Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.

Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

The RFamide neuropeptide 26RFa and its role in the control of neuroendocrine functions.

Identification of novel neuropeptides and their cognate G protein-coupled receptors is essential for a better understanding of neuroendocrine regulations. The RFamide peptides represent a family of regulatory peptides that all possess the Arg-Phe-NH2 motif at their C-terminus. In mammals, seven RFamide peptides encoded by five distinct genes have been characterized. The present review focuses on 26RFa (or QRFP) which is the latest member identified in this family. 26RFa is present in all vertebrate phyla and its C-terminal domain (KGGFXFRF-NH2), which is responsible for its biological activity, has been fully conserved during evolution. 26RFa is the cognate ligand of the orphan G protein-coupled receptor GPR103 that is also present from fish to human. In all vertebrate species studied so far, 26RFa-expressing neurons show a discrete localization in the hypothalamus, suggesting important neuroendocrine activities for this RFamide peptide. Indeed, 26RFa plays a crucial role in the control of feeding behavior in mammals, birds and fish. In addition, 26RFa up-regulates the gonadotropic axis in mammals and fish. Finally, evidence that the 26RFa/GPR103 system regulates steroidogenesis, bone formation, nociceptive transmission and arterial blood pressure has also been reported. Thus, 26RFa appears to act as a key neuropeptide in vertebrates controlling vital neuroendocrine functions. The pathophysiological implication of the 26RFa/GPR103 system in human is totally unknown and some fields of investigation are proposed.

Apelin and the proopiomelanocortin system: a new regulatory pathway of hypothalamic α-MSH release.

Neuronal networks originating in the hypothalamic arcuate nucleus (Arc) play a fundamental role in controlling energy balance. In the Arc, neuropeptide Y (NPY)-producing neurons stimulate food intake, whereas neurons releasing the proopiomelanocortin (POMC)-derived peptide α-melanocyte-stimulating hormone (α-MSH) strongly decrease food intake. There is growing evidence to suggest that apelin and its receptor may play a role in the central control of food intake, and both are concentrated in the Arc. We investigated the presence of apelin and its receptor in Arc NPY- and POMC-containing neurons and the effects of apelin on α-MSH release in the hypothalamus. We showed, by immunofluorescence and confocal microscopy, that apelin-immunoreactive (IR) neuronal cell bodies were distributed throughout the rostrocaudal extent of the Arc and that apelin was strongly colocalized with POMC, but weakly colocalized with NPY. However, there were numerous NPY-IR nerve fibers close to the apelin-IR neuronal cell bodies. By combining in situ hybridization with immunohistochemistry, we demonstrated the presence of apelin receptor mRNA in Arc POMC neurons. Moreover, using a perifusion technique for hypothalamic explants, we demonstrated that apelin-17 (K17F) increased α-MSH release, suggesting that apelin released somato-dendritically or axonally from POMC neurons may stimulate α-MSH release in an autocrine manner. Consistent with these data, hypothalamic apelin levels were found to be higher in obese db/db mice and fa/fa Zucker rats than in wild-type animals. These findings support the hypothesis that central apelin is involved in regulating body weight and feeding behavior through the direct stimulation of α-MSH release.

Myosin 1b and F-actin are involved in the control of secretory granule biogenesis.

Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.

Hypothalamic Neuropeptide 26RFa Acts as an Incretin to Regulate Glucose Homeostasis.

26RFa is a hypothalamic neuropeptide that promotes food intake. 26RFa is upregulated in obese animal models, and its orexigenic activity is accentuated in rodents fed a high-fat diet, suggesting that this neuropeptide might play a role in the development and maintenance of the obese status. As obesity is frequently associated with type 2 diabetes, we investigated whether 26RFa may be involved in the regulation of glucose homeostasis. In the current study, we show a moderate positive correlation between plasma 26RFa levels and plasma insulin in patients with diabetes. Plasma 26RFa concentration also increases in response to an oral glucose tolerance test. In addition, we found that 26RFa and its receptor GPR103 are present in human pancreatic ß-cells as well as in the gut. In mice, 26RFa attenuates the hyperglycemia induced by a glucose load, potentiates insulin sensitivity, and increases plasma insulin concentrations. Consistent with these data, 26RFa stimulates insulin production by MIN6 insulinoma cells. Finally, we show, using in vivo and in vitro approaches, that a glucose load induces a massive secretion of 26RFa by the small intestine. Altogether, the present data indicate that 26RFa acts as an incretin to regulate glucose homeostasis.

The orexin type 1 receptor is overexpressed in advanced prostate cancer with a neuroendocrine differentiation, and mediates apoptosis.

AIM: In the present study, we have examined the presence of orexins and their receptors in prostate cancer (CaP) and investigated their effects on the apoptosis of prostate cancer cells. METHODS: We have localised the orexin type 1 and 2 receptors (OX1R and OX2R) and orexin A (OxA) in CaP sections of various grades and we have quantified tumour cells containing OX1R. Expression of OX1R was evaluated in the androgeno-dependent (AD) LNCaP and the androgeno-independent (AI) DU145 prostate cancer cells submitted or not to a neuroendocrine differentiation. The effects of orexins on the apoptosis and viability of DU145 cells were also investigated. RESULTS: OX1R is strongly expressed in carcinomatous foci exhibiting a neuroendocrine differentiation, and the number of OX1R-stained cancer cells increases with the grade of the CaP. In contrast, OX2R is only detected in scattered malignant cells in high grade CaP. OX1R is expressed in the AI DU145 cells but is undetectable in the LNCaP cells. Acquisition of a neuroendocrine phenotype by the DU145 cells is associated with an overexpression of OX1R. Orexins induce the apoptosis of DU145 cells submitted to a neuroendocrine differentiation. CONCLUSION: The present data indicate that OX1R-driven apoptosis is overexpressed in AI CaP exhibiting a neuroendocrine differentiation opening a gate for novel therapies for these aggressive cancers which are incurable until now.

The neuropeptide 26RFa is expressed in human prostate cancer and stimulates the neuroendocrine differentiation and the migration of androgeno-independent prostate cancer cells.

AIM: Accumulating data suggest that neuropeptides produced by neuroendocrine cells play a crucial role in the progression and aggressiveness of hormone refractory prostate cancer (CaP). In this study, we have investigated the presence and function of the neuropeptide 26RFa in CaP. METHODS: We have localised and quantified tumour cells containing 26RFa and its receptor, GPR103, in CaP sections of various grades. In vitro experiments were performed to investigate the effects of 26RFa on the migration, proliferation and neuroendocrine differentiation of the androgeno-independent (AI) prostate cancer cell line DU145. RESULTS: 26RFa and GPR103 are present in carcinomatous foci exhibiting a neuroendocrine differentiation, and the number of 26RFa and GPR103-immunoreactive cancer cells increases with the grade of CaP. 26RFa stimulated the migration of native or transdifferentiated AI DU145 cells, but had no effect on their proliferation. Furthermore, 26RFa induced the neuroendocrine differentiation of DU145 cells as assessed by the occurrence of neurite-like extensions and the increase of the expression of the neuroendocrine marker chromogranin A. CONCLUSION: The present data indicate that 26RFa may participate to the development of CaP at the AI state by promoting the neuroendocrine differentiation and the migration of cancer cells via autocrine/paracrine mechanisms.

Orexigenic neuropeptide 26RFa: new evidence for an adaptive profile of appetite regulation in anorexia nervosa.

CONTEXT: Restrictive anorexia nervosa (AN) presents an adaptive appetite regulating profile including mainly high levels of ghrelin. Because this adaptive mechanism is not effective on food intake, other appetite-regulating peptides need to be explored. 26RFa is a hypothalamic neuropeptide that stimulates appetite, gonadotropin release, and bone metabolism. OBJECTIVE: The objective of the study was to evaluate the circadian levels of 26RFa in AN patients compared with healthy subjects, other eating disorders, and constitutional thinness (CT). DESIGN AND SETTINGS: This was a cross-sectional study performed in an endocrine unit and an academic laboratory. INVESTIGATED SUBJECTS: Five groups of age-matched young women were included in the study: 19 restrictive AN, 10 AN with bingeing/purging episodes, 14 with CT, 10 bulimic, and 10 normal-weight controls. MAIN OUTCOME MEASURES: Twelve-point circadian profiles of plasma 26RFa levels were measured in each subject. RESULTS: Significant circadian variations of 26 RFA were noticed in controls with higher values in the morning and abrupt decrease at noon. Twenty-four-hour mean 26RFa levels were significantly increased in restrictive AN and AN with bingeing/purging episodes (P < 0.001), predominantly in the afternoon and evening when compared with controls. Preprandial rises of 26 RFA were noticed in AN patients. Mean 26RFa levels trend to be higher in CT than in controls (P = 0.06) and significantly lower than in AN. The bulimic patients presented a circadian profile of 26RFa similar to that of controls. CONCLUSION: High levels of circulating 26RFa observed in AN patients might reflect an adaptive mechanism of the organism to promote energy intake and to increase fat stores in response to undernutrition.

The orexigenic activity of the hypothalamic neuropeptide 26RFa is mediated by the neuropeptide Y and proopiomelanocortin neurons of the arcuate nucleus.

26RFa is a hypothalamic RFamide neuropeptide that was identified as the endogenous ligand of the orphan G protein-coupled receptor, GPR103, and that stimulates appetite in mice. Up until now, the mechanism of action of 26RFa in the hypothalamic control of food intake remains unknown. The high density of GPR103 in the arcuate nucleus (Arc) prompted us to investigate, in the present study, the effects of 26RFa on the rat neuropeptide Y (NPY)/proopiomelanocortin (POMC) system. Intracerebroventricular injection of 26RFa stimulated NPY expression and release in the basal hypothalamus, whereas it decreased POMC expression and alpha-MSH release, and these effects were associated with an increase in food intake. A double in situ hybridization procedure indicated that the 26RFa receptor is present in NPY neurons of the Arc, but not in POMC neurons. Central administration of NPY Y1 and Y5 receptor antagonists abolished the inhibitory effects of 26RFa on POMC expression and alpha-MSH release, and reversed 26RFa-induced food consumption. Finally, 26RFa antagonized the effects of leptin on NPY expression and release, POMC expression and alpha-MSH release, and food intake. Altogether, the present data demonstrate for the first time that 26RFa exerts its orexigenic activity by stimulating the release of NPY in the Arc, which in turn inhibits POMC neurons by activating the Y1 and Y5 receptors. It is also suggested that the balance 26RFa/leptin is an important parameter in the maintenance of energy homeostasis.