Chronic Hypercortisolism Research Articles

Development of test systems for the discovery of selective human aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) inhibitors.: Discovery of a new lead compound for the therapy of congestive heart failure, myocardial fibrosis and hypertension

Two key players in adrenal steroid hormone biosynthesis are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2 that catalyze the final steps in the biosynthesis of cortisol and aldosterone, respectively. Overproduction of both hormones contributes to a number of severe diseases, as illustrated by the association of elevated aldosterone levels with hypertension and higher mortality in congestive heart failure, and by Cushing’s syndrome as the clinical correlate of chronic hypercortisolism. Thus, CYP11B1 and CYP11B2 comprise new targets for drug treatment and selective inhibitors of both enzymes are of high pharmacological interest. To facilitate the search for such compounds, we have established novel test procedures using recombinant fission yeast strains that stably express these enzymes. The aim of this study was to compare the inhibition profiles displayed by these enzymes in established mammalian cell culture test systems to those obtained with the new fission yeast assays, and to evaluate the usefulness of the Schizosaccharomyces pombe strains as screening systems for the identification of novel lead compounds. Using these test systems, we were able to identify a new and very selective CYP11B2 inhibitor (SIAS-1) that displayed no detectable interference with CYP11B1 activity.

Fisiopatologia da osteoporose induzida por glicocorticóide

O hipercortisolismo crônico é a causa mais freqüente de osteoporose secundária, acometendo principalmente o osso trabecular. Aproximadamente 30-35% dos pacientes com síndrome de Cushing apresentam fraturas de vértebras por compressão e o risco de fraturas de colo de fêmur é aumentado em 50% nessa população. Vários mecanismos têm sido propostos para explicar a ocorrência de osteoporose nessa condição, como a ação direta dos glicocorticóides nas paratireóides e nas células ósseas, alterações na produção de prostaglandinas, citocinas, interleucinas, alterações na secreção do hormônio do crescimento (GH), do fator insulina símile-I (IGF-I) e esteróides gonadais. Resultados controversos têm sido apresentados quanto à alteração na secreção do PTH nesta situação, onde níveis normais e elevados têm sido descritos. A elevação da secreção de PTH pode ser secundária a distúrbios do metabolismo mineral induzidos pelo hipercortisolismo, como diminuição na absorção intestinal, aumento da excreção renal de cálcio, diminuição no número de receptores paratireoideanos para a 1,25(OH)2D3, anormalidades no limiar de sensibilidade do cálcio (set point) para a secreção do PTH e alteração na sua atividade. Nesta revisão, são discutidos diversos aspectos fisiopatológicos e possíveis mecanismos envolvidos na associação entre hipercortisolismo e osteoporose.

Ghrelin Is No Longer Able to Stimulate Growth Hormone Secretion in Patients with Cushing’s Syndrome but Instead Induces Exaggerated Corticotropin and Cortisol Responses

Growth and growth hormone (GH) secretion are blunted or severely impeded in chronic hypercortisolism and in patients with Cushing’s syndrome. A mechanistic explanation for the effect however has yet to be provided. On the other hand, several properties of ghrelin, a new peptide recently identified as the endogenous ligand of the GH secretagogue receptor, are still largely unknown. The two aims of this study were to observe whether ghrelin-mediated GH secretion was altered, and to characterize the corticotropin (ACTH) and cortisol response to this new stimulus in patients with Cushing’s disease. Ten patients with active Cushing’s disease (6 harboring microadenomas and 4 with macroadenomas) and 10 sex- and age-matched controls were studied. Ghrelin was administered at a dose of 1 µg/kg i.v. and GH, ACTH and cortisol analyzed in duplicate. In control women, ghrelin induced GH secretion to levels of 74.4 ± 12.8 µg/l, while chronic hypercortisolism severely reduced the ghrelin-mediated GH release in all patients with Cushing’s disease (peak values 17.7 ± 5.2 µg/l). The slightly, but significantly higher adiposity of patients vs. controls may have contributed to the effect, since a significant negative correlation (r = 0.639) was found between the amplitude of the GH peak and body mass index. In control women, ghrelin increased ACTH and cortisol levels, with peaks at 57.4 ± 19.0 ng/l and 162 ± 16 µg/l, respectively. This secretion was enhanced in Cushing’s syndrome patients, with ACTH and cortisol values of 380.7 ± 109.8 ng/l and 338 ± 81 µg/l respectively, both significantly higher than in controls. In conclusion, ghrelin-induced GH secretion was severely blunted in patients with active Cushing’s syndrome, in addition to a remarkable hyper-response in ACTH and cortisol secretion. These findings could have implications for the understanding of the physiology and physiopathology of interactions between GH and ACTH regulation.

Parathyroid hormone secretion in chronic human endogenous hypercortisolism.

Osteoporosis is a common manifestation of Cushing's syndrome, but the mechanisms responsible for this abnormality have not been defined. With the objective of analyzing parathyroid hormone (PTH) secretion in chronic hypercortisolism (CH), we evaluated 11 healthy subjects and 8 patients with CH, 6 with Cushing's disease and 2 with adrenal adenoma. These volunteers were submitted to tests of PTH stimulation through hypocalcemia (EDTA), PTH suppression through hypercalcemia (iv and oral calcium), and evaluation of bone mineral density (BMD) by DEXA. During the test of PTH stimulation, the calcium and magnesium concentrations of the normal and CH groups were similar. Patients with CH showed an increased PTH response to the hypocalcemic stimulus compared to controls. PTH values were significantly higher in the CH group at 70 (17.5 +/- 3.5 vs 10.2 +/- 1.3 pmol/l, P = 0.04), and 120 min (26.1 +/- 5.9 vs 11.3 +/- 1.9 pmol/l, P = 0.008) of EDTA infusion. The area under the curve for PTH during EDTA infusion was also significantly higher in patients with CH than in normal subjects (1867 +/- 453 and 805 +/- 148 pmol l(-1) 2 h(-1), P = 0.02). During the test of PTH suppression, calcium, magnesium and PTH levels of the patients with hypercortisolism and controls were similar. BMD was decreased in patients with hypercortisolism in the spine (0.977 +/- 0.052 vs 1.205 +/- 0.038 g/cm2 in controls, P<0.01). In conclusion, our results show that subjects with CH present decreased bone mass mainly in trabecular bone. The use of dynamic tests permitted the detection of increased PTH secretion in response to a hypocalcemic stimulus in CH patients that may probably be involved in the occurrence of osteoporosis in this state.

Uncommon clinical course of multiple osteochondromatosis in a patient with a long-term history of Cushing's disease.

Cushing's disease (CD), the chronic endogenous hypercortisolism derived from an ACTH-secreting pituitary adenoma, and multiple osteochondromatosis (MO), a congenital mesoderm dyschondroplasia, represent two distinct rare neoplastic diseases. Clinical appearance of MO usually occurs during the first-second decade of life. In fact, the growth of osteochondromas parallels the patient's growth, then becoming quiescent after the closure of the epiphyses and the achievement of final stature. Here we describe an uncommon case of a patient with a long-term history of childhood-onset CD, who surprisingly developed MO during the third decade of life, after the remission of CD. Indeed, a female patient had been followed for CD from the age of 12 to the age of 24 years, when CD definitively remitted. At the age of 26 the patient complained progressively worsening backache and pain at level of hips and feet. Standard radiography of skeleton showed multiple bone dysmorphisms at level of the four limbs, spine and pelvis consistent with multiple osteochondromas and exostoses. A diagnosis of MO was performed. Total body bone scintigraphy with 99mTc-MDP revealed an increased uptake of the radioligand, suggesting an increased metabolic turnover in correspondence of the majority of the osteochondromas. However, the negativity of the majority of the lesions at 99mTc-DMSA scintigraphy and the histological diagnosis of benign osteochondroma of the only positive lesion at 99mTc-DMSA evidenced that the high metabolic activity of the osteochondromas was not due to malignant transformation. However, the activity of the lesions was highly surprising considering that they usually become quiescent after the achievement of the final stature. In last analysis, the uncommon characteristics of MO and, particularly, its occurrence after stable remission of hypercortisolism, suggests a possible role of glucocorticoids in influencing the clinical course of the skeletal disease. The inhibitory effect of hypercortisolism on bone growth and maturation could explain the block in the proliferation of skeletal lesions during the developmental age, where CD was in the active phase, and the opposite effect of stimulation of the ostochondromas growth during stable normalization of cortisol secretion, after CD remission.

Evaluation of hypothalamic-pituitary-adrenal axis in amenorrhoeic women with insulin-dependent diabetes.

Diabetes is associated with a higher incidence of secondary hypogonadotrophic amenorrhoea. In amenorrhoeic women with insulin-dependent diabetes a derangement in hypothalamic-pituitary-ovary axis has been proposed. No data exist on hypothalamic-pituitary-adrenal function in these women. Gonadotrophin releasing hormone (GnRH), corticotrophin releasing hormone (CRH), metoclopramide and thyroid releasing hormone (TRH) tests were performed in 15 diabetic women, eight amenorrhoeic (AD) and seven eumenorrhoeic (ED). Frequent blood samples were taken during 24 h to evaluate cortisol plasma concentrations. There were no differences between the groups in body mass index, duration of diabetes, insulin dose and metabolic control. The AD women had lower plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, oestradiol, androstenedione and 17-hydroxyprogesterone (17-OHP) than the ED women. The responses of pituitary gonadotrophins to GnRH, and of thyroid stimulating hormone (TSH) to TRH, were similar in both groups. The AD women had a lower prolactin response to TRH and metoclopramide, and lower ACTH and cortisol responses to CRH, than the ED women. Mean cortisol concentrations > 24 h were higher in the amenorrhoeic group. Significant differences in cortisol concentrations from 2400 to 1000 h were found between the two groups. Insulin-dependent diabetes may involve mild chronic hypercortisolism which may affect metabolic control. Stress-induced activation of the hypothalamic-pituitary-adrenal axis would increase hypothalamic secretion of CRH. This would lead directly and perhaps also indirectly by increasing dopaminergic tonus to inhibition of GnRH secretion and hence hypogonadotrophic amenorrhoea. Amenorrhoea associated with metabolically controlled insulin-dependent diabetes is a form of functional hypothalamic amenorrhoea that requires pharmacological and psychological management.

7 Interaction between body composition, leptin and growth hormone status

Administration of growth hormone (GH) induces changes in body composition, namely, increases in both bone and lean mass and a decrease in fatty tissue. However, the contrary issue, i.e. the way in which body composition affects the secretion of GH, is highly controversial. Disease states such as obesity and chronic hypercortisolism are associated with increased adiposity and/or the central distribution of fat. Ageing, characterized by excess adiposity, is also associated with impaired secretion of GH. In these states, both spontaneous and stimulated secretion of GH is severely impeded. At the other extreme, malnutrition and fasting are both associated with increased secretion of GH when confronted with most, if not all, stimuli. As the common factor in all of these situations is the increased or decreased adiposity, or the changes in energy homeostasis, it has been postulated that adipose tissue exerts a relevant role in the control of GH secretion in man. The link between adipose tissue and GH seems to be exerted through at least two signals produced by adipocytes: free fatty acids (FFA) and the recently cloned protein, leptin. An increase in FFA blocks secretion of GH, while a decrease in FFA enhances secretion. Leptin, a hormone whose main role is to regulate the intake of food and energy expenditure, seems to regulate GH secretion by acting at the hypothalamic level. In summary, body composition affects GH secretion by way of the degree of adiposity, and free fatty acids and leptin would appear to be the messages through which adipocytes participate in the regulation of GH secretion. This framework clarifies the metabolic control of GH, a hormone with profound metabolic activities.

The dexamethasone-suppressed corticotropin-releasing hormone stimulation test differentiates mild Cushing's disease from normal physiology.

The dexamethasone-suppressed CRH test (Dex-CRH test) differentiates patients with Cushing's syndrome (CS) from those with pseudo-Cushing states, who have decreased ACTH responses to CRH because of negative feedback exerted by chronic hypercortisolism. Normal subjects, however, have not been studied with the Dex-CRH test, raising concern that this test might not separate patients with CS from patients with normal adrenal function. To determine whether the criterion that separates CS from pseudo-Cushing states also would differentiate patients with Cushing's disease (CD) from individuals with eucortisolism, we studied 20 healthy volunteers during low-dose (2 mg/day) dexamethasone suppression, and then during the Dex-CRH test (CRH stimulation test performed 2 h after completion of low-dose dexamethasone suppression), and contrasted their results with those of 20 patients with surgically proven mild CD [urine free cortisol (UFC) < 1000 nmol/day). Basal UFC was significantly greater in patients with CD (P < 0.001) but within the normal range (55-250 nmol/day) in 4 patients. During low-dose dexamethasone suppression, a UFC less than 100 nmol/day (36 micrograms/day) was found in all but 1 volunteer subject, and a urine 17-hydroxycorticosteroid excretion less than 14.6 mumol/day (5.3 mg/day) was found in all but 2 subjects. During the Dex-CRH test, plasma cortisol less than 38 nmol/L was found in all 20 normal volunteers until 30 min after CRH administration. By contrast, the 15-min CRH-stimulated plasma cortisol exceeded 38 nmol/L in all patients with CD (P < 0.001). Plasma dexamethasone measured just before CRH administration was similar in normal volunteers (13.0 +/- 6.1 mumol/L) and patients with CD (16.4 +/- 6.4 mumol/L). We conclude that cortisol measurements obtained during the Dex-CRH test are suppressed in normal volunteers below those found in mild CD. These results suggest that the Dex-CRH test may be useful in the evaluation of CS in patients without significant hypercortisoluria. However, its value in patients with episodic hormonogenesis has not been tested.

Enhanced growth hormone (GH) responsiveness to GH-releasing hormone after dietary restriction in patients with Cushing's syndrome.

In patients with Cushing's syndrome, decreased growth hormone (GH) secretion is observed although the basic mechanism is not yet understood. A short-term hypocaloric diet is known to increase both spontaneous and GHRH-stimulated GH secretion in normal subjects. In order to gain further insight into the altered GH secretion in patients with Cushing's syndrome, we assessed the effect of a short-term hypocaloric diet on GH responses to GHRH in these patients. Two GHRH tests (1 microgram/kg i.v.) were performed, the first under basal conditions (normocaloric diet) and the second after a 3-day hypocaloric diet (650 cal/day). Six female patients with untreated Cushing's disease. Plasma GH levels were measured by immunoradiometric assay. GHRH-induced GH release was impaired in patients with Cushing's disease on a normal diet (mean peak 12.4 +/- 6.4 mU/l, area under the curve (AUC) 744 +/- 332 mU/l/120 min). Following a hypocaloric diet, GH responses to GHRH were markedly enhanced in the same group of patients (mean peak 46.2 +/- 14.8 mU/l, AUC 3142 +/- 1032 mU/l/120 min, P < 0.05). This study demonstrates that in patients with Cushing's disease the somatotroph hyporesponsiveness to growth hormone releasing home is improved after a short-term hypocaloric diet. Therefore, blunted growth-hormone secretion in chronic hypercortisolism is a potentially reversible state and the secretory capacity of the somatotroph appears not to be severely compromised in patients with Cushing's disease.

Resistance to vasopressin action on the kidney in patients with Cushing's disease.

To assess the plasma levels and action of arginine vasopressin (AVP) in patients with Cushing's disease. There are many reports that patients with Addison's disease have increased AVP levels associated with hyponatraemia and hypoosmolality, but none on the dynamics of secretion of this neurohormone during osmolality-based stimulation in patients with chronic hypercortisolism. The plasma AVP concentration and the urinary and plasma osmolality after a 7.5-h water deprivation test (WDT) were evaluated in 13 patients with Cushing's disease and 15 normal (control) individuals. In patients with Cushing's disease we also assessed the urinary osmolality in response to 10 micrograms i.v. desmopressin (DDAVP) administered at the end of the WDT. At the end of the WDT, urinary osmolality was significantly lower in patients with Cushing's disease (511.5 +/- 148.5 mOsm/l) than in the normal subjects (981.1 +/- 107.1 mOsm/l, P < 0.001), whereas plasma osmolality did not differ between the two groups. Consequently, the urine/plasma osmolality ratio (Uosm/Posm) was lower in patients with Cushing's disease than in normal individuals (1.8 +/- 0.5 compared with 3.4 +/- 0.4, P < 0.001). The AVP concentration also was greater (7.3 +/- 3.1 pmol/l) in those with Cushing's disease than in the controls (3.9 +/- 2.3 pmol/l, P < 0.005). After administration of DDAVP to the hypercortisolaemic patients, the urinary osmolality attained (718.0 +/- 200.0 mOsm/l) was still lower than that in the normal group at the end of WDT (P < 0.005). Patients with Cushing's disease presented higher AVP levels and smaller Uosm/Posm ratios than normal subjects. After DDAVP, the patients with Cushing's disease were unable to concentrate the urine adequately. These data suggest that the kidney shows resistance to the action of both endogenous and exogenous AVP in patients with Cushing's disease.

Plasma leptin levels do not change in patients with Cushing's disease shortly after correction of hypercortisolism.

In the present study, we characterized the changes in plasma leptin levels in patients with pituitary Cushing's disease and in age- and sex-matched controls. Plasma levels of ACTH, cortisol, and leptin were measured before and after iv administration of ovine CRH in controls once and in patients twice (while they had active hypercortisolism and 10 days after successful surgery). Cushing's patients had elevated body mass indexes (34 +/- 1.9 vs. 22.9 +/- 0.8) and plasma leptin levels (35.6 +/- 3.4 vs. 9.2 +/- 1.9 ng/mL) compared to controls, which remained unchanged 10 days after successful transsphenoidal surgery and directly proportional to the body mass index. Plasma leptin levels were not affected by CRH infusion in either the controls or the patients despite clear-cut elevations in plasma ACTH and cortisol. These findings suggest that although acute changes in plasma cortisol do not affect plasma leptin, chronic hypercortisolism results in elevated leptin levels, probably by causing visceral obesity.

Glucocorticoid-Phenylethanolamine-N-methyltransferase Interactions in Humans

Publisher Summary Phenylethanolamine-N-methyltransferase (PNMT) is highly concentrated in the adrenal medulla but has also been demonstrated, although to a much limited extent, in other human tissues, such as lung, heart, kidney, liver, spleen, and pancreas. From in vitro and in vivo studies performed in several animal species, it is well known that in adrenal medulla PNMT activity is dependent on the high levels of glucocorticoids received from the cortex. In vivo data from studies performed in humans are scanty. In children with hypocortisolism because of adrenocotricotropic hormone (ACTH) deficiency, plasma Epi levels are lower. Stimulation of Epi secretion was achieved by intravenous glucagon. Patients with HP were studied while receiving thyroid, glucocorticoid, and sexual steroid replacement therapy. Obtained data confirm that in humans, adrenal PNMT activity seems to depend on the normal cortisol supply to the medulla from the cortex. In fact, in adults affected by HP because of ACTH deficiency, the adrenal medulla secretion is impaired in basal as well as in stimulated conditions. The deficiency of Epi secretion is very probably one of the most important factors causing an impaired response to hypoglycemia in these patients. In fact, the similar Epi/NE ratio found in the adrenal vein of patients with and without chronic hypercortisolism suggests that in humans, a normal cortisol secretion is sufficient to activate PNMT maximally. These results confirm the finding that in the adrenal medullas obtained at surgery from patients with cushing's syndrome (CS), the PNMT activity resulted similar to that found in control human adrenal glands obtained at autopsy.

Growth hormone axis in cushing's syndrome.

All levels of the growth hormone (GH), GH binding protein (GHBP), insulin-like growth factor (IGF) and IGF binding protein (IGFBP) axis are influenced by chronic hypercortisolism. Thus, there is a blunted response to GHRH alone or together with other stimuli associated with a marked suppression of endogenous GH secretion but accompanied by normal GHBP, normal to low IGF-1 and GHBPs 1 and 3 with the correspondent 41.5 and 38.5-kD molecular forms of the latter presenting values similar to normal. These findings may suggest enhanced GH sensitivity with normal or increased IGF-1 bioavailability to the correspondent tissue receptors. In conclusion, the glucocorticoid (GC)-induced target tissue resistance can neither be attributed to the suppression of the GH axis nor to changes in circulating GHBPs 1 and 3. However, it may be related either to the described 12-to-20-kD inhibitor(s) which antagonizes postbinding IGF-1 bioactivity (gene expression) and/or by the downmodulation of activator protein-1 (Fos/Jun) activity by the GC-GC receptor complex.

Regulation of hypothalamic somatostatin by glucocorticoids.

Glucocorticoids (GCs) play a key role in the physiology of the hypothalamic-somatotroph axis, since these steroids enhance growth hormone (GH) gene transcription and increase GHRH receptor synthesis. However, GC excess inhibits normal growth in all species studied. This is mainly due to the impaired GH secretion observed during hypercortisolism, a situation in which GH responses to a number of stimuli, including GHRH, are blunted. The inhibitory effect of GCs on GH secretion seems to be dependent on enhanced hypothalamic SS secretion. Since SS release is stimulated by beta-adrenergic agonism we tested the possibility that GC inhibition of GH secretion would depend on increased beta-adrenoceptor activity in SS-producing neurons. The experimental design consisted in evaluating the GH response to GHRH in normal subjects after having induced hypercortisolism, with DEX, and blocking beta-adrenoceptors with propranolol (PRO). Moreover, to investigate the specificity of this mechanism, GHRH-induced GH release was tested after inducing hypercortisolism and enhancing alpha 2-adrenergic or muscarinic cholinergic tone, by giving clonidine (CLO) or pyridostigmine (PD), respectively. As expected, nocturnal DEX administration inhibited the GH response to GHRH. In this situation of hypercortisolism, both PRO and CLO, but not PD, were able to reverse the inhibitory effect of DEX on GHRH-elicited release. However, the potentiating effect of these drugs on the GHRH-induced GH secretion was only observed for PRO. These data confirm that GC excess inhibits GH release by increasing hypothalamic SS secretion, and that the mechanism is mediated by GC-induced enhanced beta-adrenergic responsiveness. Therefore, the defective GHRH secretion observed in chronic hypercortisolism must be a consequence of the continuous blockade that SS excess exerts on GHRH-producing neurons. Our postulate agrees with other data in the literature showing that GCs modulate the secretion of some hypothalamic peptides by changing the responsiveness of the producing neurons from alpha 2-adrenoceptors to that of beta-adrenoceptors.

Chronic glucocorticoid excess and impaired osmoregulation of vasopressin release in dogs with hepatic encephalopathy

Chronic liver disease may be accompanied by disturbed sodium and water homeostasis. There is usually sodium retention and ascites. However, spontaneous natriuresis has also been reported in humans and experimental animals with liver cirrhosis. Chronic hypercortisolism, which may occur in dogs with advanced liver disease, is known to induce the inhibition of the osmostimulation of vasopressin (AVP) release. We have therefore investigated the osmoregulation of AVP release in 11 dogs with chronic hypercortisolism associated with advanced liver dysfunction and hepatic encephalopathy and in 10 control dogs. Basal pituitary-adrenocortical activity was investigated by measuring the concentration in multiple plasma samples of adrenocorticotropin (ACTH), α-melanocyte-stimulating hormone (MSH), and cortisol and the cortisol:creatinine ratio in 24-hr urine. Urine specific gravity was also measured. The feedback regulation of the system was investigated by measuring these hormones in plasma after an intravenous (iv) injection of 0.01 mg/kg of dexamethasone. The osmoregulation of the release of AVP was investigated by the intravenous infusion of a 20% NaCl solution at a flow rate of 0.03 ml/kg for 2 hr and the measurement of AVP in plasma sampled at 20-min intervals. The AVP release was analyzed in terms of the threshold osmolality at which it commenced and the sensitivity, which reflects the magnitude of the response. All dogs had highly increased urinary cortisol:creatinine ratios, ranging from 21 to 210 × 10 −6 (normally <10 × 10 −6). The mean basal plasma concentrations of the three pituitary-adrenocortical hormones were significantly increased. ACTH values were 35 to 146 ng/l (normally, 14 to 68), MSH values were 26 to 118 ng/l (normally, 10 to 36), and cortisol values were 88 to 194 nmol/l (normally, 23 to 112). The feedback inhibition of the secretion of ACTH and cortisol in response to dexamethasone was unaffected. Urine specific gravity was significantly decreased. The regulation of AVP release was found to be abnormal in all dogs with hepatic encephalopathy. The osmotic threshold at which the release of AVP was induced was abnormally high in seven of the dogs with liver disease and in the normal range in one. It could not be determined in three dogs. The sensitivity of AVP release in response to increasing plasma hypertonicity was normal in two dogs and decreased in nine. In three dogs, there was no increase in AVP release. None of the dogs had normal values for both the sensitivity and the threshold. We conclude that dogs with liver disease associated with hepatic encephalopathy have hyper-adrenocorticism due to a resetting of the pituitary-adrenocortical axis, associated with profoundly impaired osmoregulation of the release of AVP by the posterior pituitary. These neuroendocrine derangements may contribute to impaired electrolyte homeostasis in chronic liver disease.

Adrenal medulla secretion in Cushing's syndrome.

To investigate whether chronic endogenous hypercortisolism might alter adrenomedullary phenylethanolamine N-methyltransferase activity, we measured epinephrine/norepinephrine (E/NE) ratios in the adrenal venous blood of 8 patients undergoing surgery for Cushing's syndrome and in 12 control subjects undergoing surgery for left kidney diseases. To investigate the adrenomedullary secretory activity in Cushing's syndrome, we measured basal E plasma levels in 24 patients and 32 age- and sex-matched normal control subjects, and we evaluated the adrenomedullary response to glucagon in 9 patients and in 22 age- and sex-matched normal subjects. Last, to clarify whether chronic endogenous hypercortisolism might modify E plasma levels through a modification of E metabolism, we measured the E MCR in four patients and four age-matched controls. Mean (+/- SEM) E/NE ratio in adrenal venous blood was similar in patients with Cushing's syndrome (4.61 +/- 0.78) and in the control group (4.71 +/- 0.74). Mean (+/- SEM) basal plasma E was significantly lower in patients with Cushing's syndrome (98.2 +/- 10.9 vs. 184 +/- 25.1 pmol/L, P < 0.01) than in the control group. Similarly, plasma NE also was reduced (0.75 +/- 0.09 vs. 1.10 +/- 0.07 nmol/L, P < 0.01). In patients with Cushing's syndrome the E response to glucagon was significantly reduced (P < 0.01). E MCR was almost identical in patients with Cushing's syndrome (1.48 +/- 0.10 L/min.m2) and in control subjects (1.51 +/- 0.10 L/min.m2). Our data demonstrate that: 1) chronic endogenous hypercortisolism is not able to change adrenomedullary phenylethanolamine N-methyltransferase activity and therefore the quality of adrenomedullary secretion; and 2) chronic endogenous hypercortisolism causes a decrease in basal and stimulated adrenomedullary activity without altering E MCR significantly. Therefore the adrenal medulla does not seem to play a pathogenetic role in the hypertension of Cushing's syndrome.

The ovine corticotropin-releasing hormone-stimulation test in type I diabetic patients and controls: Suggestion of mild chronic hypercortisolism

We examined hypothalamic-pituitary-adrenal (HPA) axis function in insulin-dependent diabetic outpatients (N = 22) and age-, sex-, and weight-matched normal controls (N = 22). The evaluation included measurements of 9:00 am fasting plasma cortisol and cortisol-binding globulin (CBG) levels, 24-hour urinary free cortisol (UFC) excretion, and plasma corticotropin and cortisol responses to intravenously administered ovine corticotropin-releasing hormone ([CRH] 1 μg/kg given as a bolus at 8:00 pm). Diabetic patients had significantly elevated 9:00 am plasma cortisol levels (mean ± SE, 300.7 ± 99.3 v 237.3 ± 99.3 nmol/L, P < .04), higher 24-hour UFC excretion ( 313.2 ± 112.6 v 244.2 ± 69.3 nmol 24 h , P < .02 ), and greater cortisol responses to CRH infusion (time-integrated values: 49,408.2 ± 11,289.8 v 40,217.9 ± 7,228.6 nmol/L · 120 min, P < .004; peak cortisol values: 529.7 ± 107.6 v 438.7 ± 77.3 nmol/L, P < .002) than controls. UFC excretion values were positively correlated with both 5-year averaged hemoglobin A 1c level ( P = .03) and total number of insulin units administered per day ( P = .03). These results suggest that insulin-dependent diabetic outpatients have mild chronic hypercortisolism, which might influence the control of the disease and play a role in the development of its chronic complications.

Growth hormone releasing hormone priming increases growth hormone secretion in patients with Cushing's syndrome.

In patients with Cushing's syndrome, decreased growth hormone (GH) secretion is observed though the basic mechanism is unknown. In states of chronic deficiency of hypothalamic growth hormone releasing hormone (GHRH) release, a blunted GH response to exogenous GHRH has been reported; such impairment can be partially normalized by repetitive GHRH administration (priming). In order to clarify whether a deficit in hypothalamic release of GHRH is the basis of the decreased GH secretion in patients with Cushing's syndrome, GHRH plus pyridostigmine tests were undertaken, both before and after GHRH priming. GHRH (200 micrograms/day as a single s.c. injection) was given daily over 7 days. Two pyridostigmine (120 mg p.o.) plus GHRH (100 micrograms i.v.) tests were performed before and after priming to assess GH response. Eight patients (seven women, one man), with untreated Cushing's syndrome (six Cushing's disease, one autonomous bilateral adrenal hyperplasia, one adrenal adenoma), were studied. Plasma GH levels were measured by immunoradiometric assay. GHRH plus pyridostigmine-induced GH release was impaired in patients with untreated Cushing's syndrome (mean peak 5.2 +/- 1.4 mU/l, area under the curve (AUC) 472 +/- 96). Repetitive administration of GHRH over 7 days partially restored the GH response to the second pyridostigmine-GHRH test (mean peak 15.0 +/- 2.1 mU/l. AUC 1016 +/- 104), both P < 0.05. All of the eight Cushing's syndrome patients studied presented a higher GHRH plus pyridostigmine-induced GH secretion after priming. Repetitive administration of GHRH increases the pyridostigmine-GHRH-induced GH secretion in patients with Cushing's syndrome. This suggests that impaired hypothalamic release of GHRH is a contributing factor to the decreased GH secretion observed in chronic hypercortisolism.

Unrestrained production of proopiomelanocortin (POMC) and its peptide fragments by pituitary corticotroph adenomas in Cushing's disease

The hallmark of ACTH oversecretion in Cushing's disease is its partial resistance to the normal suppressive effect of glucocorticoids. Because ACTH secretion by the pituitary tumor is not normally restrained ACTH is overproduced with subsequent chronic hypercortisolism. Since peripheral tissues have retained their normal sensitivity to the action of cortisol they appropriately develop the features of Cushing's disease. The question of whether a collection of corticotroph cells, eventually arranged in an adenomatous-like fashion, is a primary pituitary event or is corticotropin-releasing factor driven has had no response so far. Clonal composition of such lesions has been determined by X chromosome inactivation using DNA probes which detect multiallelic polymorphism in females. A monoclonal pattern is found in all macroadenomas. ACTH is co-secreted with other peptide fragments derived from their common polypeptide precursor, proopiomelanocortin (POMC). As a rule POMC processing in pituitary tumors is qualitatively unaltered: plasma values of the N-terminal fragment, the joining peptide, the β-and γ-lipotropins, and β-endorphin all are valid alternate markers of the tumor activity. Tumor POMC peptides including ACTH and its phosphorylated form usually show no peculiar or unexpected molecular forms in contrast with what is often found when POMC expression occurs in a non-pituitary tumor.