STAT3-Ser/Hes3 Signaling: A New Molecular Component of the Neuroendocrine System?

The endocrine system involves communication among different tissues in distinct organs, including the pancreas and components of the Hypothalamic-Pituitary-Adrenal Axis. The molecular mechanisms underlying these complex interactions are a subject of intense study as they may hold clues for the progression and treatment of a variety of metabolic and degenerative diseases. A plethora of signaling pathways, activated by hormones and other endocrine factors have been implicated in this communication. Recent advances in the stem cell field introduce a new level of complexity: adult progenitor cells appear to utilize distinct signaling pathways than the more mature cells in the tissue they co-reside. It is therefore important to elucidate the signal transduction requirements of adult progenitor cells in addition to those of mature cells. Recent evidence suggests that a common non-canonical signaling pathway regulates adult progenitors in several different tissues, rendering it as a potentially valuable starting point to explore their biology. The STAT3-Ser/Hes3 Signaling Axis was first identified as a major regulator of neural stem cells and, subsequently, cancer stem cells. In the endocrine/neuroendocrine system, this pathway operates on several levels, regulating other types of plastic cells: (a) it regulates pancreatic islet cell function and insulin release; (b) insulin in turn activates the pathway in broadly distributed neural progenitors and possibly also hypothalamic tanycytes, cells with important roles in the control of the adrenal gland; (c) adrenal progenitors themselves operate this pathway. The STAT3-Ser/Hes3 Signaling Axis therefore deserves additional research in the context of endocrinology.

Anti-inflammatory properties of bone morphogenetic protein 4 in human adipocytes.

BACKGROUND: Obesity is characterized by increased adipocyte number and size as well as white adipose tissue (WAT) inflammation, which is fundamental for the development of insulin resistance and type-2 diabetes. These processes, regulated by various endocrine, paracrine and autocrine factors, are extensively studied with the hope to interfere and to inhibit weight gain and related complications in obese patients. Recent data suggest an important role of bone morphogenic protein 4 (BMP4) in the regulation of adipogenesis and development of obesity. BMP4 is a growth factor of the transforming growth factor-ß superfamily. Initially, BMPs were identified as inducers of ectopic bone formation. It is now apparent, however, that these proteins have different pleiotropic developmental actions and including playing a role in white adipogenesis. METHODS AND RESULTS: Here, we demonstrate that the expression of BMP4 in human WAT is negatively correlated to body mass index and to the expression of pro-inflammatory cytokines. In vitro, BMP4 expression in cultured human adipocytes is upregulated after induction of differentiation. Cells treated with exogenous BMP4 increased peroxisome proliferator-activated receptor γ (PPARγ) expression and significantly reduced the expression of pro-inflammatory cytokines including tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein 1. TNF-α treatment of fully differentiated adipocytes resulted in downregulation of the expression of adipogenic genes and elevated expression of pro-inflammatory cytokines. Exogenous BMP4 addition significantly reduced the negative effect of TNF-α on the expression profile of adipocytes. Finally, treatment of human adipocytes with exogenous BMP4 reduced the adipocytes' chemoattractant potential and the migration of monocytes toward adipocyte-conditioned medium. CONCLUSIONS: These results indicate that BMP4 is an important anti-inflammatory molecule, which may act through PPARγ and reduces TNF-α-mediated pro-inflammatory cytokine production in human adipocytes. Through its anti-inflammatory potential, BMP4 may serve as a protective factor for inflammation-related diseases such as insulin-tolerance or type-2 diabetes.

Valproic acid enhances neuronal differentiation of sympathoadrenal progenitor cells.

The antiepileptic drug valproic acid (VPA) has been shown to influence the neural differentiation and neurite outgrowth of neural stem cells. Sympathoadrenal progenitor cells share properties with neural stem cells and are considered a potential cell source in the treatment of neurodegenerative diseases. The present study therefore aims at modulating the neural differentiation potential of these cells by treatment with the histone deacetylase inhibitor VPA. We studied the epigenetic effects of VPA in two culture conditions: suspension conditions aimed to expand adrenomedullary sympathoadrenal progenitors within free-floating chromospheres and adherent cell cultures optimized to derive neurons. Treatment of chromospheres with VPA may launch neuronal differentiation mechanisms and improve their neurogenic potential upon transplantation. However, also transplantation of differentiated functional neurons could be beneficial. Treating chromospheres for 7 days with clinically relevant concentrations of VPA (2 mm) revealed a decrease of neural progenitor markers Nestin, Notch2 and Sox10. Furthermore, VPA initiated catecholaminergic neuronal differentiation indicated by upregulation of the neuronal marker ß-III-tubulin, the dopaminergic transcription factor Pitx3 and the catecholaminergic enzymes TH and GTPCH. In adherent neural differentiation conditions, VPA treatment improved the differentiation of sympathoadrenal progenitor cells into catecholaminergic neurons with significantly elevated levels of nor- and epinephrine. In conclusion, similar to neural stem cells, VPA launches differentiation mechanisms in sympathoadrenal progenitor cells that result in increased generation of functional neurons. Thus, data from this study will be relevant to the potential use of chromaffin progenitors in transplantation therapies of neurodegenerative diseases.

Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.

Metagenomic sequencing of the human gut microbiome before and after bariatric surgery in obese patients with type 2 diabetes: correlation with inflammatory and metabolic parameters.

Roux-en-Y gastric bypass (RYGB) has become a prominent therapeutic option for long-term treatment of morbid obesity and type 2 diabetes mellitus (T2D). Cross talk and pathogenetic consequences of RYGB-induced profound effects on metabolism and gut microbiome are poorly understood. The aim of the present study therefore was to characterize intra-individual changes of gut microbial composition before and 3 months after RYGB by metagenomic sequencing in morbidly obese patients (body mass index (BMI)>40 kg m(-)(2)) with T2D. Subsequently, metagenomic data were correlated with clinical indices. Based on gene relative abundance profile, 1061 species, 729 genera, 44 phyla and 5127 KO (KEGG Orthology) were identified. Despite high diversity, bacteria could mostly be assigned to seven bacterial divisions. The overall metagenomic RYGB-induced shift was characterized by a reduction of Firmicutes and Bacteroidetes and an increase of Proteobacteria. Twenty-two microbial species and 11 genera were significantly altered by RYGB. Using principal component analysis, highly correlated species were assembled into two common components. Component 1 consisted of species that were mainly associated with BMI and C-reactive protein. This component was characterized by increased numbers of Proteobacterium Enterobacter cancerogenus and decreased Firmicutes Faecalibacterium prausnitzii and Coprococcus comes. Functional analysis of carbohydrate metabolism by KO revealed significant effects in 13 KOs assigned to phosphotransferase system. Spearmen's Rank correlation indicated an association of 10 species with plasma total- or low-density lipoprotein cholesterol, and 5 species with triglycerides. F. prausnitzii was directly correlated to fasting blood glucose. This is the first clinical demonstration of a profound and specific intra-individual modification of gut microbial composition by full metagenomic sequencing. A clear correlation exists of microbiome composition and gene function with an improvement in metabolic and inflammatory parameters. This will allow to develop new diagnostic and therapeutic strategies based on metagenomic sequencing of the human gut microbiome.

ERK1/2 MAPKs and Wnt signaling pathways are independently involved in adipocytokine-mediated aldosterone secretion.

Obesity is one major risk factor for the development of arterial hypertension, and the development of obesity-related hypertension has been associated with increased plasma aldosterone levels. Our previous work shows a direct stimulatory effect of adipokines on aldosterone secretion from human adrenocortical cells, mediated via ERK1/2-dependent upregulation of steroid acute regulatory protein (StAR) activity. Recent evidence also indicates the involvement of the Wnt-signaling pathway in fat cell-mediated aldosterone secretion. Wnt-signaling molecules are secreted by adipocytes and regulate the activity of SF-1, a key transcription factor in adrenal steroidogenesis. The goal of this study was to investigate the cellular mechanisms of adipocyte-induced aldosterone secretion in detail, and to evaluate effects and possible interactions of the ERK1/2 MAPK- and the Wnt-signaling pathways on adipocyte-induced adreno-cortical aldosterone secretion. Our results show that, similar to adipocyte-conditioned medium (ACM), ß-catenin, which is an intracellular mediator of canonical Wnt-signaling, induced StAR promotor activity in human NCI-H295R adrenocortical cells, and ACM-induced StAR promotor activity depended on intact SF-1 binding sites. Wnt antagonist sFRP-1 inhibited adipokine-mediated StAR activity, but did not affect ERK1/2 MAPK activation. Accordingly, Wnt did not stimulate ERK1/2 phosphorylation in adrenocortical cells, indicating that ERK1/2 MAPK and Wnt signaling pathways are independently involved in adipocyte-mediated aldosterone secretion.

Adipocyte-derived factors suppress heart contraction.

BACKGROUND: Obesity is strongly associated with cardiovascular diseases including systemic hypertension, coronary artery disease and heart failure. Despite several investigations the pathophysiological mechanisms involved remain unclear. We have previously shown that adipose tissue exerts a highly potent activity with an acute depressant effect on cardiomyocytes, thus suggesting direct involvement of adipose tissue in the development of heart dysfunction. OBJECTIVE AND DESIGN: This study investigates the effects of adipocyte factors obtained from subcutaneous adipose tissue on the whole cardiac function by using isolated perfused rat hearts in a Langendorff mode. We recorded changes in coronary flow, developed isovolumetric left ventricular pressure, contraction rate and relaxation rate. RESULTS: We observed a significant decrease in heart contractility parameters as well as in coronary flow within a few seconds of incubation with adipocyte factors. The cardiodepressant effects could not be blocked by the nonselective cyclooxygenase-inhibitor indomethacin. Human adipocytes release tumor necrosis factor-α, interleukin-6 (IL-6) and IL-1ß into extracellular medium. These cytokines were tested for their potential effect but were, however, not responsible for the cardiodepressant effect observed. CONCLUSION: These data indicate that human adipocytes secrete factors with a strong acute depressant effect on cardiac force generation and coronary flow due to contraction of the coronary vessels, thus suggesting a direct role of adipose tissue in the pathogenesis of cardiac dysfunction.

Expression and function of endocannabinoid receptors in the human adrenal cortex.

Endogenous cannabinoids are important signaling molecules in neuroendocrine control of homeostatic and reproductive functions including stress response and energy metabolism. The hypothalamic paraventricular and supraoptic nuclei have been shown to release endocannabinoids, which act as retrograde messengers to modulate the synaptic release of glutamate during stress response. This study endeavors to elucidate possible interaction of the endocannabinoid system with the regulation of adrenocortical function at the adrenal level. Human adrenocortical NCI-H295R cells and normal human adrenal glands were used to study the possible effects of anandamide and cannabinoid receptor 1 (CB1) antagonist SR141716A on aldosterone and cortisol secretion. Our data indicate the expression of CB1 in human adrenal cortex and adrenocortical NCI-H295R cells; CB2 was not expressed. Furthermore, anandamide inhibited basal release and stimulated release of adrenocortical steroids (corticosterone and aldosterone); this effect was reversed by CB1 antagonist (SR141716A). Therefore, the endocannabinoid system at the level of the adrenal, can directly influence adrenocortical steroidogenesis.

Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: antiproliferative effects of peptide analogues.

Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors.

Anandamide injected into the lateral ventricle of the brain inhibits submandibular salivary secretion by attenuating parasympathetic neurotransmission

Our objective was to determine the effect of arachidonylethanolamide (anandamide, AEA) injected intracerebroventricularly (icv) into the lateral ventricle of the rat brain on submandibular gland (SMG) salivary secretion. Parasympathetic decentralization (PSD) produced by cutting the chorda tympani nerve strongly inhibited methacholine (MC)-induced salivary secretion while sympathetic denervation (SD) produced by removing the superior cervical ganglia reduced it slightly. Also, AEA (50 ng/5 µL, icv) significantly decreased MC-induced salivary secretion in intact rats (MC 1 µg/kg: control (C), 5.3 ± 0.6 vs AEA, 2.7 ± 0.6 mg; MC 3 µg/kg: C, 17.6 ± 1.0 vs AEA, 8.7 ± 0.9 mg; MC 10 µg/kg: C, 37.4 ± 1.2 vs AEA, 22.9 ± 2.6 mg). However, AEA did not alter the significantly reduced salivary secretion in rats with PSD, but decreased the slightly reduced salivary secretion in rats with SD (MC 1 µg/kg: C, 3.8 ± 0.8 vs AEA, 1.4 ± 0.6 mg; MC 3 µg/kg: C, 14.7 ± 2.4 vs AEA, 6.9 ± 1.2 mg; P < 0.05; MC 10 µg/kg: C, 39.5 ± 1.0 vs AEA, 22.3 ± 0.5 mg; P < 0.001). We showed that the inhibitory effect of AEA is mediated by cannabinoid type 1 CB1 receptors and involves GABAergic neurotransmission, since it was blocked by previous injection of the CB1 receptor antagonist AM251 (500 ng/5 µL, icv) or of the GABA A receptor antagonist, bicuculline (25 ng/5 µL, icv). Our results suggest that parasympathetic neurotransmission from the central nervous system to the SMG can be inhibited by endocannabinoid and GABAergic systems.

Pam3CSK4 and LTA-TLRs ligands associated with microdomains induce IL8 production in human adrenocortical cancer cells.

Bacterially derived ligands, Pam3CSK4 and LPS, can directly impact adrenal glands steroidogenesis through microdomain-related TLR1/2 and 4, respectively, and indirectly via immune cell-derived cytokines. The bilateral immunoadrenal relationship plays an important role in the proper functioning of both systems. CXC chemokine-dependent immune cell infiltration into adrenocortical carcinomas (ACC), which correlates with poor prognosis, is a common phenomenon. Recently, IL8 was identified in ACC and NCI-H295R cells, and was found to contribute to ACC tumour growth. The aim of this study was to clarify the role of different TLR ligands in IL8 production in NCI-H295R cells. This is the first study to demonstrate the expression of several TLRs including TLR1, 3, 6, 7 and 9 in human adrenocortical cells by using the RT-PCR approach. Only stimulation with TLR1/6 together with TLR2 ligands resulted in IL8 peptide and mRNA induction in a dose and time-dependent manner. Our data suggest that gram-positive bacteria-related TLR1/2/6 ligands might contribute to adrenal gland tumorigenesis via IL8 production.

Human adipocytes induce an ERK1/2 MAP kinases-mediated upregulation of steroidogenic acute regulatory protein (StAR) and an angiotensin II-sensitization in human adrenocortical cells.

OBJECTIVES: Hypertension is a major complication of overweight with frequently elevated aldosterone levels in obese patients. Our previous work suggests a direct stimulation of adrenal aldosterone secretion by adipocytes. Owing to aldosterone's important role in maintaining blood pressure homeostasis, its regulation in obesity is of major importance. One objective was to determine the signaling mechanisms involved in adipocyte-induced aldosterone secretion. In addition to a direct stimulation, a sensitization toward angiotensin II (AngII) might be involved. The second objective was to determine a possible adipokines-induced sensitization of human adrenocortical cells to AngII. DESIGN: Human subcutaneous adipocytes and adrenocortical cells, and the adrenocortical cell line NCI-H295R were used. Adrenocortical cells were screened for signal transduction protein expression and phosphorylation. Subsequently, steroidogenic acute regulatory protein (StAR), cAMP response element-binding protein (CREB), cAMP and phosphorylated extracellular regulated kinase were analyzed by Western blot, enzyme-linked immunosorbent assay, quantitative PCR, reporter gene assay and confocal microscopy to investigate their role in adipocyte-mediated aldosterone secretion. RESULTS: AngII-mediated aldosterone secretion was largely increased by preincubating H295R cells with adipocyte secretory products. StAR mRNA and StAR protein were upregulated in a time-dependent way. This steroidogenic effect was independent of the cAMP-protein kinase A (PKA) pathway as cellular cAMP was unaltered and inhibition of PKA by H89 failed to reduce aldosterone secretion. However, CREB reporter gene activity was moderately elevated. Upregulation of StAR was accompanied by ERK1/2 MAP kinase activation and nuclear translocation of the kinases. Inhibition of MAP kinase by UO126 abolished adipokine-stimulated aldosterone secretion from primary human adrenocortical and H295R cells, and inhibited StAR gene activity. Adipokines stimulated steroidogenesis also in primary human adrenocortical cells, supporting a role in human physiology and/or pathology. CONCLUSIONS: Adipokines induce aldosterone secretion from human adrenocortical cells and sensitization of the cells to stimulation by AngII, possibly mediated via ERK1/2-dependent upregulation of StAR activity. This stimulation of aldosterone secretion could be one link between overweight and inappropriately elevated aldosterone levels.

Adipocyte-derived products induce the transcription of the StAR promoter and stimulate aldosterone and cortisol secretion from adrenocortical cells through the Wnt-signaling pathway.

CONTEXT: Obesity is associated with hypersecretion of cortisol and aldosterone and a high prevalence of arterial hypertension. At the cellular level, a direct effect of adipocytes on the expression of the steroidogenic acute regulatory (StAR) protein, a regulator of cortisol and aldosterone synthesis, and on aldosterone and cortisol secretion has been shown. However, the molecular mechanisms mediating this effect are not known. OBJECTIVE: Wnt-signaling molecules are secreted by adipocytes and regulate the activity of SF-1, a key transcription factor in adrenal steroidogenesis. Therefore, we investigated whether adipocytes stimulate adrenal steroidogenesis through the activation of Wnt-signaling. RESULTS: Using immunohistochemistry, we detected the expression of frizzled and beta-catenin in the adult human adrenal cortex. Transient transfection of a Wnt-dependent reporter-gene into adrenal NCI-H295R cells showed an induction of Wnt-mediated transcription to 308% after treatment with human fat cell-conditioned medium (FCCM). This finding was paralleled by an induction of StAR promoter activity (420%) by FCCM. The induction of StAR promoter activity by FCCM was inhibited by 49% when Wnt-signaling was blocked by the soluble Wnt-antagonist secreted Frizzled-Related-Protein-1 (sFRP-1). Overexpression of a constitutively active mutant of beta-catenin induced the transcription of the StAR promoter (440%). beta-Catenin and FCCM induced SF-1-mediated transcription at a SF-1-driven reporter gene (420 and 402%, respectively). Furthermore, the secretion of aldosterone and cortisol by NCI-H295R cells induced by FCCM was significantly inhibited by the Wnt-antagonist sFRP-1. CONCLUSION: These data indicate that the Wnt-signaling pathway is one of the mechanisms mediating the effects of fat cells on adrenal StAR transcription and aldosterone and cortisol secretion.

Vascular-adrenal niche--endothelial cell-mediated sensitization of human adrenocortical cells to angiotensin II.

Alterations in both vasculature and renin-angiotensin-aldosterone system are a consistent finding in the metabolic syndrome. Adrenal tissue is highly vascularized and encounters blood flow, exceeding by far the volume expected for its size. Endothelial cells in the adrenal vasculature are therefore a major cellular component of adrenocortical tissue. The aim of the study was to analyze the cellular interaction between endothelial and steroid producing cells, focusing on endothelial cell-factor-mediated activation of aldosterone synthesis. The interaction between human endothelial (HUVECs) cell-conditioned medium and human adrenocortical (NCI-H295R) cells IN VITRO induced a significant surge in aldosterone secretion. The endothelial cell-conditioned medium together with angiotensin II and forskolin also potentiated aldosterone release by 1.5-fold and 2.6-fold, respectively, while preincubation of NCI-H295R cells for 24 h with endothelial cell-conditioned medium enhanced and sensitized the response of NCI-H295R to subsequent angiotensin II and forskolin stimuli by 2.5-fold and 2.2-fold, respectively. The increase in aldosterone release after preincubation with endothelial cell-conditioned medium was sensitive to cycloheximide and KN-93. Cellular conditioning with endothelial-cell factors exerts a hitherto unknown paracrine regulation of aldosterone production in human adrenocortical cells. This interaction may contribute to altered basal aldosterone release and have a role in patients with hypertension.

Human adipocytes secrete mineralocorticoid-releasing factors.

Obesity has become an epidemic problem in western societies, contributing to metabolic diseases, hypertension, and cardiovascular disease. Overweight and obesity are frequently associated with increased plasma levels of aldosterone. Recent evidence suggests that human fat is a highly active endocrine tissue. Therefore, we tested the hypothesis that adipocyte secretory products directly stimulate adrenocortical aldosterone secretion. Secretory products from isolated human adipocytes strongly stimulated steroidogenesis in human adrenocortical cells (NCI-H295R) with a predominant effect on mineralocorticoid secretion. Aldosterone secretion increased 7-fold during 24 h of incubation. This stimulation was comparable to maximal stimulation of these cells with forskolin (2 x 10(-5) M). On the molecular level, there was a 10-fold increase in the expression of steroid acute regulatory peptide mRNA. This effect was independent of adipose angiotensin II as revealed by the stimulatory effect of fat cell-conditioned medium even in the presence of the angiotensin type 1 receptor antagonist, valsartan. None of the recently defined adipocytokines accounted for the effect. Mineralocorticoid-stimulating activity was heat sensitive and could be blunted by heating fat cell-conditioned medium to 99 degrees C. Centrifugal filtration based on molecular mass revealed at least two releasing factors: a heat sensitive fraction (molecular mass >50 kDa) representing 60% of total activity, and an inactive fraction (molecular mass <50 kDa). However, the recovery rate increased to 92% when combining these two fractions, indicating the interaction of at least two factors. In conclusion, human adipocytes secrete potent mineralocorticoid-releasing factors, suggesting a direct link between obesity and hypertension.

Basic and clinical aspects of intraadrenal regulation of steroidogenesis.

The adrenal gland combines essential components of the autonomic nervous system and the HPA axis in close contact. From morphological analyses employing immunohistochemistry, in situ hybridization, the novel technique of laser capture microdissection, and electron microscopy, it has been has shown that the chromaffin cells of the adrenal medulla and the steroid-producing cells of the adrenal cortex are extensively intermingled and functionally interrelated. In in vitro studies, a variety of regulatory factors produced and released by the adrenal medulla were identified as playing an important role in modulating adrenocortical function. An isolated adrenocortical cell deprived of its tissue integrity, input from the nervous system, or intercellular communication with chromaffin, vascular, and immune cells of the adrenal gland, loses its normal capacity to produce glucocorticoids and to adequately respond to the homeostatic challenges of stress. Adrenocortical cells in co-culture with chromaffin cells produced ten times more glucocorticoids than the same number of pure adrenocortical cells and demonstrated marked up-regulation in the mRNA expression of cytochrome p450 enzymes and STAR in the co-culture, while this expression was down-regulated in isolated cells. Transgenic animal models of over-expression or deletion of enzymes involved in catecholamine synthesis, as well as of altered function of components of the HPA axis, provide evidence that the mutual interdependence of the sympatho-adrenal system and the HPA axis at the level of the adrenal gland is of physiologic relevance in vivo. Alterations in intercellular communications, local production of neuropeptides, growth factors and cytokines, and aberrant expression of ectopic receptors on adrenal cells have been implicated in adrenal cell growth, development, hyperplasia, tumor formation, autonomous hormone production, and autoimmune disease. Moreover, we described a direct cellular interaction of lymphocytes with adrenal cells as a novel non-cytokine mediated mechanism of immune endocrine interactions. Highlighting the importance of the extra-pituitary mechanisms of adrenocortical regulation, be them neural or immune, is a worthwhile starting point for a more complete analysis of the human stress system in vivo.

Lack of leptin suppression in response to hypersecretion of catecholamines in pheochromocytoma patients.

Leptin is a major regulator of body weight and energy balance and is subject to a variety of regulatory inputs. From several previous studies, catecholamines have been suggested to exert an inhibitory influence on leptin production in animals. In the present study, we analyzed leptin levels in relation to catecholamine hypersecretion in 27 human pheochromocytoma patients. A 10-fold increase in circulating norepinephrine (P < .0001) did not result in suppression of plasma leptin in the patients compared with normal controls (median and interquartile range, 4.3 ng/mL [2.4 to 6.8] v 2.2 ng/mL [1.9 to 3.0] in men and 18.6 [12.3 to 27.0] v 11.4 [10.1 to 15.9] in women). Correlation analysis indicated a significant association of leptin with epinephrine in normal subjects (r = -.81, P < .0001), but not in pheochromocytoma patients. Leptin was not related to norepinephrine in either group. In conclusion, our data suggest that a chronic elevation of catecholamines does not cause suppression of leptin secretion in patients with pheochromocytoma. This lack of effect may be attributable to the development of tolerance of adipose tissue leptin production to catecholamines.

Local renin-angiotensin system is involved in K+-induced aldosterone secretion from human adrenocortical NCI-H295 cells.

NCI-H295, a human adrenocarcinoma cell line, has been proposed as a model system to define the role of the renin-angiotensin system in the regulation of aldosterone production in humans. Because the precise cellular localization of the components of the renin-angiotensin system in human adrenal cortical cells remains unclear, we investigated their localization in this defined cell system. NCI-H295 cells expressed both angiotensinogen and renin as shown by reverse transcriptase polymerase chain reaction and immunohistochemistry. Human angiotensin-converting enzyme (ACE) was not detectable by immunocytochemistry, ACE binding, or reverse transcriptase polymerase chain reaction. However, 3.5 mmol/L K+ stimulated the formation of both angiotensin I and angiotensin II 1. 9- and 2.5-fold, respectively, and increased aldosterone release 3. 0-fold. The K+-induced stimulation of aldosterone release was decreased by captopril and enalaprilat (24% and 26%, respectively) and by the angiotensin type 1 (AT1)-receptor antagonist losartan (28%). Angiotensin II-induced stimulation of aldosterone release was abolished by losartan treatment. Specific [125I]Sar1-angiotensin II binding was detected by receptor autoradiography. The binding of [125I]Sar1-angiotensin II was completely displaced by the AT1 antagonist losartan but not by the AT2 receptor ligand PD 123319, confirming the expression of angiotensin II AT1 receptors in NCI-H295 cells. Our results demonstrate that NCI-H295 cells express most of the components of the renin-angiotensin system. Our failure to detect ACE, however, suggests that the production of angiotensin II in NCI-H295 cells may be ACE independent. NCI-H295 cells are able to produce angiotensin II, and K+ increases aldosterone secretion in part through an angiotensin-mediated pathway. The production of angiotensin II in NCI-H295 cells demonstrates that this human cell line can be useful to characterize the role of locally produced angiotensin II in the regulation of aldosterone release.

Synaptophysin immunoreactivity in primary pigmented nodular adrenocortical disease: neuroendocrine properties of tumors associated with Carney complex.

Carney complex (CNC) is characterized by lentiginosis and myxomatosis together with a variety of endocrine, neural crest-derived, and other tumors, including primary pigmented nodular adrenocortical disease (PPNAD). PPNAD is characterized by lipofuscin-containing, autonomously functioning, cortisol-producing nodules surrounded by mostly atrophic adrenocortical and normal adrenomedullary tissue. The nature and origin of the tumors, including the myxomas and PPNAD, are unclear. In this study, seven paraffin-embedded PPNAD tumors, one skin myxoma, and two cell lines (one myxoma and one PPNAD) established from patients with CNC were stained with antisera for synaptophysin (SYN), neuron-specific enolase, chromogranin A, tyrosine hydroxylase, and the neural cell adhesion molecule (NCAM). In addition, one PPNAD specimen and one myxoma were analyzed by electron microscopy. The results showed that chromogranin A and tyrosine hydroxylase stained adrenomedullary tissue, but not the PPNAD nodules or the extranodular adrenal cortex. SYN, neuron-specific enolase, and NCAM also stained the medulla. PPNAD nodules and the PPNAD cell line, but not the extranodular adrenal cortex, stained intensely for SYN. The myxoma cell line, but not normal fibroblasts, stained for SYN and NCAM. Ultrastructural analysis of a PPNAD tumor and a skin myxoma revealed a well developed rough endoplasmic reticulum, prominent mitochondria, and vesicle-like structures dispersed throughout the cytoplasm. We conclude that immunostaining for SYN, a marker protein for neuroendocrine cells, clearly distinguishes PPNAD nodules from surrounding adrenocortical tissue and can be helpful in the detection of small nodules in apparently unaffected cortex. The cells of a cutaneous myxoma were also stained positive by two of the three neuroendocrine markers. Finally, both PPNAD and myxoma cells demonstrated ultrastructural features suggestive of neuroendocrine properties. These results support the previously suggested hypothesis that the genetic mechanism leading to CNC involves genes with a neuroendocrine role.