Elise P. Gomez-Sanchez

Adipocytes Produce Aldosterone Through Calcineurin-Dependent Signaling Pathways: Implications in Diabetes Mellitus–Associated Obesity and Vascular Dysfunction

We reported aldosterone as a novel adipocyte-derived factor that regulates vascular function. We aimed to investigate molecular mechanisms, signaling pathways, and functional significance of adipocyte-derived aldosterone and to test whether adipocyte-derived aldosterone is increased in diabetes mellitus–associated obesity, which contributes to vascular dysfunction. Studies were performed in the 3T3-L1 adipocyte cell line and mature adipocytes isolated from human and mouse (C57BL/6J) adipose tissue. Mesenteric arteries with and without perivascular fat and mature adipocytes were obtained from obese diabetic db/db and control db/+ mice. Aldosterone synthase (CYP11B2; mRNA and protein) was detected in 3T3-L1 and mature adipocytes, which secrete aldosterone basally and in response to angiotensin II (Ang II). In 3T3-L1 adipocytes, Ang II stimulation increased aldosterone secretion and CYP11B2 expression. Ang II effects were blunted by an Ang II type 1 receptor antagonist (candesartan) and inhibitors of calcineurin (cyclosporine A and FK506) and nuclear factor of activated T-cells (VIVIT). FAD286 (aldosterone synthase inhibitor) blunted adipocyte differentiation. In candesartan-treated db/db mice (1 mg/kg per day, 4 weeks) increased plasma aldosterone, CYP11B2 expression, and aldosterone secretion were reduced. Acetylcholine-induced relaxation in db/db mesenteric arteries containing perivascular fat was improved by eplerenone (mineralocorticoid receptor antagonist) without effect in db/+ mice. Adipocytes possess aldosterone synthase and produce aldosterone in an Ang II/Ang II type 1 receptor/calcineurin/nuclear factor of activated T-cells–dependent manner. Functionally adipocyte-derived aldosterone regulates adipocyte differentiation and vascular function in an autocrine and paracrine manner, respectively. These novel findings identify adipocytes as a putative link between aldosterone and vascular dysfunction in diabetes mellitus–associated obesity.

Steroidogenic enzyme gene expression in the human brain

UNLABELLED mRNA, protein and activity for the enzymes required for the synthesis of adrenal corticosteroids have been demonstrated in rat brains by several laboratories. In this study real-time RT-PCR was used to determine whether mRNA for these enzymes are expressed in the human amygdala, caudate nucleus, cerebellum, corpus callosum, hippocampus, spinal cord, and thalamus. Published sequences for the human adrenal enzymes were used to construct primers. RESULTS mRNAs encoding cholesterol side-chain cleavage enzyme (CYP11A gene), 17beta-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 21-hydroxylase (CYP21), 11beta-hydroxysteroid dehydrogenase (11beta-HSD2) and glucocorticoid and mineralocorticoid receptors were detectable in all anatomical regions evaluated. The 11beta-hydroxylase mRNA was detected in all except cerebellum and hippocampus. The aldosterone synthase mRNA was not found in amygdala, cerebellum or hippocampus. Levels of transcripts were 10(-1)-10(-7)-fold lower than those in the adrenal, with corpus callosum and spinal cord having the highest concentrations. Enzyme activity or relevance is yet unknown.

Potassium Channel Mutant KCNJ5 T158A Expression in HAC-15 Cells Increases Aldosterone Synthesis

Primary aldosteronism is the most common cause of secondary hypertension, most frequently due to an aldosterone-producing adenoma or idiopathic hyperaldosteronism. Somatic mutations of the potassium channel KCNJ5 in the region of the selectivity filter have been found in a significant number of aldosterone-producing adenomas. There are also familial forms of primary aldosteronism, one of which, familial hyperaldosteronism type 3 which to date has been found in one family who presented with a severe abnormality in aldosterone and 18-oxocortisol production and hypertrophy and hyperplasia of the transitional zone of the adrenal cortex. In familial hyperaldosteronism type 3, there is a genomic mutation causing a T158A change of amino acids within the selectivity filter region of the KCNJ5 gene. We are reporting our studies demonstrating that lentiviral-mediated expression of a gene carrying the T158A mutation of the KCNJ5 in the HAC15 adrenal cortical carcinoma cell line causes a 5.3-fold increase in aldosterone secretion in unstimulated HAC15-KCNJ5 cells and that forskolin-stimulated aldosterone secretion was greater than that of angiotensin II. Expression of the mutated KCNJ5 gene decreases plasma membrane polarization, allowing sodium and calcium influx into the cells. The calcium channel antagonist nifedipine and the calmodulin inhibitor W-7 variably inhibited the effect. Overexpression of the mutated KCNJ5 channel resulted in a modest decrease in HAC15 cell proliferation. These studies demonstrate that the T158A mutation of the KCNJ5 gene produces a marked stimulation in aldosterone biosynthesis that is dependent on membrane depolarization and sodium and calcium influx into the HAC15 adrenal cortical carcinoma cells.

Does Aldosterone Upregulate the Brain Renin-Angiotensin System in Rats With Heart Failure?

The brain renin-angiotensin system (RAS) contributes to increased sympathetic drive in heart failure (HF). The factors upregulating the brain RAS in HF remain unknown. We hypothesized that aldosterone (ALDO), a downstream product of the systemic RAS that crosses the blood-brain barrier, signals the brain to increase RAS activity in HF. We examined the relationship between circulating and brain ALDO in normal intact rats, in adrenalectomized rats receiving subcutaneous infusions of ALDO, and in rats with ischemia-induced HF and sham-operated controls. Brain ALDO levels were proportional to plasma ALDO levels across the spectrum of rats studied. Compared with sham-operated controls rats, HF rats had higher plasma and hypothalamic tissue levels of ALDO. HF rats also had higher expression of mRNA and protein for angiotensin-converting enzyme and angiotensin type 1 receptors in the hypothalamus, increased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and superoxide generation in the paraventricular nucleus of the hypothalamus, increased excitation of paraventricular nucleus neurons, and increased plasma norepinephrine. HF rats treated for 4 weeks with intracerebroventricular RU28318 (1 &mgr;g/h), a selective mineralocorticoid receptor antagonist, had less hypothalamic angiotensin-converting enzyme and angiotensin type 1 receptor mRNA and protein, less reduced nicotinamide-adenine dinucleotide phosphate–induced superoxide in the paraventricular nucleus, fewer excited paraventricular nucleus neurons, and lower plasma norepinephrine. RU28318 had no effect on plasma ALDO or on angiotensin-converting enzyme or angiotensin type 1 receptor expression in brain cortex. The data demonstrate that ALDO of adrenal origin enters the hypothalamus in direct proportion to plasma levels and suggest that ALDO contributes to the upregulation of hypothalamic RAS activity and sympathetic drive in heart failure.

Development of monoclonal antibodies against human CYP11B1 and CYP11B2

1. The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate. 2. Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme. 3. The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.

Central hypertensive effects of aldosterone.

The soluble mineralocorticoid receptor bound to an agonist acts as a transcription factor for several genes relevant to ion transport by kidney and colon epithelial cells and is a major regulator of electrolyte and fluid homeostasis. Mineralocorticoids, the most prominent of which is aldosterone, also influence the activity of nonepithelial target cells, including vascular smooth muscle cells, by altering intracellular ion transport and content. Evidence is summarized for mineralocorticoid modulation of neuronal activity in a center or centers within the brain, probably in the periventricular area of the anterior hypothalamus, where information on electrolyte, fluid, and cardiovascular status is received and integrated, resulting in alterations in central sympathetic efferent activity. These functions are distinct from central aldosterone effects on salt appetite and peripheral trophic effects on cardiovascular tissue. The isolated mineralocorticoid receptor binds several adrenal steroids, including aldosterone and the major glucocorticoids, with equal affinity. Ligand specificity for the mineralocorticoid receptor differs between tissues, including different organs in the brain. Specificity is conferred extrinsically by the 11-beta-hydroxysteroid dehydrogenase enzymes in transport epithelia, but mechanisms for mineralocorticoid ligand specificity have not been completely defined in the brain. The functional interaction between the mineralocorticoid receptor bound to different ligands and between the mineralocorticoid and glucocorticoid receptors is complex and as yet unresolved. Evidence is presented for the de novo synthesis of adrenal corticosteroids in the brain which may, by paracrine regulation of central control mechanisms, be relevant for certain clinical and experimental forms of hypertension characterized by low circulating levels of mineralocorticoids which respond to mineralocorticoid receptor antagonists.

Aldosterone synthesis in the brain contributes to Dahl salt-sensitive rat hypertension.

The enzymes required for aldosterone synthesis from cholesterol are expressed in rat and human brains. The hypertension of Dahl salt‐sensitive (SS) rats is mitigated by the intracerebroventricular (i.c.v.) infusion of antagonists of the mineralocorticoid receptor (MR) and downstream effectors of mineralocorticoid action, as well as ablations of brain areas that also abrogate mineralocorticoid–salt excess hypertension in normotensive rats. We used real time RT‐PCR to measure mRNA of aldosterone synthase and 11β‐hydroxylase, the requisite enzymes for the last step in the synthesis of aldosterone and corticosterone, respectively, MR and the determinants of MR ligand specificity, 11β‐hydroxysteroid dehydrogenase types 1 and 2 (11β‐HSD1&2) and hexose‐6‐phosphate dehydrogenase (H6PDH). A combination of extraction and ELISA was used to measure aldosterone concentrations in tissue and urine of SS and Sprague–Dawley (SD) rats. Aldosterone synthase mRNA expression was higher in the brains and lower in the adrenal glands of SS compared with SD rats. The amounts of mRNA for MR, 11β‐hydroxylase, 11β‐HSD1&2 and H6PD were similar. Aldosterone concentrations were greater in brains of SS than SD rats, yet, in keeping with the literature, the circulating and total aldosterone production of aldosterone in SS rats were not. The selective inhibitor of aldosterone synthase, FAD286, was infused i.c.v. or subcutaneously in a cross‐over blood pressure study in hypertensive SS rats further challenged by a high‐salt diet. The i.c.v. infusion of FAD286, at a dose that had no effect systemically, significantly and reversibly lowered blood pressure in SS rats. Aldosterone synthesis in brains of SS rats is greater than in SD rats and is important in the genesis of their salt‐sensitive hypertension.

Mineralocorticoids, salt and high blood pressure.

Essential hypertensive patients often respond to treatments mitigating mineralocorticoid action, even though circulating levels of these steroids are within normal ranges. In addition to the kidney, mineralocorticoid or Type I receptors are found in the brain and vascular smooth muscle where they mediate effects associated with several forms of experimental hypertension. Studies in which discrete anatomic or functional areas of the brain have been ablated demonstrate that the periventricular areas of the hypothalamus and the central sympathetic and baroreceptor systems are crucial for the development of hypertension in the renoprival, DOCA salt, and Dahl salt-sensitive rat. Intracerebroventricular (i.c.v.) infusion of aldosterone in both rats and dogs at doses that do not raise serum levels above normal produce hypertension. The hypertension produced by systemic mineralocorticoid excess, adrenal regeneration, and i.c.v. or oral administration of glycyrrhetinic acid or carbenoxolone in genetically normotensive rats and by dietary salt in the Dahl salt-sensitive rat is inhibited by the i.c.v. infusion of a mineralocorticoid receptor antagonist or a Na+ channel-selective amiloride analog. Recent data demonstrate the extraadrenal synthesis of steroids in aortic endothelial cells, smooth muscle cells and the brain. The role of the extraadrenal synthesis of steroids raises new avenues for research into the causes of hypertension.

CORTICOSTEROID SYNTHESIS IN THE CENTRAL NERVOUS SYSTEM

The possibility that adrenocorticosteroids might be synthesized in the central nervous system was assessed by RT-PCR using primers for the CYP11B1 gene which codes for 11 beta-hydroxylase, the enzyme responsible for corticosterone and cortisol formation in the zona fasciculata, incubation of minces of several areas of the brain with 3H-DOC and measuring steroid metabolites, and determining the effect of the intracerebroventricular infusion of the 11 beta-hydroxylase mechanism-based inhibitor 19-ethynyldeoxycorticosterone upon the salt-induced increase in blood pressure in SS/jr rats. Significant, though small relative to the adrenal, amounts of mRNA for 11 beta-hydroxylase was found in the aorta, cerebrum, cerebellum, hippocampus, hypothalamus and amygdala, but not in the heart. Brain minces converted 3H-DOC to corticosterone and 11-dehydrocorticosterone to a greater degree than to 18-OH-DOC. The effect of 19-ethynyldeoxycorticosterone was dose dependent, with the lower doses preventing salt-induced hypertension and the higher doses having no effect or increasing the blood pressure.

18-Hydroxycorticosterone, 18-Hydroxycortisol, and 18-Oxocortisol in the Diagnosis of Primary Aldosteronism and Its Subtypes

CONTEXT Diagnosis of primary aldosteronism (PA) is made by screening, confirmation testing, and subtype diagnosis (computed tomography scan and adrenal vein sampling). However, some tests are costly and unavailable in most hospitals. OBJECTIVE The aim of the study was to evaluate the role of serum 18-hydroxycorticosterone (s18OHB), urinary and serum 18-hydroxycortisol (u- and s18OHF), and urinary and serum 18-oxocortisol (u- and s18oxoF) in the diagnosis of PA and its subtypes, aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). PATIENTS The study included 62 patients with low-renin essential hypertension (EH), 81 patients with PA (20 APA, 61 BAH), 24 patients with glucocorticoid-remediable aldosteronism, 16 patients with adrenal incidentaloma, and 30 normotensives. INTERVENTION AND MAIN OUTCOME MEASURES We measured s18OHB, s18OHF, and s18oxoF before and after saline load test (SLT) and 24-h u18OHF and u18oxoF. RESULTS PA patients displayed significantly higher levels of s18OHB, u18OHF, and u18oxoF compared to EH and normal subjects; APA patients displayed s18OHB, u18OHF, and u18oxoF levels significantly higher than BAH patients. Similar results were obtained for s18OHF and s18oxoF. SLT significantly reduced s18OHB, s18OHF, and s18oxoF in all groups, but steroid reduction was much less for APA patients compared to BAH and EH. The s18OHB/aldosterone ratio after SLT more than doubled in EH but remained unchanged in APA patients. CONCLUSIONS u18OHF, u18oxoF, and s18OHB measurements in patients with a positive aldosterone/plasma renin activity ratio correlate with confirmatory tests and adrenal vein sampling in PA patients. If verified, these steroid assays would refine the diagnostic workup for PA.