Morphologic and Molecular Characterization of Adrenals and Adrenal Rest Affected by Congenital Adrenal Hyperplasia.

Introduction: Adrenocortical hyperplasia and adrenal rest tumor (ART) formation are common in congenital adrenal hyperplasia (CAH). Although driven by excessive corticotropin, much is unknown regarding the morphology and transformation of these tissues. Our study objective was to characterize CAH-affected adrenals and ART and compare with control adrenal and gonadal tissues. Patients/Methods: CAH adrenals, ART and control tissues were analyzed by histology, immunohistochemistry, and transcriptome sequencing. We investigated protein expression of the ACTH receptor (MC2R), steroidogenic (CYP11B2, CYP11B1, CYB5A) and immune (CD20, CD3, CD68) biomarkers, and delta-like 1 homolog (DLK1), a membrane bound protein broadly expressed in fetal and many endocrine cells. RNA was isolated and gene expression was analyzed by RNA sequencing (RNA-seq) followed by principle component, and unsupervised clustering analyses. Results: Based on immunohistochemistry, CAH adrenals and ART demonstrated increased zona reticularis (ZR)-like CYB5A expression, compared to CYP11B1, and CYP11B2, markers of zona fasciculata and zona glomerulosa respectively. CYP11B2 was mostly absent in CAH adrenals and absent in ART. DLK1 was present in CAH adrenal, ART, and also control adrenal and testis, but was absent in control ovary. Increased expression of adrenocortical marker MC2R, was observed in CAH adrenals compared to control adrenal. Unlike control tissues, significant nodular lymphocytic infiltration was observed in CAH adrenals and ART, with CD20 (B-cell), CD3 (T-cell) and CD68 (macrophage/monocyte) markers of inflammation. RNA-seq data revealed co-expression of adrenal MC2R, and testis-specific INSL3, HSD17B3 in testicular ART indicating the presence of both gonadal and adrenal features, and high expression of DLK1 in ART, CAH adrenals and control adrenal. Principal component analysis indicated that the ART transcriptome was more similar to CAH adrenals and least similar to control testis tissue. Conclusions: CAH-affected adrenal glands and ART have similar expression profiles and morphology, demonstrating increased CYB5A with ZR characteristics and lymphocytic infiltration, suggesting a common origin that is similarly affected by the abnormal hormonal milieu. Immune system modulators may play a role in tumor formation of CAH.

Metabolic profiling of serum from dogs with pituitary-dependent hyperadrenocorticism.

Hyperadrenocorticism (HAC) is one of the most common endocrine diseases in dogs characterized by excessive cortisol production caused by an adrenocorticotropic hormone (ACTH)-secreting tumor, namely pituitary-dependent HAC (PDH) or cortisol-secreting adrenal tumor. Metabolomics presents the ability to identify small molecule metabolites. Thus, the use of metabolomics techniques in canine PDH can provide information about the pathophysiology and metabolic changes in this disease. This study aimed to identify and compare differences in serum metabolites between dogs with PDH and healthy dogs. The metabolomic profile of 20 dogs diagnosed with PDH was compared with 20 healthy dogs using liquid chromatography/mass spectrometry (LC/MS), and metabolite discrimination was performed using partial least squares-discriminant analysis (PLS-DA), the variable important in projection (VIP) and fold changes (FC) group-wise comparisons. The hypergeometric test identified the significantly altered pathways. A total of 21 metabolites were found to be significantly different between the two groups. The major alterations were found in arachidonic and decanoic acid, and phospholipids related to phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI). These metabolites are related to insulin resistance and other complications (i.e. hypertension). Our results indicate that PDH produces changes in serum metabolites of dogs, and the knowledge of these changes can aid to better understanding of pathophysiological processes involved and contribute to potentially detect new biomarkers for this disease.

Impaired dexamethasone resorption in two patients with pseudo-Cushing after bariatric surgery: Implications for immunosuppressive treatment.

Two cases of middle-aged female patients treated by gastric bypass surgery for weight loss presented to our clinic for a follow-up examination 3-6 months after the surgical procedure (a mini gastric bypass and a modified single anastomosis sleeve-ileostomy). In both patients increased ACTH levels and either high serum cortisol or an increased urinary cortisol excretion was apparent and triggered further endocrine testing. Serum cortisol could not be suppressed adequately by 2 and 4 mg dexamethasone in the standardized oral overnight suppression test while midnight salivary cortisol dropped well below the desired cut-off. This led to the hypothesis of an impaired dexamethasone resorption and could be further substantiated by suppression of serum cortisol below the cut-off by an intravenous dexamethasone application. The data presented point to an impairment of enteral synthetic corticosteroid resorption in patients after gastric bypass surgery and could be of importance for individuals in need for immunosuppressive treatment. In view of the growing number of bariatric procedures, pharmacokinetics of corticosteroids and other drugs should be tested in clinical trials.

Age-dependent effects of Armc5 haploinsufficiency on adrenocortical function.

Inactivating mutations in the Armadillo repeat-containing 5 (ARMC5) gene have recently been discovered in primary macronodular adrenal hyperplasia (PMAH), a cause of Cushing syndrome. Biallelic ARMC5 inactivation in PMAH suggested that ARMC5 may have tumor suppressor functions in the adrenal cortex. We generated and characterized a new mouse model of Armc5 deficiency. Almost all Armc5 knockout mice died during early embryonic development, around 6.5 and 8.5 days. Knockout embryos did not undergo gastrulation, as demonstrated by the absence of mesoderm development at E7.5. Armc5 heterozygote mice (Armc5+/-) developed normally but at the age of 1 year, their corticosterone levels decreased; this was associated with a decrease of protein kinase A (PKA) catalytic subunit α (Cα) expression both at the RNA and protein levels that were also seen in human patients with PMAH and ARMC5 defects. However, this was transient, as corticosterone levels normalized later, followed by the development of hypercorticosteronemia in one-third of the mice at 18 months of age, which was associated with increases in PKA and Cα expression. Adrenocortical tissue analysis from Armc5+/- mice at 18 months showed an abnormal activation of the Wnt/ß-catenin signaling pathway in a subset of zona fasciculata cells. These data confirm that Armc5 plays an important role in early mouse embryonic development. Our new mouse line can be used to study tissue-specific effects of Armc5. Finally, Armc5 haploinsufficiency leads to Cushing syndrome in mice, but only later in life, and this involves PKA, its catalytic subunit Cα, and the Wnt/ß-catenin pathway.

Carbenoxolone Disodium Treatment for Canine Pituitary-Dependent Hyperadrenocorticism.

Pituitary-dependent hyperadrenocorticism (PDH) is mainly caused by pituitary corticotroph tumors in dogs. A characteristic feature of corticotroph tumors is their resistance to negative feedback by glucocorticoids. In some animal species, including dogs, the aberrant expression of 11ß-hydroxysteroid dehydrogenase (11HSD), a cortisol metabolic enzyme, is observed in corticotroph tumors. We previously reported that carbenoxolone (CBX), an inhibitor of 11HSD, suppressed ACTH secretion from the pituitary gland, and decreased cortisol concentrations in healthy dogs. Therefore, the aim of this study was to investigate the therapeutic effects of CBX on dogs with PDH. Six dogs with PDH were treated with 60 to 80 mg/kg/day of CBX for 6 weeks, followed by trilostane, which is a commonly used agent for canine PDH. CBX treatment led to a gradual decrease in both basal and in corticotropic releasing hormone (CRH)-stimulated plasma ACTH concentrations and CRH-stimulated serum cortisol concentrations, without side effects. However, basal and stimulated ACTH and cortisol concentrations remained higher than those of healthy dogs, and clinical symptoms such as polydipsia and polyuria were not ameliorated. After a 2-week wash-out interval, trilostane was administered for 2 weeks. Although basal plasma ACTH concentrations were higher after trilostane treatment than CBX treatment, polydipsia and polyuria resolved in all six dogs. The reason for the lack of improvement in polydipsia and polyuria with CBX treatment is unclear. Other mechanisms, in addition to a partial decrease in ACTH secretion, are likely to be involved. In conclusion, this is the first study to report the in vivo effects of CBX in dogs with PDH. The findings suggest that CBX inhibits ACTH secretion from canine pituitary tumors, resulting in lower cortisol concentrations.

Prediction of Vertebral Fractures in Patients With Monolateral Adrenal Incidentalomas.

CONTEXT: Subtle hypercortisolism is associated with an increased risk of vertebral fracture (VFx). OBJECTIVE: The objective was to determine the best parameters of cortisol secretion for detecting the VFx risk in patients with adrenal incidentalomas (AI). DESIGN: This was a retrospective (cross-sectional arm) and prospective (longitudinal arm) design. In the cross-sectional arm, we assessed the accuracy of the cortisol secretion indexes in identifying the patients with VFx (prevalent VFx). In the longitudinal arm, we tested the cortisol secretion parameters, which were able to identify the prevalent VFx, for the prediction of the occurrence of a new VFx (incident VFx) in AI patients followed-up for at least 2 years. SETTING: Four referral Italian endocrinology units participated in this study. PATIENTS: A total of 444 and 126 AI patients without symptoms of hypercortisolism enrolled in the cross-sectional arm and longitudinal arm, respectively. MAIN OUTCOME MEASURES: Serum cortisol after a 1-mg dexamethasone suppression test (1 mg DST), urinary free cortisol, ACTH, bone mineral density at lumbar spine and femoral neck (by dual-energy x-ray absorptiometry), and the VFx presence (by x-ray). RESULTS: The cortisol levels after 1 mg DST that were greater than 2.0 µg/dl (55 nmol/liter) were the best criteria for detecting patients with both prevalent (73.6% sensitivity, 70.5% specificity) and incident VFx (80% sensitivity, 68.8% specificity) and were associated with a 10-fold increased risk of a new VFx (odds ratio,10.27; 95% confidence interval, 3.39-31.12; P < .0001), regardless of age, gender, bone mineral density at lumbar spine, and prevalent VFx. CONCLUSIONS: In AI patients without symptoms of overt hypercortisolism, the cortisol levels after 1 mg DST greater than 2.0 µg/dl (55 nmol/liter) represent the best criterion for detecting prevalent and incident VFx.

Influence of adrenal hyperandrogenism on the clinical and metabolic phenotype of women with polycystic ovary syndrome.

OBJECTIVE: To study the impact of adrenal hyperandrogenism (AH; defined as DHEAS concentration >95th percentile of a healthy female control population) on cardiometabolic risk factors associated with polycystic ovary syndrome (PCOS). DESIGN: Cross-sectional study. SETTING: Academic hospital. PATIENT(S): Two-hundred ninety-eight consecutive women with PCOS, of whom 120 were obese (body mass index [BMI] ≥30 kg/m(2)) and 178 nonobese (BMI <30 kg/m(2)). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Comprehensive evaluation of cardiovascular risk factors, including 75-g oral glucose tolerance test, office blood pressure, lipid profile, and low-grade inflammatory markers. RESULT(S): Patients with AH (AH-PCOS) had higher insulin circulating levels and lower insulin sensitivity than their counterparts without AH (non-AH-PCOS). Obesity, but not AH, was the main contributor to the presence of glucose tolerance disorders. Both obesity and AH increased the prevalence of prehypertension and hypertension. AH diminished high-density lipoprotein (HDL) levels in nonobese PCOS women in parallel with a decrease in total cholesterol levels, leading to a total to HDL cholesterol ratio similar to that of nonobese non-AH-PCOS patients. Furthermore, AH blunted the deleterious effect of obesity on the total cholesterol/HDL ratio, with the ratio of obese AH-PCOS patients being similar to that of nonobese PCOS patients with or without AH. CONCLUSION(S): The presence of AH in women with PCOS is associated with reduced insulin sensitivity and increased blood pressure but may have beneficial impact on the lipid profile. Obesity is the main determinant of the clustering of cardiovascular risk factors in PCOS women.

Decreased gene expressions of insulin signal molecules in canine hyperadrenocorticism.

Hyperadrenocorticism (HAC) is a common endocrine disorder in dogs, in which excess glucocorticoid causes insulin resistance. Disturbance of insulin action may be caused by multiple factors, including transcriptional modulation of insulin signal molecules which lie downstream of insulin binding to insulin receptors. In this study, gene expressions of insulin signal molecules were examined using neutrophils of the HAC dogs (the untreated dogs and the dogs which had been treated with trilostane). Insulin receptor substrate (IRS)-1, IRS-2, phosphatidylinositol 3-kinase (PI3-K), protein kinase B/Akt kinase (Akt)-2 and protein kinase C (PKC)-lambda were analyzed in the HAC dogs and compared with those from normal dogs. The IRS-1 gene expressions decreased by 37% and 35% of the control dogs in the untreated and treated groups, respectively. The IRS-2 gene expressions decreased by 61% and 72%, the PI3-K gene expressions decreased by 47% and 55%, and the Akt-2 gene expressions decreased by 45% and 56% of the control dogs, similarly. Collectively, gene expressions of insulin signal molecules are suppressed in the HAC dogs, which may partially contribute to the induction of insulin resistance.

Expression of sfrp1 and activation of the Wnt pathway in the adrenal glands of healthy ferrets and neutered ferrets with hyperadrenocorticism.

Gonadectomy induces the pathogenesis of luteinising hormone receptor positive, androgen and oestrogen producing tumours in the adrenal cortex of ferrets. In mice, the castration-dependent appearance of adrenocortical tumours has been attributed to loss of expression of the tumour suppressor gene Secreted Frizzled Related Protein 1 (sfrp1), a dominant inhibitor of the Wnt pathway, which controls cell proliferation and 'cell faith' decisions. This study investigated whether sfrp1 and the Wnt pathway play a similar role in the pathogenesis of hyperadrenocorticism in ferrets. The expression of sfrp1 and three target genes of the Wnt pathway (c-myc, axin2 and cyclinD1) in seven adrenal glands from healthy ferrets and in 13 adrenocortical tumours were studied by quantitative real-time PCR. Nuclear ß-catenin staining was assessed by immunohistochemistry. Sfrp1 mRNA expression was up-regulated and axin2 and cyclinD1 were down-regulated in the tumour group in comparison with the control group. Decreased nuclear ß-catenin staining supported the decrease in active Wnt signalling in adrenocortical tumours in ferrets. Therefore, it is unlikely that the involvement of sfrp1 and the Wnt pathway in the pathogenesis of adrenocortical tumours in ferrets is similar to that described in mice.

Activation of cyclic AMP signaling leads to different pathway alterations in lesions of the adrenal cortex caused by germline PRKAR1A defects versus those due to somatic GNAS mutations.

CONTEXT: The overwhelming majority of benign lesions of the adrenal cortex leading to Cushing syndrome are linked to one or another abnormality of the cAMP or protein kinase pathway. PRKAR1A-inactivating mutations are responsible for primary pigmented nodular adrenocortical disease, whereas somatic GNAS activating mutations cause macronodular disease in the context of McCune-Albright syndrome, ACTH-independent macronodular hyperplasia, and, rarely, cortisol-producing adenomas. OBJECTIVE AND DESIGN: The whole-genome expression profile (WGEP) of normal (pooled) adrenals, PRKAR1A- (3) and GNAS-mutant (3) was studied. Quantitative RT-PCR and Western blot were used to validate WGEP findings. RESULTS: MAPK and p53 signaling pathways were highly overexpressed in all lesions against normal tissue. GNAS-mutant tissues were significantly enriched for extracellular matrix receptor interaction and focal adhesion pathways when compared with PRKAR1A-mutant (fold enrichment 3.5, P < 0.0001 and 2.1, P < 0.002, respectively). NFKB, NFKBIA, and TNFRSF1A were higher in GNAS-mutant tumors (P < 0.05). Genes related to the Wnt signaling pathway (CCND1, CTNNB1, LEF1, LRP5, WISP1, and WNT3) were overexpressed in PRKAR1A-mutant lesions. CONCLUSION: WGEP analysis revealed that not all cAMP activation is the same: adrenal lesions harboring PRKAR1A or GNAS mutations share the downstream activation of certain oncogenic signals (such as MAPK and some cell cycle genes) but differ substantially in their effects on others.

Blindness in dogs with pituitary dependent hyperadrenocorticism: relationship with glucose, cortisol and triglyceride concentration and with ophthalmic blood flow.

Pituitary dependent hyperadrenocorticism (PDH) shows a high morbidity and blindness is one of its complications. Compression of the optic chiasm (OC) by the hypophysis adenoma is one of the causes. Another cause could be due to vascular and metabolic alterations of the PDH. Out of a total of 70 dogs with confirmed diagnosis of PDH, 12/70 showed blindness. In only 2/12 the OC was compromised. Electroretinography in dogs without the OC being compromised showed altered A and B wave patterns. Ophthalmological Doppler showed an alteration of the blood flow only in blind dogs without OC compression. Cortisol concentrations (Co), triglycerides (Tg) and glycaemia (G) were greater in 10 dogs with non-compressive blindness vs. dogs with conserved vision. Loss of vision correlated with the increase in these variables. Blindness in dogs with PDH would be related to changes in retinal blood flow, associated to higher Co, Tg and G concentrations.

Increased clearance of cortisol by 5beta-reductase in a subgroup of women with adrenal hyperandrogenism in polycystic ovary syndrome.

OBJECTIVE: Increased peripheral metabolism of cortisol may explain compensatory ACTH-dependent adrenal steroidogenesis and hence hyperandrogenism in polycystic ovary syndrome (PCOS). Previous studies have described an increased 5alpha-reduction of cortisol or impaired regeneration of cortisol by 11beta-HSD1 in PCOS. However, these observations may be confounded by obesity. Moreover, the relationship between alterations in cortisol metabolism and the extent of adrenal androgen hyper-secretion in response to ACTH has not been established. This study aimed to examine the association between cortisol metabolism and ACTH-dependent adrenal hyperandrogenism in PCOS, independently of obesity. DESIGN: We compared 90 PCOS women (age 18-45 yr) stratified by adrenal androgen responses to ACTH1-24 and 45 controls matched for age and body weight. METHODS: PCOS women were stratified as normal responders (NR), intermediate responders (IR), and high responders (HR) to 250 microg ACTH1-24: NR (no.=27) had androstenedione and DHEA responses within 2 SD of the mean in controls; IR (no.=43) had DHEA responses >2 SD above controls; HR (no.=20) had both androstenedione and DHEA responses >2 SD above controls. RESULTS: All groups were similar for age, body weight, and body fat distribution. Basal testosterone, androstenedione, and 5alpha-dihydrotestosterone plasma levels were similarly elevated among the 3 groups of PCOS compared with controls, whereas basal DHEA-S was higher in HR (2.8+/-1.2 microg/ml) and IR (2.4+/-1.1 microg/ml) than in NR (1.8+/-0.8 microg/ml) and controls (1.7+/-0.6 microg/ml). The HR group had the lowest basal plasma cortisol levels (101+/-36 ng/ml vs IR 135+/-42 ng/ml, NR 144+/-48 ng/ml, and controls 165+/-48 ng/ml; all p<0.01), but the greatest cortisol response to ACTH1-24 (Delta(60-0)cortisol 173+/-60 ng/ml vs IR 136+/-51 ng/ml, NR 114+/-50 ng/ml, and controls 127+/-50 ng/ml; all p<0.01), and the highest urinary excretion of total and 5beta-reduced cortisol metabolites (eg 5beta-tetrahydrocortisol/ cortisol ratio 25.2+/-15.3 vs IR 18.8+/-10.7, NR 19.7+/-11.4, and controls 17.2+/-13.7; all p<0.05). There were no differences in urinary excretion of 5alpha-reduced cortisol metabolites or in 5alpha-dihydrotestosterone/testosterone ratio between groups. CONCLUSIONS: Adrenal androgen excess in PCOS is associated with increased inactivation of cortisol by 5beta-reductase that may lower cortisol blood levels and stimulate ACTH-dependent steroidogenesis.

Pituitary tumour-transforming gene (PTTG) and pituitary senescence.

Pituitary tumours account for 15% of intracranial neoplasms and are benign monoclonal neoplasms that may be clinically silent or secrete hormones, including prolactin, growth hormone, adrenocorticotrophic hormone or, rarely, thyroid-stimulating hormone or gonadotrophins. These adenomas account for clinical infertility, growth disorders and hypercortisolism or metabolic dysfunctions associated with hypopituitarism. We explored the role of disordered pituitary cell proliferation control in the pathogenesis of these invariably benign adenomas, studying the mechanisms underlying pituitary aneuploidy, premature proliferative arrest (senescence), markers of cell proliferation and tumorigenesis in single, double or triply mutant transgenic mice with mutations of Rb, Pttg and/or p21. Our results provide further insights into the role of cell-cycle control and growth constraints on experimental and human pituitary tumours, which underlie their failure to progress to malignancy. These results improve our understanding of pituitary syndromes associated with infertility, growth disorders, hypercortisolism or adrenal, thyroid and gonadal failure due to abrogated pituitary function.

The limited role of midnight salivary cortisol levels in the diagnosis of subclinical hypercortisolism in patients with adrenal incidentaloma.

OBJECTIVE: The criteria for defining subclinical hypercortisolism (SH) are debated and a real gold standard test or combination of tests is lacking. Recently, late-night salivary cortisol (MSC) has been described as a sensitive and easy-to-perform marker for diagnosing overt hypercortisolism. No data are available on the role of MSC in the diagnosis of SH. The aim of this study was to evaluate the sensitivity and specificity of MSC levels in the diagnosis of SH in patients with adrenal incidentalomas (AI). METHODS: In 103 (females/males, 69/34) patients with AI, MSC levels were studied. One milligram overnight dexamethasone suppression test (DST), urinary-free cortisol (UFC), and ACTH plasma levels were also evaluated. Patients were defined as affected by SH if they showed two of the following criteria: DST>83 nmol/l, ACTH <2.2 pmol/l, and UFC >193 nmol/24 h. RESULTS: No difference in MSC levels in patients with SH (3.1+/-3.1 nmol/l) compared with patients without SH (2.2+/-2.8 nmol/l) was observed. In patients with SH, MSC levels were significantly correlated with DST (r=0.4, P<0.05). Using the cut-off of 5.1 nmol/l, the sensitivity and specificity of MSC levels for diagnosis of SH is 22.7 and 87.7% respectively. CONCLUSION: In patients with AI, normal levels of MSC do not exclude SH, whereas high levels may suggest the presence of SH identified by conventional tests. Thus, MSC is not suitable as a screening test, although it may be used in conjunction with other tests as the confirming test in selected patients.

Major depression in late life is associated with both hypo- and hypercortisolemia.

BACKGROUND: In younger adults, depression has been associated with hypercortisolemia. In older depressed patients, however, both low and high cortisol levels have been reported. We examined the possibility of a U-shaped association between depression and cortisol in older people, suggesting both hypo- and hyperactivity of the hypothalamic-pituitary-adrenal axis. We also examined whether this might represent different depression subtypes. METHODS: This population-based study included 1185 subjects aged 65 and older. Depression was measured at both diagnostic (major depression) and symptomatic (subthreshold depression) levels of caseness. Plasma concentrations of cortisol (CORT) and corticosteroid binding globulin (CBG) were determined. From these (CORT/CBG), a free cortisol index (FCI) was computed. RESULTS: The association between cortisol and major depression was U-shaped (B CORT = -9.50 [SE 3.85] p = .014; B CORT(2) = .008 [SE .003] p = .021). Hypocortisolemic depression (lower cortisol tertile) was associated with female sex, joint diseases, and smoking. Hypercortisolemic (upper cortisol tertile) depression was associated with older age, male sex, cardiovascular diseases, nonsteroidal antiinflammatory use, and (borderline significant) cognitive impairment. CONCLUSIONS: In older people, the association between cortisol and major depression is U-shaped. Hypo- and hypercortisolemic depression may represent different depression subtypes, requiring different clinical management.

Secretion of sex hormones in dogs with adrenal dysfunction.

OBJECTIVE: To evaluate adrenal sex hormone concentrations in response to ACTH stimulation in healthy dogs, dogs with adrenal tumors, and dogs with pituitary-dependent hyperadrenocorticism (PDH). DESIGN: Prospective study. ANIMALS: 11 healthy control dogs, 9 dogs with adrenal-dependent hyperadrenocorticism (adenocarcinoma [ACA] or other tumor); 11 dogs with PDH, and 6 dogs with noncortisol-secreting adrenal tumors (ATs). PROCEDURE: Hyperadrenocorticism was diagnosed on the basis of clinical signs; physical examination findings; and results of ACTH stimulation test, low-dose dexamethasone suppression test, or both. Dogs with noncortisol-secreting ATs did not have hyperadrenocorticism but had ultrasonographic evidence of an AT. Concentrations of cortisol, androstenedione, estradiol, progesterone, testosterone, and 17-hydroxyprogesterone were measured before and 1 hour after i.m. administration of 0.25 mg of synthetic ACTH. RESULTS: All dogs with ACA, 10 dogs with PDH, and 4 dogs with ATs had 1 or more sex hormone concentrations greater than the reference range after ACTH stimulation. The absolute difference for progesterone, 17-hydroxyprogesterone, and testosterone concentrations (value obtained after ACTH administration minus value obtained before ACTH administration) was significantly greater for dogs with ACA, compared with the other 3 groups. The absolute difference for androstenedione was significantly greater for dogs with ACA, compared with dogs with AT and healthy control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs with ACA secrete increased concentrations of adrenal sex hormones, compared with dogs with PDH, noncortisol-secreting ATs, and healthy dogs. Dogs with noncortisol-secreting ATs also have increased concentrations of sex hormones. There is great interdog variability in sex hormone concentrations in dogs with ACA after stimulation with ACTH.

Effects of growth hormone-releasing peptides in healthy dogs and in dogs with pituitary-dependent hyperadrenocorticism.

The aim of this study is to investigate the effects of ghrelin and GH-releasing peptide-6 (GHRP-6) on the release of growth hormone (GH), adrenocorticotrophic hormone (ACTH), and cortisol in dogs with pituitary-dependent hyperadrenocorticism (PDH) and in healthy dogs of comparable age. In eight healthy dogs, the responses to ghrelin and GHRP-6 were compared to those of GH-releasing hormone (GHRH) and NaCl 0.9% (control). In seven dogs with PDH, the effects of ghrelin and GHRP-6 were compared with their effects in healthy dogs. In the healthy dogs, GHRH, GHRP-6, and ghrelin caused a significant rise in plasma GH concentrations. GHRH administration elicited significantly higher plasma GH concentrations than administration of ghrelin and GHRP-6. In the dogs with PDH, the GHRP-6-induced release of GH was significantly lower than in healthy dogs. Administration of ghrelin elicited a GH release that did not differ significantly between dogs with PDH and healthy dogs. Ghrelin and GHRP-6 did not cause a significant rise in plasma ACTH and cortisol concentrations in either the healthy dogs or the dogs with PDH. It is concluded that in comparison with GHRH, GHRP-6 and ghrelin have a low GH-releasing potency in healthy dogs. In dogs with PDH, the GH release in response to GHRP-6 is impaired. Neither GHRP-6 nor ghrelin activates the pituitary-adrenocortical axis in healthy elderly dogs and dogs with PDH.

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.

Steroidogenic response of adrenal tissues after administration of ACTH to dogs with hypercortisolemia.

OBJECTIVE: To evaluate adrenal sex hormone concentrations in neutered dogs with hypercortisolemia. DESIGN: Case series. ANIMALS: 11 neutered dogs with hypercortisolemia. PROCEDURE: Serum samples obtained before and 1 hour after administration of ACTH were evaluated for concentrations of cortisol, progesterone, testosterone, dehydroepiandrosterone sulfate or androstenedione or both, and 17-hydroxyprogesterone. RESULTS: For all dogs, concentrations of 1 or more adrenal sex hormones were substantially greater than reference range values before or after administration of ACTH. Testosterone concentration was not greater than reference range values in any of the dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results emphasize the importance of ruling out hypercortisolemia before measuring adrenal sex hormone concentrations as a means of diagnosing adrenal hyperplasia syndrome (alopecia X) in dogs.