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.

The effects of stress on brain and adrenal stem cells.

The brain and adrenal are critical control centers that maintain body homeostasis under basal and stress conditions, and orchestrate the body's response to stress. It is noteworthy that patients with stress-related disorders exhibit increased vulnerability to mental illness, even years after the stress experience, which is able to generate long-term changes in the brain's architecture and function. High levels of glucocorticoids produced by the adrenal cortex of the stressed subject reduce neurogenesis, which contributes to the development of depression. In support of the brain-adrenal connection in stress, many (but not all) depressed patients have alterations in the components of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis, with enlarged adrenal cortex and increased glucocorticoid levels. Other psychiatric disorders, such as post-traumatic stress disorder, bipolar disorder and depression, are also associated with abnormalities in hippocampal volume and hippocampal function. In addition, hippocampal lesions impair the regulation of the LHPA axis in stress response. Our knowledge of the functional connection between stress, brain function and adrenal has been further expanded by two recent, independent papers that elucidate the effects of stress on brain and adrenal stem cells, showing similarities in the way that the progenitor populations of these organs behave under stress, and shedding more light into the potential cellular and molecular mechanisms involved in the adaptation of tissues to stress.

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.

Calcium-dependent release of adipocyte fatty acid binding protein from human adipocytes.

BACKGROUND: Fatty acid binding protein 4 (FABP4) is a predominantly cytosolic protein of the adipocytes, but also abundantly present in human plasma; its plasma concentrations were linked to obesity and metabolic syndrome. Recent studies have suggested a direct extracellular effect of FABP4 in the regulation of glucose metabolism and heart function independently of its effect as a carrier protein. Interestingly, FABP4 has no secretory signal sequence; hence, the mechanisms how FABP4 is released from adipocytes are unclear. METHODS AND RESULTS: In this study we investigated the mechanisms for FABP4 secretion from human adipocytes by using isolated primary pre-adipocytes (PAs) and the human adipocyte cell strain Simpson-Golabi-Behmel syndrome. In undifferentiated PAs, FABP4 expression was barely detectable and increased continuously during differentiation. The increase in FABP4 mRNA expression was accompanied by high levels of FABP4 secretion. In differentiated human adipocytes, FABP4 secretion was not abolished by blocking the Golgi-dependent secretory pathway in vitro, supporting a non-classical secretion mechanism for FABP4. However, raising intracellular Ca(2+) levels enhanced FABP4 secretion in a concentration-dependent manner. CONCLUSION: This study shows that FABP4 is actively released from human adipocytes in vitro via a non-classical, calcium-dependent mechanism.

Lipoprotein apheresis reduces adipocyte fatty acid-binding protein serum levels.

BACKGROUND AND METHODS: Adipocyte fatty acid-binding protein (FABP4) is a member of the intracellular lipid-binding protein family highly expressed in adipocytes and macrophages. Recent studies indicate a key role for circulating FABP4 in the pathogenesis of atherosclerosis and type 2 diabetes. We described an additional role for FABP4 in the development of cardiac dysfunction in obesity. Therefore, FABP4 seems to be a target in the prevention and treatment of metabolic and cardiovascular disorders in obesity with high potential for future therapeutic applications. However, a safe pharmacological therapy is not yet available. Lipoprotein apheresis is an established therapy for severe and otherwise untreatable hypercholesterolemia which increases life expectancy in patients at high-risk for cardiovascular events. We therefore investigated the acute effect of lipoprotein apheresis on FABP4 serum levels in 64 high-risk patients (25 women, 39 men) under regular apheresis treatment. RESULTS: FABP4 levels were significantly reduced by 23.2 ± 1.8% by apheresis treatment. Although women had higher FABP4 levels than men (53.5 ± 8.3 ng/ml vs. 30.7 ± 4.3 ng/ml), reduction rate after lipoprotein apheresis was similar in both genders. Among the apheresis methods investigated, immunoadsorption of lipoproteins was most effective in lowering circulating FABP4. CONCLUSION: These data suggest that the reduction of FABP4 serum levels may contribute to the preventive effect of lipoprotein apheresis on cardiovascular events.

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.

Chromaffin cells: the peripheral brain.

Chromaffin cells probably are the most intensively studied of the neural crest derivates. They are closely related to the nervous system, share with neurons some fundamental mechanisms and thus were the ideal model to study the basic mechanisms of neurobiology for many years. The lessons we have learned from chromaffin cell biology as a peripheral model for the brain and brain diseases pertain more than ever to the cutting edge research in neurobiology. Here, we highlight how studying this cell model can help unravel the basic mechanisms of cell renewal and regeneration both in the central nervous system (CNS) and neuroendocrine tissue and also can help in designing new strategies for regenerative therapies of the CNS.

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.

Adrenal gland responses to lipopolysaccharide after stress and ethanol administration in male rats.

All forms of stress, including restraint stress (RS) and lipopolysaccharide (LPS) administration, activate the hypothalamic-pituitary-adrenal (HPA) axis. LPS binds to a recognition protein (CD14) and toll-like receptor 2/4 in different cells and tissues, including the adrenal gland, to induce the production of cytokines and cause upregulation of cyclooxygenase and nitric oxide synthase (NOS) enzymes. Acute ethanol exposure activates the HPA axis, but in some conditions prolonged administration can dampen this activation as well as decrease the inflammatory responses to LPS. Therefore, this study was designed to evaluate the adrenal response to a challenge dose of LPS (50 µg/kg) injected i.p., after submitting male rats to RS, twice a day (2 h each time) for 5 days and/or ethanol administration (3 g/kg) by gavage also for 5 days, twice daily. At the end of the experiment, plasma corticosterone concentrations and adrenal gland content of prostaglandin E (PGE) and NOS activity were measured as stress mediators. The results showed that repetitive ethanol administration attenuated the adrenal stress response to LPS challenge alone and after RS, by preventing the increase in plasma corticosterone concentrations and by decreasing the PGE content and NOS activity in the adrenal gland. Therefore, we conclude that moderate alcohol consumption could attenuate the effects of psychophysical stress and impair an inflammatory response.

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.

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 microL, icv) significantly decreased MC-induced salivary secretion in intact rats (MC 1 microg/kg: control (C), 5.3 +/- 0.6 vs AEA, 2.7 +/- 0.6 mg; MC 3 microg/kg: C, 17.6 +/- 1.0 vs AEA, 8.7 +/- 0.9 mg; MC 10 microg/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 microg/kg: C, 3.8 +/- 0.8 vs AEA, 1.4 +/- 0.6 mg; MC 3 microg/kg: C, 14.7 +/- 2.4 vs AEA, 6.9 +/- 1.2 mg; P < 0.05; MC 10 microg/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 microL, icv) or of the GABA A receptor antagonist, bicuculline (25 ng/5 microL, icv). Our results suggest that parasympathetic neurotransmission from the central nervous system to the SMG can be inhibited by endocannabinoid and GABAergic systems.