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EM66 is a conserved 66-amino acid peptide derived from secretogranin II (SgII), a member of the granin protein family. EM66 is widely distributed in secretory granules of endocrine and neuroendocrine cells, as well as in hypothalamic neurones. Although EM66 is abundant in the hypothalamus, its physiological function remains to be determined. The present study aimed to investigate a possible involvement of EM66 in the hypothalamic regulation of feeding behaviour. We show that i.c.v. administration of EM66 induces a drastic dose-dependent inhibition of food intake in mice deprived of food for 18 hours, which is associated with an increase of hypothalamic pro-opiomelanocortin (POMC) and melanocortin-3 receptor mRNA levels and c-Fos immunoreactivity in the POMC neurones of the arcuate nucleus. By contrast, i.c.v. injection of EM66 does not alter the hypothalamic expression of neuropeptide Y (NPY), or that of its Y1 and Y5 receptors. A 3-month high-fat diet (HFD) leads to an important decrease of POMC and SgII mRNA levels in the hypothalamus, whereas NPY gene expression is not affected. Finally, we show that a 48 hours of fasting in HFD mice decreases the expression of POMC and SgII mRNA, which is not observed in mice fed a standard chow. Taken together, the present findings support the view that EM66 is a novel anorexigenic neuropeptide regulating hypothalamic feeding behaviour, at least in part, by activating the POMC neurones of the arcuate nucleus., (© 2017 British Society for Neuroendocrinology.)

EM66 is a secretogranin II-derived peptide strongly expressed within hypothalamic neuroendocrine areas such as the parvocellular aspect of the paraventricular nucleus (pPVN) as well as the median eminence (ME), suggesting a hypophysiotropic role for this neuropeptide. The aim of the present study was to explore such a role in the corticotrope and thyrotrope axes. We analyzed EM66 occurrence respectively in CRH and TRH neurosecretory cells of the rat pPVN by double immunohistochemistry. Functionally, we studied the effect of acute stress (immobilization for 2 h or cold exposure at 5°C for 4 h) and hypothyroidism (induced by 1-week thyroidectomy) on EM66 immunoreactivity (IR) within the pPVN. Double immunohistochemical labeling revealed that EM66-IR colocalized with CRH or TRH labelings within pPVN hypophysiotropic neurons as well as the axon terminals of the external layer of the ME. Because TRH neuronal population of the pPVN is completely distinct from the CRH neurosecretory system, our data demonstrate the existence of at least two distinct EM66 neuronal populations in the rat pPVN. Acute immobilization or cold exposure stresses did not affect EM66 expression as evaluated by the number of EM66-IR neurons within the pPVN. These results suggest that EM66 does not participate to the phenotypic plasticity of hypothalamic parvocellular neurons in response to acute stress. In addition, short-term hypothyroidism did not provoke any significant variation of the number of intraparaventricular EM66 neurons, indicating that EM66 expression would be insensitive to short-term hypothyroidism despite its occurrence within TRH neurons. Thus, the present data show the occurrence of EM66 in distinct areas of the rat PVN but its expression is not coregulated with those of CRH and TRH during acute stress and hypothyroidism., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives and Methods: Neuropeptides, as the main neuroendocrine system effectors, regulate notably the response to different stressors via a secretory plasticity within their respective hypothalamic neuronal populations. The aim of the present study was to explore by immunocytochemistry the occurrence and the potential expression plasticity of the novel neuropeptide EM66 in the CRH neurones of stressed rats., Results: The secretogranin II (SgII)-derived peptide EM66 is strongly expressed within hypothalamic neuroendocrine areas such as the parvocellular aspect of the paraventricular nucleus (pPVN) as well as the median eminence, suggesting a probable hypophysiotropic effect of this peptide. As a first approach to investigate such a role, we evaluated by immunohistochemistry EM66 expression within the pPVN following acute immune stress induced by lipopolysaccharide (LPS) or interleukin-1β (IL-1β) injection in rat. This study showed that EM66 is present in the pPVN but the number of EM66 immunolabeled cells did not fluctuate in this structure following LPS peripheral injection. In line with this observation, an intracerebroventricular injection of IL-1β did not provoke any significant variation of the number of intraparaventricular EM66 neurones., Conclusion: The present data revealed for the first time that EM66 expression would be insensitive to the central and peripheral cytokines within the neurosecretory hypothalamic pPVN. This result indicates that EM66 does not participate to the phenotypic plasticity of hypothalamic parvicellular neurones in response to acute inflammatory stress.

Context: Neurotensin (NT) modulates corticosteroid secretion from the mammalian adrenal gland., Objective: The objective of this study was to investigate the possible involvement of NT in the control of cortisol secretion in the human adrenal gland., Design: In vitro studies were conducted on cultured human adrenocortical cells., Setting: This study was conducted in a university research laboratory., Patients: Adrenal explants from patients undergoing expanded nephrectomy for kidney cancer were studied., Main Outcome Measure: Cortisol secretion from cultured adrenocortical cells was measured., Results: NT1-11, the N-terminal fragment of NT, dose-dependently inhibited basal and ACTH-stimulated cortisol production by human adrenocortical cells in primary culture. In contrast, NT had no influence on cortisol output at concentrations up to 10(-6) m. HPLC and RT-PCR analyses failed to detect any significant amounts of NT and NT mRNA, respectively, in adrenal extracts. Molecular and pharmacological studies were performed to determine the type of NT receptor involved in the corticostatic effect of NT1-11. RT-PCR analysis revealed the expression of NT receptor type (NTR) 3 mRNA but not NTR1 and NTR2 mRNAs in the human adrenal tissue. However, the pharmacological profile of the adrenal NT1-11 receptor was different from that of NTR3, indicating that this receptor type is not involved in the action of NT1-11 on corticosteroidogenesis., Conclusion: Our results indicate that NT1-11 may act as an endocrine factor to inhibit cortisol secretion through activation of a receptor distinct from the classical NTR1, NTR2, and NTR3.

Three overlapping peptides have been isolated from the whole brain of the European green frog, Rana ridibunda and identified as fragments of the cytoplasmic domain of the synaptic vesicle membrane protein, synaptophysin. The primary structures of the peptides define the amino acid sequence of the 58 residues at the COOH-terminus of the protein and indicate that fragments arose from proteolytic cleavages at the COOH-terminal side of aspartyl residues. The overall conservation of structure of the cytoplasmic domain between the mammalian and amphibian proteins is relatively poor (58% sequence identity between frog and rat synaptophysin). However, the pattern of repeating tyrosine residues that is present in mammalian and Torpedo synaptophysins is also found in the frog protein suggesting that this structural motif is necessary for the, as yet unknown, function of the protein. The cytoplasmic region of frog synaptophysin is also rich in glutamine and glycine residues, but the putative consensus repeating sequence Tyr-Gly-Pro/Gln-Gln-Gly in mammalian synaptophysins does not occur in the frog protein. The data raise the possibility that synaptophysin, like the chromogranins and secretogranin II, may function as the precursor of biologically active peptides.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been isolated from the frog brain and the sequence of the peptide appears to be strikingly similar to that of mammalian PACAP. In the present study, we have investigated the localization of PACAP in the frog interrenal (adrenal) gland by immunocytochemistry using antisera directed against PACAP 38 or PACAP 27. Two types of PACAP-immunoreactive fibres were observed: thick varicose fibres coursing between adrenal cells and thin processes located in the walls of blood vessels irrigating the gland. Bilateral transection of the splanchnic nerves did not affect the intensity and distribution of PACAP immunoreactivity. The mean +/- S.E.M. concentration of PACAP, measured by radioimmunoassay in crude adrenal extracts, was 0.65 +/- 0.16 nmol/g wet tissue. Two molecular forms of PACAP in the adrenal gland were characterized by reversed phase high-performance liquid chromatography combined with radioimmunoassay quantification. The elution profiles revealed the existence of two peaks exhibiting the same retention times as synthetic frog PACAP 38 (fPACAP 38) and PACAP 27, the predominant form being PACAP 38. The possible involvement of PACAP in the regulation of adrenal steroidogenesis was investigated in vitro using a perifusion system for frog adrenal slices. Graded doses of fPACAP 38 (0.1-10 mumol/l) increased the secretion of both corticosterone and aldosterone in a dose-dependent manner. Administration of repeated pulses of fPACAP 38 (1 mumol/l), at 120-min intervals, led to a reproducible stimulation of corticosteroid secretion without any tachyphylaxis. Prolonged infusion (2 h) of the peptide induced a rapid increase in corticosterone and aldosterone output, followed by a gradual decline in the secretion rate, suggesting the occurrence of a desensitization phenomenon. Synthetic porcine vasoactive intestinal peptide, which is structurally related to PACAP, was about ten times less potent than fPACAP 38 in stimulating steroidogenesis while the [Des-His1]-fPACAP 38 analogue was 100 times less effective. These results demonstrate that a peptide closely related to fPACAP 38 is present in fibres innervating the frog adrenal gland and could participate in the regulation of corticosteroid secretion, particularly during neurogenic stress.