrations in the medium by a specific RIA; the acute effects of cytokines on ACTH release from rat dispersed pituitary cells were also measured. IL-6 increased hypothalamic CRH-41 secretion in the range 10-100 U/ml, but had no effect on isolated median eminences incubated in vitro under the same conditions. IL-6 (1-1000 U/ml) also had no effect on the secretion of ACTH from freshly dispersed rat anterior pituitary cells when administered in 10-min pulses. The effects of both IL-1 and IL-6 were antagonized by blockade of the eicosanoid cyclooxygenase pathway, but not by lipooxygenase blockade. Neither IL-2 (1-10000 U/ml), IL-8 (0.1-10 nM), tumor necrosis factor (10-1000 U/ml), interferon-alpha 2 (10-1000 U/ml) nor interferon-gamma (10-1000 U/ml) had any effect on hypothalamic CRH-41 release or pituitary ACTH release. It is therefore concluded that IL-6, like IL-1, can exert a potent enhancing effect on the HPA by acutely stimulating the secretion of CRH-41 from the hypothalamus at a site above the level of the median eminence, at concentrations known to occur in human plasma and cerebrospinal fluid. These effects are probably mediated by cyclooxygenase products. Acute stimulatory effects of the other cytokines investigated on the HPA are unlikely to be exerted through changes in either CRH-41 or ACTH directly.

Interleukin-1 beta modulates the acute release of growth hormone-releasing hormone and somatostatin from rat hypothalamus in vitro, whereas tumor necrosis factor and interleukin-6 have no effect.

There is clear evidence for communication between the immune and neuroendocrine systems. However, the effect of cytokines as major immune mediators on the hypothalamic growth peptides, GHRH and somatostatin (SRIH), is not well established. To investigate a possible hypothalamic action of the cytokines interleukin-1 beta (IL-1), interleukin-6, and tumor necrosis factor-alpha, on the release of GHRH and SRIH, we used a previously validated acute rat hypothalamic explant system. IL-1 caused a pronounced dose-dependent stimulation of SRIH in the dose-range 1-100 U/ml (P less than 0.01). GHRH showed a slight, but significant, increase in response to IL-1 tested in the dose-range 10-100 U/ml. Similar studies with mediobasal hypothalamic (GHRH and SRIH) or median eminence (SRIH) fragments produced no change in either GHRH or SRIH release. The effects of IL-1 were antagonized by the cyclo-oxygenase inhibitor, indomethacin (10 micrograms/ml). Stimulation of GHRH and SRIH could not be blocked by the CRH-antagonist alpha-helical CRH (9-41) at 10(-6) M. Interleukin-6, in the dose range 10-100 U/ml, and tumor necrosis factor-alpha, in the dose range 10-10,000 U/ml, had no effect on the acute hypothalamic release of either GHRH or SRIH. It is concluded that IL-1 stimulates the acute hypothalamic release of GHRH and SRIH, and that this effect is mediated by cyclo-oxygenase products. The marked IL-1 stimulation of hypothalamic SRIH release may override the minor increase of GHRH increase, and may thus contribute to disturbances in growth seen in the presence of chronic inflammation.

Food-dependent androgen and cortisol secretion by a gastric inhibitory polypeptide-receptor expressive adrenocortical adenoma leading to hirsutism and subclinical Cushing's syndrome: in vivo and in vitro studies.

Aberrant gastric inhibitory polypeptide (GIP) receptor expression in bilaterally hyperplastic adrenals or unilateral adrenal adenomas is a rare form of adrenal hyperfunction. So far, only few cases have been described. In all these cases, cortisol was the predominant steroid released in a food-dependent manner, leading to the development of non-ACTH-dependent Cushing's syndrome. In the present study, we describe a novel case of a GIP receptor-expressive adrenocortical adenomatous nodule, detected incidentally by computed tomography scanning in a 41-yr-old lady with hirsutism but no clinical signs of Cushing's syndrome, on physical examination. Hormonal investigations in morning fasting samples showed slightly elevated androgen levels, low-normal baseline cortisol, normal suppression of cortisol after dexamethasone administration, and ACTH levels that were not suppressed and did stimulate after CRH administration. The elevated urinary free cortisol excretion, in conjunction with an atypical cortisol diurnal rhythm, raised the possibility of an aberrant stimulation of cortisol production by the adrenal tumor. Further studies demonstrated food-dependent secretion of cortisol, which was abolished by prior octreotide administration. Notably, substantial amounts of adrenal androgens were also secreted after food consumption. Removal of the tumor resulted in undetectable cortisol and androgen levels that did not respond to food consumption. Histological examination of the excised tumor revealed an adrenocortical adenomatous nodule originating from the inner zona reticularis, consisting mainly of compact cells. A steroidogenic secretory pattern, indicating the concomitant release of adrenal androgens and cortisol, was also observed in vitro from tumor cells cultured in the presence of GIP. The in vitro secretory response to GIP was higher for the adrenal androgen DHEA, compared with cortisol. The expression of the GIP receptor in tumor cells, but not in the adjacent normal adrenal, was demonstrated by RT-PCR), using specific oligonucleotide probes for this receptor. In summary, we describe a patient with a GIP-expressive cortisol and androgen oversecreting adrenocortical nodule with the unusual presentation of hirsutism and not the typical clinical signs of Cushing's syndrome. It is of note that food intake in this patient provoked a substantial increase in both adrenal androgen and cortisol levels that, together with the histological appearance of this nodule, was compatible with a zona reticularis-derived tumor. Thus, aberrant expression of the GIP receptor does not exclusively involve cells of a zona fasciculata phenotype, as previously reported, but may also occur in other types of differentiated adrenocortical cells.

A reappraisal of the utility of somatostatin receptor scintigraphy in patients with ectopic adrenocorticotropin Cushing's syndrome.

Ectopic ACTH hypersecretion is a rare cause of Cushing's syndrome. Bronchial carcinoids are the most common neoplasms causing the occult ectopic ACTH syndrome (EAS). Localization of these tumors is often difficult. The diagnostic utility of somatostatin receptor scintigraphy (SRS) in EAS has been studied in a limited number of patients with conflicting results. Herein we report our experience with 12 consecutive cases. Histological confirmation was obtained in nine patients, the majority being bronchial carcinoids. Among the seven patients with histologically confirmed bronchial carcinoids, SRS was performed in six patients. In three patients SRS correctly localized a bronchial carcinoid tumor at presentation. In the remaining three it became positive after 8, 22, and 27 months during follow-up. In two patients SRS was positive without any finding in the corresponding conventional imaging study. In two patients positive computed tomography/magnetic resonance imaging preceded SRS localization. There was no false positive SRS. Among three patients with highly suspected EAS, SRS was positive in one. Both patients with EAS due to medullary thyroid carcinoma had focal positive uptake. In summary, in this study a substantial number of patients had positive tumor localization by SRS. Therefore, SRS is a useful tool in the evaluation of patients with EAS.

The desmopressin and combined CRH-desmopressin tests in the differential diagnosis of ACTH-dependent Cushing's syndrome: constraints imposed by the expression of V2 vasopressin receptors in tumors with ectopic ACTH secretion.

The role of desmopressin, alone or in combination with CRH, in the differential diagnosis between Cushing's disease (CD) and ectopic ACTH secretion (EAS) still remains uncertain. Based on existing data, the desmopressin test is regarded as an alternative to the CRH stimulation test and, when given in combination with CRH, it has been suggested to completely discriminate between patients with CD and EAS. However, assessment of these tests has been limited in only a small number of patients with EAS. Desmopressin is a relatively specific V2 vasopressin receptor (V2R) agonist. Although expression of V3 vasopressin receptor (V3R) is common in tumors with EAS, the expression of V2R has not been extensively investigated. In the present study, we report our findings of the desmopressin and the combined CRH-desmopressin test in a series of patients with CD and EAS; also, the expression of V2R and V3R was investigated in tumors with EAS by a RT-PCR method. We assessed a cohort of 31 patients with ACTH-dependent Cushing's syndrome, including 26 patients with CD and five cases with histologically confirmed EAS. To avoid bias of predetermined criteria, univariate curves of the receiver operating characteristics (ROC) were constructed by plotting the sensitivity against 1-specificity at each level of the percent cortisol (F) and ACTH responses to these tests. Following desmopressin administration there was an overlap of the percent F and ACTH responses among patients with CD and EAS, and the area under the ROC curve for both these responses was not significantly different than that occurring by chance. This was also true for the percent F response following the combined CRH-desmopressin test. However, the area under the ROC curve for the percent ACTH rise following the combined test was significantly different; the point of the ROC curve closest to 1 corresponded to a percent ACTH rise of 218% (88% sensitivity and 80% specificity). Expression of V2R and V3R mRNA was investigated in four of the five excised tumors with EAS and revealed the presence of the V2R in all, whereas the V3R mRNA was expressed in three of these cases. In conclusion, in this series the desmopressin test produced a significant overlap of responses between CD and patients with EAS and, therefore, is of limited value in the differential diagnosis of the ACTH-dependent Cushing's syndrome. This is most probably due to the expression of the V2R in tumors with EAS. Moreover, following the combined CRH-desmopressin test only the ACTH but not the F responses were diagnostically useful, but still far from completely discriminating patients with CD and EAS.

Involvement of calmodulin in depolarization-induced release of corticotrophin-releasing hormone-41 from the rat hypothalamus in vitro.

We have employed an acute explant system of the rat hypothalamus in vitro, as previously described, to examine the role of calcium and calmodulin in the release of corticotrophin-releasing hormone-41 (CRH-41). Release of CRH-41, as determined by radioimmunoassay, was stimulated in a dose-dependent manner by the membrane-depolarizing agents KCl and veratridine. Stimulation was also observed with the calcium ionophore A23187. The calcium channel blocker verapamil (1-100 mumol/l) inhibited both KCl- and veratridine-induced release in a dose-dependent manner (maximum inhibition of 75% and 60% respectively), thus providing further evidence that calcium entry is required for secretion of CRH-41 following membrane depolarization. Trifluoperazine (1-100 mumol/l), an inhibitor of calmodulin-calcium interaction, decreased both KCl- and veratridine-evoked CRH-41 secretion in a dose-dependent fashion (maximum inhibition of 50% and 30% respectively). Similarly, phenytoin, a calmodulin-dependent kinase inhibitor, in the concentration range of 1-100 mumol/l, also decreased depolarization-induced CRH-41 release in a dose-dependent manner. The basal release of CRH-41 was unaffected by either treatment. Finally, both calmodulin inhibitors (10 mumol/l) decreased CRH-41 release induced by the calcium ionophore A23187 (10 mumol/l). These data provide evidence for the role of calcium in membrane depolarization-induced stimulus-secretion coupling of rat hypothalamic CRH-41. Furthermore, inhibition of the stimulatory responses by two separate classes of calmodulin inhibitors suggests a role for calmodulin, at least in part, in this process.