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Recently, the genes of components of the renin - angiotensin system (RAS), namely angiotensinogen (AGT), angiotensin converting enzyme and angiotensin II receptor have been described in adipose tissue. In animal models the angiotensinogen in adipose tissue has been implicated in the pathogenesis of metabolic alterations and hypertension associated with obesity. The aim of our study was to evaluate the AGT gene expression both in visceral and subcutaneous adipose tissue in obese patients and lean subjects. AGT mRNA levels were measured by reverse transcriptase polymerase chain reaction (RT-PCR) using specific primers. AGT mRNA was expressed at variable levels in obese patients. It was significantly greater in visceral than in subcutaneous adipose tissue. Positive and significant correlation was found between the expression of AGT in visceral adipose tissue and BMI. These data suggest that angiotensinogen may be determinant of fat distribution and may be involved in the plurimetabolic syndrome of central obesity. International Journal of Obesity (2000) 24, Suppl 2, S142–S143. [ABSTRACT FROM AUTHOR]

Treatment options are insufficient in patients with adrenocortical carcinoma (ACC). Based on the efficacy of sorafenib, a tyrosine kinase inhibitor, and everolimus, an inhibitor of the mammalian target of rapamycin in tumors of different histotype, we aimed at testing these drugs in adrenocortical cancer models. The expression of vascular endothelial growth factor and its receptors (VEGFR1-2) was studied in 18 ACCs, 33 aldosterone-producing adenomas, 12 cortisol-producing adenomas, and six normal adrenal cortex by real-time PCR and immunohistochemistry and by immunoblotting in SW13 and H295R cancer cell lines. The effects of sorafenib and everolimus, alone or in combination, were tested on primary adrenocortical cultures and SW13 and H295R cells by evaluating cell viability and apoptosis in vitro and tumor growth inhibition of tumor cell line xenografts in immunodeficient mice in vivo. VEGF and VEGFR1-2 were detected in all samples and appeared over-expressed in two-thirds of ACC specimens. Dose-dependent inhibition of cell viability was observed particularly in SW13 cells after 24 h treatment with either drug; drug combination produced markedly synergistic growth inhibition. Increasing apoptosis was observed in tumor cells treated with the drugs, particularly with sorafenib. Finally, a significant mass reduction and increased survival were observed in SW13 xenograft model undergoing treatment with the drugs in combination. Our data suggest that an autocrine VEGF loop may exist within ACC. Furthermore, a combination of molecularly targeted agents may have both antiangiogenic and direct antitumor effects and thus could represent a new therapeutic tool for the treatment of ACC.

Objectives: Aldosterone production in the adrenal glomerulosa is mainly regulated by angiotensin II and K+. Adrenal glomerulosa cells are uniquely sensitive to extracellular K+. Genetic deletion of subunits of K+-selective leak-channels (KCNK), TASK1 and/or TASK3, in mice generates animals with hyperaldosteronism and histological changes in the adrenal cortex. Herein, we studied the expression of TASK1 in human adrenocortical cells, as well as its role in aldosterone production in H295R cells., Design: TASK1 expression was investigated by comparative microarray analysis of aldosterone-producing adenomas (APA) and normal adrenals (NAs). The effects of TASK1 knockdown by siRNA transfection were investigated in H295R cells. Fluo-4 fluorescent measurements of intracellular Ca2+ and pharmacological inhibition of Ca2+ -dependent calmodulin kinases (CaMK) were performed to better define the effects of TASK1 on Ca2+ signalling pathways., Results: Microarray analysis of APA and NA showed similar expression of TASK1 between these two groups. However, in APA, NA and H295R cells the expression of TASK1 was predominant when compared with other KCNK family members. Knockdown of TASK1 (with siRNA) induced the expression of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2), and also stimulated pregnenolone and aldosterone production. Cells transfected with siTASK1 had increased intracellular Ca2+, leading to activation of CaMK and increased expression of CYP11B2., Conclusions: Our study reveals the predominant expression of TASK1 over other KCNK family genes in the human adrenal cortex. Herein, we also described the role of TASK1 in the regulation of human aldosterone production through regulation of intracellular Ca2+ and CaMK signalling pathways.

The impact of genetics and genomics on clinical medicine is becoming more and more important. Endocrinology pioneered the development of molecular medicine, but also the study of adrenal tumors had a great impact in this field. Particularly important was the detection of genetics of tumors derived from the adrenal medulla, as well as that of those derived from the sympathetic and parasympathetic paraganglia. The identification of mutations in one of the several pheochromocytoma/paraganglioma susceptibility genes may indicate a specific clinical management drive. Less well understood is the genetics of adrenal cortex tumors, in particular adrenocortical carcinoma, a rare and particularly aggressive disease. There are only a few examples of hereditary transmission of adrenocortical carcinoma, but the analysis of low penetrance genes by genome wide association study may enable us to discover new genetic mechanisms responsible for adrenocortical-derived tumors.

Objective: Autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED) is a rare syndrome characterized by chronic candidiasis, chronic hypoparathyroidism and Addison's disease. APECED has been associated with mutations in autoimmune regulator (AIRE) gene. Our aim is to perform a genetic analysis of the AIRE gene in Italian APECED patients and in their relatives. Design AIRE mutations were determined by DNA sequencing in all subjects. Patients were tested for clinical autoimmune or non-autoimmune diseases, or for organ and non-organ specific autoantibodies., Patients: A total of 24 Italian patients with APECED (15 from the Venetian region, 2 from Southern-Tyrol, 4 from Apulia, 3 from Sicily), 25 relatives and 116 controls were studied., Results: Ten out of the 15 Venetian patients (66%) were homozygous for R257X or compound heterozygous with 1094-1106del13. One patient was homozygous for 1094-1106del13 and another for R139X. A novel mutation (1032-1033delGT) in combination with 1094-1106del13 was identified in one patient. No mutations were found in two cases. Two patients from Southern Tyrol were homozygous for R257X and for 1094-1106del13bp. All patients from Apulia were homozygous or heterozygous for W78R combined with Q358X. The patients from Sicily were homozygous for R203X or compound heterozygous with R257X. The analysis of the genotype-phenotype revealed that patients carrying 1094-1106del13 at the onset of Addison's disease were significantly older than those carrying other mutations. The genetic study of 25 relatives identified 20 heterozygous subjects. They suffered from various autoimmune and non-autoimmune diseases but no major disease of APECED was found., Conclusion: These data demonstrate the great genetic heterogeneity for the AIRE mutations in Italian APECED patients, and that the heterozygosity for AIRE mutations do not produce APECED.

Introduction: We report the clinical and immunological features of the autoimmune regulator gene (AIRE) in two Sicilian brothers with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). They were compound heterozygotes with R203X/R257X. Both had oral candidiasis since the first year of life and later developed hypoparathyroidism and Addison disease. The elder brother had experienced recurrent lower respiratory infections since 5 years of age and over the years developed severe obstructive lung disease with bronchiectasis, which led to death at 18 years of age. Both brothers had circulating autoantibodies against tryptophan hydroxylase and serotonin-producing cells were absent in the duodenal mucosa. This was associated with intestinal dysfunction in only the elder brother., Conclusion: (1) In the first Sicilian family with APECED reported up to now we found a heterozygous mutation that had been previously reported only once. (2) In the older brother of this family we observed a severe and lethal lung disease; this case adds to a growing literature describing this association between APECED and respiratory illnesses. (3) Tryptophan hydroxylase antibodies might be hypothesized to be the marker of an autoimmune gastrointestinal illness possibly associated with APECED.

The source of aldosterone in 30-40% of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined the expression of G-protein-coupled receptors (GPCRs) in APA and demonstrated that when compared with normal adrenals, there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs; n = 13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least 1 out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than threefold when compared with normal adrenals, suggesting a general increase in expression when compared with normal adrenal glands. Four GPCR transcripts exhibited a > 15-fold increase of expression in one or more of the APA samples when compared with normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be LH receptor, serotonin receptor 4, GnRH receptor, glutamate receptor metabotropic 3, endothelin receptor type B-like protein, and ACTH receptor. There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study.

Objective: To evaluate the expression of 11beta-hydrxysteroid dehydrogenase type 1 (11beta-HSD1) in omental adipose tissue of patients with Cushing's syndrome and simple obesity, compared with normal weight controls., Design and Methods: We have performed a case-control study and studied omental adipose tissue from a total of 24 subjects (eight obese subjects, ten patients with Cushing's syndrome due to adrenal adenoma, and six normal weight controls). Body mass index, blood pressure, plasma glucose, plasma insulin, plasma cortisol, urinary free cortisol and post dexamethasone plasma cortisol were measured with standard methods. 11beta-HSD1 mRNA and protein expression were evaluated in real-time PCR and western blot analysis respectively., Results: 11beta-HSD1 mRNA was 13-fold higher in obese subjects compared with controls (P=0.001). No differences were found between Cushing's patients and controls. Western blot analysis supported the mRNA expression results., Conclusions: Our data show the involvement of 11beta-HSD1 enzyme invisceral obesity, which is more evident in severely obese patients than in Cushing's syndrome patients. The lack of increase of 11beta-HSD1 expression in Cushing's syndrome could suggest downregulation of the enzyme as a result of long-term overstimulation.

Acromegaly is characterized by major cardiovascular alterations. Although the underlying mechanisms of these vascular modifications have not been elucidated, recent studies have focused on endothelial dysfunction. Nitric oxide (NO) may contribute to increased vascular resistance, reduced platelet aggregation, inhibition of smooth muscle cell proliferation, and reduction of lipoxygenase activity. At present, no data on NO production in acromegalics are available. The aim of this study was to evaluate the effect of high levels of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) present in acromegaly on NO pathway to investigate the role played by this molecule in the cardiovascular changes experienced by these patients. We studied 13 acromegalics and 12 sex- and age-matched normotensive controls. Platelet NO levels were measured in the supernatant of lysed platelets. Endothelial NO synthase (eNOS) was determined by Western blot analysis of platelets. NO concentrations were significantly reduced in patients (p<0.0001). There were no differences between male and female patients, nor were platelet NO levels and the presence/absence of hypertension related in acromegalics; by contrast, NO concentrations inversely correlated with GH (p=0.03) and IGF-1 (p=0.04) levels, and with disease duration (p=0.04). eNOS protein concentrations were significantly reduced in the platelets of patients compared with controls (p<0.0001). This study demonstrates for the first time a strong reduction in platelet NO concentrations in acromegalic patients due to reduced eNOS expression. Moreover the inverse correlation of NO levels with GH, IGF-1 and disease duration suggests that reduced levels of platelet NO linked to GH excess may contribute to the vascular alterations affecting patients with acromegaly.