Catia Pilon

Prevalence, Clinical, and Molecular Correlates of KCNJ5 Mutations in Primary Aldosteronism

Primary aldosteronism is the most common form of secondary hypertension. Mutations in the KCNJ5 gene have been described recently in aldosterone-producing adenomas (APAs). The aim of this study was to investigate the prevalence of KCNJ5 mutations in unselected patients with primary aldosteronism and their clinical, biological and molecular correlates. KCNJ5 sequencing was performed on somatic (APA, n=380) and peripheral (APA, n=344; bilateral adrenal hyperplasia, n=174) DNA of patients with primary aldosteronism, collected through the European Network for the Study of Adrenal Tumors. Transcriptome analysis was performed in 102 tumors. Somatic KCNJ5 mutations (p.Gly151Arg or p.Leu168Arg) were found in 34% (129 of 380) of APA. They were significantly more prevalent in females (49%) than males (19%; P<10−3) and in younger patients (42.1±1.0 versus 47.6±0.7 years; P<10−3) and were associated with higher preoperative aldosterone levels (455±26 versus 376±17 ng/L; P=0.012) but not with therapeutic outcome after surgery. Germline KCNJ5 mutations were found neither in patients with APA nor those with bilateral adrenal hyperplasia. Somatic KCNJ5 mutations were specific for APA, because they were not identified in 25 peritumoral adrenal tissues or 16 cortisol-producing adenomas. Hierarchical clustering of transcriptome profiles showed that APAs with p.Gly151Arg or p.Leu168Arg mutations were indistinguishable from tumors without KCNJ5 mutations. In conclusion, although a large proportion of sporadic APAs harbors somatic KCNJ5 mutations, germline mutations are not similarly causative for bilateral adrenal hyperplasia. KCNJ5 mutation carriers are more likely to be females; younger age and higher aldosterone levels at diagnosis suggest that KCNJ5 mutations may be associated with a more florid phenotype of primary aldosteronism.

Evidence for osteocalcin production by adipose tissue and its role in human metabolism.

CONTEXT The adipose tissue (AT), which is an endocrine organ, is linked to several metabolic abnormalities. Undercarboxylated osteocalcin (ucOCN) regulates insulin and adiponectin secretion. OBJECTIVE Our objective was to investigate the involvement of OCN in obesity and to evaluate, in vitro and ex vivo, the role of AT in the modulation of this endocrine circuit. DESIGN, PATIENTS, AND SETTING This transversal study involved 83 male subjects, divided according to the World Health Organization body mass index classification, evaluated at Padovas Obesity Outpatient Clinic. METHODS OCN, both undercarboxylated (ucOCN) and carboxylated (cOCN) forms, was measured in serum by ELISA. OCN mRNA expression and protein production were measured by quantitative RT-PCR and immunohistochemistry during in vitro adipogenesis and in sc AT (SAT) and omental AT (OAT) from normal adult men. cOCN and ucOCN release by AT in a simple growth medium was verified by ELISA. RESULTS Overweight and obese patients had a lower ucOCN and ucOC/OCN ratio. In the whole cohort, ucOCN/OCN ratio was negatively correlated to body mass index (rho = -0.233; P < 0.05). OCN mRNA was present in SAT and OAT and during all stages of adipogenesis, with higher expression in the first steps. Immunohistochemistry confirmed the expression of OCN protein. Both SAT and OAT were able to release cOCN and ucOCN. CONCLUSIONS Our data support a pathophysiological link between ucOCN and cOCN balance and obesity. OCN is present in the first phases of adipogenesis but also in human AT ex vivo. AT releases, in vitro, both ucOCN and cOCN, suggesting a possible link between AT and OCN in the regulation of metabolism.

CYP11B2 Gene Polymorphisms in Idiopathic Hyperaldosteronism

Primary aldosteronism is characterized by autonomous production of aldosterone and arterial hypertension, and it occurs in 2 principal forms: aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA). APA can be cured through removal of the adenoma, whereas IHA leads to hypertension that must be treated with medication. The origin of the autonomous aldosterone production in IHA is poorly understood, but genetic factors may contribute to its cause. To test the hypothesis that variants of the aldosterone synthase gene may contribute to susceptibility to IHA, we compared genotypes at 3 polymorphic sites in the CYP11B2 gene in patients with IHA (n=90) with those found in patients with APA (n=38), in patients with essential hypertension (n=72), and in normotensive individuals (n=102). We observed significant linkage disequilibrium among the 3 polymorphisms with 2 frequent haplotypes in all groups studied. One haplotype (C2R) was found to be increased in frequency in the IHA group (47%) compared with the other groups, which had a similar haplotype frequency (36%). The 3 polymorphisms studied have been implicated in either essential hypertension or excess aldosterone production in previous studies. Because of the strong linkage disequilibrium, the observed results could be due to the action of any 1 of the 3 alleles or to another allele in linkage disequilibrium with them. Our results suggest that variations in the CYP11B2 gene may contribute to dysregulation of aldosterone synthesis and lead to susceptibility to IHA.

Rosiglitazone modifies the adipogenic potential of human muscle satellite cells.

Aims/hypothesisSatellite cells are responsible for postnatal skeletal muscle regeneration. It has been demonstrated that mouse satellite cells behave as multipotent stem cells. We studied the differentiation capacities of human satellite cells and evaluated the effect of the insulin sensitiser rosiglitazone, a well known peroxisome proliferative activated receptor gamma (PPARG) agonist, on their adipogenic conversion.Subjects, materials and methodsWe obtained human satellite cells from human muscle biopsies of healthy subjects by single-fibre isolation and cultured them under myogenic, osteogenic and adipogenic conditions. Moreover, we compared the morphological features and the adipose-specific gene expression profiling, as assessed by quantitative PCR, between adipocytes differentiated from human satellite cells and those obtained from the stromal vascular fraction of human visceral fat.ResultsWe proved by morphological analysis, mRNA expression and immunohistochemistry that human satellite cells are able to differentiate into myotubes, adipocytes and osteocytes. The addition of rosiglitazone to the adipogenic medium strongly activated PPARG expression and enhanced adipogenesis in human satellite cells, but did not in itself trigger the complete adipogenic programme. Moreover, we observed a decrease in wingless-type MMTV integration site family member 10B and an upregulation of growth differentiation factor 8 expression, both being independent of PPARG activation.Conclusions/interpretationHuman satellite cells possess a clear adipogenic potential that could explain the presence of mature adipocytes within skeletal muscle in pathological conditions such as obesity, type 2 diabetes and ageing-related sarcopenia. Rosiglitazone treatment, while enhancing adipogenesis, induces a more favourable pattern of adipocytokine expression in satellite-derived fat cells. This could partially counteract the worsening effect of intermuscular adipose tissue depots on muscle insulin sensitivity.

Loss-of-Function Mutation of the GPR40 Gene Associates with Abnormal Stimulated Insulin Secretion by Acting on Intracellular Calcium Mobilization

BACKGROUND Free fatty acids (FFAs) acutely stimulate but chronically impair glucose-stimulated insulin secretion from beta-cells. The G protein-coupled transmembrane receptor 40 (GPR40) mediates both acute and chronic effects of FFAs on insulin secretion and plays a role in glucose homeostasis. Limited information is available on the effect of GPR40 genetic abnormalities on insulin secretion and metabolic regulation in human subjects. STUDY DESIGN AND RESULTS For in vivo studies, we screened 734 subjects for the coding region of GPR40 and identified a new single-nucleotide mutation (Gly180Ser). The mean allele frequency was 0.75%, which progressively increased (P < 0.05) from nonobese subjects (0.42%) to moderately obese (body mass index = 30-39.9 kg/m2, 1.07%) and severely obese patients (body mass index > or = 40 kg/m2, 2.60%). The relationship between the GPR40 mutation, insulin secretion, and metabolic alterations was studied in 11 Gly/Ser mutation carriers. In these subjects, insulin secretion (insulinogenic index derived from oral glucose tolerance test) was significantly lower than in 692 Gly/Gly carriers (86.0 +/- 48.2 vs. 183.7 +/- 134.4, P < 0.005). Moreover, a case-control study indicated that plasma insulin and C-peptide responses to a lipid load were significantly (P < 0.05) lower in six Gly/Ser than in 12 Gly/Gly carriers. In vitro experiments in HeLa cells cotransfected with aequorin and the mutated Gly/Ser GPR40 indicated that intracellular Ca2+ concentration increase after oleic acid was significantly lower than in Gly/Gly GPR40-transfected cells. This fact was confirmed using fura-2 acetoxymethyl ester. CONCLUSIONS This newly identified GPR40 variant results in a loss of function that prevents the beta-cell ability to adequately sense lipids as an insulin secretory stimulus because of impaired intracellular Ca2+ concentration increase.

Inhibition of Zac1, a New Gene Differentially Expressed in the Anterior Pituitary, Increases Cell Proliferation

Zac1 is a new zinc finger protein that concomitantly controls apoptosis and cell cycle arrest through separate pathways. The mouse Zac1 gene is mainly expressed in the pituitary gland and in different brain areas. In this study regional and cellular expression of Zac1 in the pituitary gland was determined by in situ hybridization. Zac1 messenger RNA was abundantly expressed in the anterior pituitary lobe compared with that in the intermediate and posterior lobes. Zac1 transcripts were found in all hormone-secreting cell types, with the highest levels in GH- and PRL-producing cells. To investigate the impact of Zac1 in pituitary cell proliferation, we ablated the endogenous Zac1 gene by antisense treatment in two murine cell types, AtT-20 and TtT/GF, that are representative of granular and agranular cell lineages, respectively. The decline in Zac1 protein levels under antisense treatment was accompanied by increased DNA synthesis in clonal corticotroph and folliculo-stellate cells, as demonstrated by enhan...

Primary aldosteronism and metabolic syndrome.

Hypertension is frequently associated with interrelated risk factors of metabolic origin, including abdominal obesity, dyslipidemia, and alterations in glucose homeostasis, all promoting the pathogenesis of arteriosclerosis. Clustering of these risk factors, defined as metabolic syndrome, is associated with an overall high cardiovascular risk profile. This article reviews current knowledge regarding the prevalence and characteristics of the metabolic syndrome in primary aldosteronism, and discusses a possible pathophysiological link between aldosterone and its individual components other than hypertension. An abnormal glucose metabolism due to insulin resistance appears to be linked to aldosterone overproduction, and seems the major contributor to metabolic dysfunction in primary aldosteronism. Impairment of insulin action may be also due to concurrent environmental factors (hypokalemia?), and/or it might occur in compartments other than fat tissue (liver? skeletal muscle?). Higher rates of cardiovascular events reported in primary aldosteronism could be due in part to the increased prevalence of the metabolic syndrome in this disorder. Regression of glucometabolic complications after the cure of aldosterone excess should be confirmed by larger studies, and the influence on the natural history of primary aldosteronism by using agents potentially able to correct metabolic abnormalities should be further explored.

Teratocarcinoma-Derived Growth Factor-1 Is Upregulated in Aldosterone-Producing Adenomas and Increases Aldosterone Secretion and Inhibits Apoptosis In Vitro

Aldosterone-producing adenomas (APA) are a frequent cause of secondary hypertension characterized by autonomous hypersecretion of aldosterone. However, the molecular mechanisms involved in adrenal tumorigenesis and deregulated aldosterone secretion are currently unknown. To identify putative functional genes, a transcriptional screening was performed on 8 APA and 3 normal adrenals (NA) using oligonucleotide microarrays. Data were next validated on an expanded set of samples by real-time PCR (APA, n=19; NA, n=10). The epidermal growth factor–like teratocarcinoma-derived growth factor-1 (TDGF-1) was upregulated in APA compared with NA (14.7-fold and 21.4-fold by microarray and real-time PCR, respectively). In vitro studies and Western blot analysis using the NCI H295R adrenocortical cell line showed that TDGF-1 increased Akt phosphorylation on Thr308 and Ser473, consistent with activation of phosphatidylinositol 3-kinase/Akt signaling, and also demonstrated a concomitant inactivation of the Akt substrate glycogen synthesis kinase-3&bgr; via Ser9 phosphorylation. Furthermore, TDGF-1 mediated a 3.8±0.4-fold increase in aldosterone secretion (n=4) that was specifically blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin (50 nmol/L) and LY294002 (20 &mgr;mol/L). Finally, TDGF-1 protected H295R cells from apoptosis induced by staurosporine, causing a decrease in caspase-3 activity, a reduction in the inactivation of poly(ADP-ribose) polymerase, and an inhibition of DNA fragmentation, detected by the TUNEL reaction and fluorescence microscopy that was blocked by LY294002. Taken together, our data suggest that TDGF-1, which is significantly upregulated in APA and mediates aldosterone hypersecretion and deregulated growth in adrenocortical cells in vitro, may represent a key player in the development and pathophysiology of primary aldosteronism.

Effects of taxol on the human NCI-H295 adrenocortical carcinoma cell line.

We investigated the effects of taxol, an antimicrotubule agent active in different cancers, on the human NCI-H295 steroid-secreting adrenocortical carcinoma cell line. Cells were incubated for 48, 72 or 96 h with taxol 10(-10)-10(-4) M. Cell viability was evaluated by MTT assay with IC50 calculation. Apoptosis was investigated by measuring DNA fragmentation with ELISA assay after cell exposure to taxol at IC50 for 24 h. For secretion studies, aldosterone, cortisol, testosterone and dehydroepiandrosterone-sulphate (DHEA-S) were measured by RIA in the conditioned medium after 96 h exposure to taxol 10(-10)-10(-6) M, and expressed as percentage of steroid production by control cells. By MTT, taxol induced a dose-dependent inhibition of cell proliferation, with ICs50 at 72-96 h corresponding to blood levels achieved in vivo in patients with other types of cancer. Nuclear fragmentation, morphologically confirmed at electron microscopy, showed a 4-fold increase after exposure to taxol. With 10(-6) M taxol, aldosterone decreased to 48%, cortisol to 61%, testosterone to 76% and DHEA-S to 89% of steroid production by control cells. Taxol is an effective cytotoxic and antiproliferative agent in a human adrenocortical steroid-secreting carcinoma cell line. Apoptosis induced by the drug is involved in neoplastic cell death.

Analysis of insulin sensitivity in adipose tissue of patients with primary aldosteronism

OBJECTIVE The objective of the study was to assess the effect of high aldosterone levels on insulin sensitivity of adipose tissue in humans. METHODS Visceral adipose tissue (VAT) was obtained from patients with aldosterone-producing adenoma (APA; n=14) and, as controls, nonfunctioning adenoma (NFA; n=14) undergoing laparoscopic adrenalectomy. Homeostasis model assessment index was higher and potassium was lower in APA than NFA (P<0.05). Immunohistochemistry, Western blotting, and real-time PCR were used to detect and quantify mineralocorticoid receptor (MR) expression. Transcript levels of peroxisome proliferative-activated receptor-gamma, insulin receptor, glucose transporter 4, insulin receptor substrate-1 and -2, leptin, adiponectin, IL-6, monocyte chemoattractant protein-1, glucocorticoid receptor (GR)-alpha, 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1, and HSD11B2 were quantified. The effect of increasing aldosterone concentrations on 2-deoxy-[3H]d-glucose uptake was tested in human sc abdominal adipocytes. RESULTS Expression of MR was demonstrated in VAT, with no difference between APA and NFA as to mRNA levels of MR, GRalpha, HSD11B1, and glucose metabolism and inflammation factors. In cultured adipocytes, basal and insulin-stimulated glucose uptake were unaffected by 1-100 nM (normal/hyperaldosteronism) and impaired only by much higher, up to 10 microM, aldosterone concentrations. The impairment was prevented by RU486 but not by eplerenone. CONCLUSIONS Gene expression of insulin signaling/inflammatory molecules was similar in VAT of APA and NFA patients, not supporting an effect of aldosterone excess on insulin sensitivity of adipose tissues. Only at pharmacological concentrations and through GR activation, aldosterone reduced glucose uptake in adipocytes. Insulin resistance in primary aldosteronism might occur in compartments other than fat and/or depend on concurrent environmental factors.