Spexin2 Is a Novel Food Regulator in Gallus gallus.

Spexin2 (SPX2), a paralog of SPX1, is a newly identified gene in non-mammalian vertebrates. Limited studies in fish have evidenced its important role in food intake and energy balance modulation. However, little is known about its biological functions in birds. Using the chicken (c-) as a model, we cloned the full-length cDNA of SPX2 by using RACE-PCR. It is 1189 base pair (bp) in length and predicted to generate a protein of 75 amino acids that contains a 14 amino acids mature peptide. Tissue distribution analysis showed that cSPX2 transcripts were detected in a wide array of tissues, with abundant expression in the pituitary, testis, and adrenal gland. cSPX2 was also observed to be ubiquitously expressed in chicken brain regions, with the highest expression in the hypothalamus. Its expression was significantly upregulated in the hypothalamus after 24 or 36 h of food deprivation, and the feeding behavior of chicks was obviously suppressed after peripheral injection with cSPX2. Mechanistically, further studies evidenced that cSPX2 acts as a satiety factor via upregulating cocaine and amphetamine regulated transcript (CART) and downregulating agouti-related neuropeptide (AGRP) in hypothalamus. Using a pGL4-SRE-luciferase reporter system, cSPX2 was demonstrated to effectively activate a chicken galanin II type receptor (cGALR2), a cGALR2-like receptor (cGALR2L), and a galanin III type receptor (cGALR3), with the highest binding affinity for cGALR2L. Collectively, we firstly identified that cSPX2 serves as a novel appetite monitor in chicken. Our findings will help clarify the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.

Spexin-expressing neurons in the magnocellular nuclei of the human hypothalamus.

Neuropeptides are involved in numerous brain activities being responsible for a wide spectrum of higher mental functions. The purpose of this concise, structural and qualitative investigation was to map the possible immunoreactivity of the novel neuropeptide spexin (SPX) within the human magnocellular hypothalamus. SPX is a newly identified peptide, a natural ligand for the galanin receptors (GALR) 2/3, with no molecular structure similarities to currently known regulatory factors. SPX seems to have multiple physiological functions, with an involvement in reproduction and food-intake regulation recently revealed in animal studies. For the first time we describe SPX expressing neurons in the supraoptic (SON) and paraventricular (PVN) nuclei of the human hypothalamus using immunohistochemical and fluorescent methods, key regions involved in the mechanisms of osmotic homeostasis, energy expenditure, consummatory behaviour, reproductive processes, social recognition and stress responses. The vast majority of neurons located in both examined neurosecretory nuclei show abundant SPX expression and this may indirectly implicate a potential contribution of SPX signalling to the hypothalamic physiology in the human brain.

Treatment with celastrol protects against obesity through suppression of galanin-induced fat intake and activation of PGC-1α/GLUT4 axis-mediated glucose consumption.

Overweight and obesity may cause several metabolic complications, including type 2 diabetes mellitus and hyperlipidemia. Despite years of progress in medicine, there are no highly effective pharmacological treatments for obesity. The natural compound celastrol, a pentacyclic triterpene extracted from the roots of Tripterygium Wilfordi (thunder god vine) plant, exerts various bioactivities including anti-diabetic and anti-obese effects. Although celastrol could decrease food intake and obesity, the detailed mechanism for celastrol is still unclear as yet. Herein, we intended to determine the effect of celastrol on obesity and the underlying mechanisms. In the present study, diet-induced obese mice were treated with 100 µg/kg/d celastrol for the last 21 days, and 3T3-L1 cells were treated with celastrol for 6 h. The present findings showed that celastrol suppresses fat intake, and leads to weight loss by inhibiting galanin and its receptor expression in the hypothalamus of mice fed a high-fat diet. More importantly, in addition to these direct anti-obesity activities, celastrol augmented the PGC-1α and GLUT4 expression in adipocytes and skeletal muscles to increase glucose uptake through AKT and P38 MAPK activation. Celastrol also inhibited gluconeogenic activity through a CREB/PGC-1α pathway. In conclusion, the weight-lowering effects of celastrol are driven by decreased galanin-induced food consumption. Thus, this study contributes to our understanding of the anti-obese role of celastrol, and provides a possibility of using celastrol to treat obesity in clinic.

Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax).

The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates.

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization.

5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

[Galanin-like peptide (GALP): localization, receptors and biological function]. / Peptyd galaninopodobny (GALP): wystepowanie, receptory i funkcja biologiczna.

Galanin-like peptide (GALP) was isolated from porcine hypothalamus in 1999 on the basis of its ability to activate galanin receptors in vitro. Extensive studies carried out since the discovery of GALP contributed to the significant progress in our knowledge regarding this neuropeptide. GALP is synthesized mainly in the neurons of the hypothalamic arcuate nucleus and in the pituicytes of the posterior pituitary. The effects of GALP are mediated by well-characterized G-protein coupled galanin receptor subtypes. The fact that GALP shares homology only with 13 amino acids of the galanin sequence suggests that it might also interact with its own specific receptor. This relatively small 60-amino acid peptide belongs to a growing list of neuropeptides that play a crucial role in the regulation of food intake, energy balance and the reproductive axis. This peptide appears to be involved in integrating energy balance control and reproduction. The paper presents the current state of knowledge about the biosynthesis, structure, localization of GALP and its receptors, with particular emphasis on its role. This review will attempt to summarize the significant body of in vitro and in vivo studies conducted so far, concerning the effects of GALP.

The environmental regulation of maturation in goldfish, Carassius auratus: effects of various LED light spectra.

While there have been a number of studies on the effects of photoperiod and duration of light and dark exposure, much less information is available on the importance of light intensity. This study investigated the effects of exposure of goldfish, Carassius auratus exposed to white fluorescent bulbs, and red (peak at 630nm), and green (530nm) light emitting diodes (LEDs) at approximately 0.9W/m(2) (12-h light:12-h dark) for four months on a number of hormones of the hypothalamus-pituitary-gonad (HPG) axis, in vivo and in vitro. We investigated the effects of native GnRH molecules (gonadotropin-releasing hormones; salmon GnRH, sGnRH; and chicken GnRH-II, cGnRH-II), gonadotropin hormones (GTHα; follicle-stimulating hormone, FSH-ß; luteinizing hormone, LH-ß2), kisspeptin 1 (Kiss1) and G protein-coupled receptor 54 (GPR54) mRNA levels. Furthermore, we measured LH and 17α-hydroxypregnenolone levels in plasma and we performed gonad histological observations. GnRHs, Kiss1, GPR54 and GTH mRNA and plasma LH and 17α-hydroxypregnenolone levels in the in vivo and in vitro groups exposed to green LEDs were significantly higher than the other groups. Histological analysis revealed the presence of oocytes in the yolk stage in fish exposed to green light. These results suggest that green wavelengths regulate the HPG axis and enhance sexual maturation in goldfish.

Galanin regulation of LH release in male rats.

The present study examines the role of cerebroventricular administered (IIIrd ventricle) galanin on LHRH and LH release in adult and immature male rats. In both age groups, galanin stimulated LHRH synthesis and release from the hypothalamus, leading to a higher release of pituitary LH which in turn increased plasma LH levels. Galantide, a galanin receptor blocker, on the other hand, drastically reduced hypothalamic LHRH and plasma LH while increasing pituitary LH. In vitro incubation of anterior pituitary cells with galanin followed by LHRH resulted in increased release of pituitary LH but not by galanin alone. Galantide exhibited no such effect either alone or with LHRH. These results indicate that galanin is an important regulator for both hypothalamic LHRH and hypophysial LH and its role is independent of age in the case of male rats.

Alarin stimulates food intake and gonadotrophin release in male rats.

BACKGROUND AND PURPOSE: Alarin is a recently discovered member of the galanin peptide family encoded by a splice variant of galanin-like peptide (GALP) mRNA. Galanin and GALP regulate energy homeostasis and reproduction. We therefore investigated the effects of alarin on food intake and gonadotrophin release. EXPERIMENTAL APPROACH: Alarin was administered into the third cerebral ventricle (i.c.v.) of rats, and food intake or circulating hormone levels were measured. The effect of alarin on the hypothalamo-pituitary-gonadal axis was investigated in vitro using hypothalamic and anterior pituitary explants, and immortalized cell lines. Receptor binding assays were used to determine whether alarin binds to galanin receptors. KEY RESULTS: The i.c.v. administration of alarin (30 nmol) to ad libitum fed male rats significantly increased acute food intake to 500%, and plasma luteinizing hormone (LH) levels to 170% of responses to saline. In vitro, 100 nM alarin stimulated neuropeptide Y (NPY) and gonadotrophin-releasing hormone (GnRH) release from hypothalamic explants from male rats, and 1000 nM alarin increased GnRH release from GT1-7 cells. In vivo, pretreatment with the GnRH receptor antagonist cetrorelix prevented the increase in plasma LH levels observed following i.c.v. alarin administration. Receptor binding studies confirmed alarin did not bind to any known galanin receptor, or compete with radiolabelled galanin for hypothalamic binding sites. CONCLUSIONS AND IMPLICATIONS: These results suggest alarin is a novel orexigenic peptide, and that it increases circulating LH levels via hypothalamic GnRH. Further work is required to identify the receptor(s) mediating the biological effects of alarin.

Expression of mRNA for galanin, galanin-like peptide and galanin receptors 1-3 in the ovine hypothalamus and pituitary gland: effects of age and gender.

The neurotransmitters/neuromodulators galanin (GAL) and galanin-like peptide (GALP) are known to operate through three G protein-coupled receptors, GALR1, GALR2 and GALR3. The aim of this study was to investigate changes in expression of mRNA for galanin, GALP and GALR1-3 in the hypothalamus and pituitary gland, of male and female sheep, to determine how expression changed in association with growth and the attainment of reproductive competence. Tissue samples from the hypothalami and pituitary glands were analysed from late foetal and pre-pubertal lambs and adult sheep. Although mRNA for galanin and GALR1-3 was present in both tissues, at all ages and in both genders, quantification of GALP mRNA was not possible due to its low levels of expression. mRNA expression for both galanin and its receptors was seen to change significantly in both tissues as a function of age. Specifically, hypothalamic galanin mRNA expression increased with age in the male, but decreased with age in the female pituitary gland. mRNA expression for all receptors increased between foetal and pre-pubertal age groups and decreased significantly between pre-pubertal and adult animals. The results indicate that the expression of mRNA for galanin and its receptors changes dynamically with age and those significant differences exist with regard to tissue type and gender. These changes suggest that galaninergic neuroendocrine systems could be involved in the regulation of ovine growth and or the development of reproductive competence. The roles played by these systems in the sheep, however, may differ from other species, in particular the neuroendocrine link between nutrition and reproduction and GALR1's role in pituitary signalling.

The role of galanin receptors in anticonvulsant effects of low-frequency stimulation in perforant path-kindled rats.

Low-frequency stimulation (LFS) has antiepileptogenic effects on kindled seizures. In the present study, the role of galanin receptors in the inhibitory effect of LFS on perforant path kindling acquisition was investigated in rats. Animals were kindled by perforant path stimulation in a rapid kindling manner (six stimulations per day). LFS (0.1 ms pulses at 1 Hz, 600 pulses, and 80-150 microA) was applied immediately after termination of each kindling stimulation. M35 (0.5 and 1.0 nM per site), a nonselective galanin receptor antagonist and M871 (1.0 microM per site), a selective galanin receptor type 2 (GalR2) antagonist, were daily microinjected into the dentate gyrus before starting the stimulation protocol. The expression of GalR2 in the dentate gyrus was also investigated using semi-quantitative RT-PCR. Application of LFS significantly retarded the kindling acquisition and delayed the expression of different kindled seizure stages. In addition, LFS significantly reduced the increment of daily afterdischarge duration during kindling development. Intra-dentate gyrus microinjection of both M35 and M871 significantly prevented the inhibitory effects of LFS on kindling parameters. During the focal kindled seizure stages (1-3) M871 had no significant effect. However, during generalized seizure stages (4 and 5), M871 had the same effect as M35. Semi-quantitative RT-PCR also showed that after kindling acquisition, the GalR2 mRNA level decreased in the dentate gyrus but application of LFS prevented this decrease. Obtained results show that activation of galanin receptors by endogenous galanin has a role in mediating the inhibitory effect of LFS on perforant path-kindled seizures. This role is exerted through GalR1 during focal- and through GalR2 during generalized-kindled seizures.

Region specific galanin receptor/neuropeptide Y Y1 receptor interactions in the tel- and diencephalon of the rat. Relevance for food consumption.

The aim of this work was to determine the interactions between NPY and GAL receptor (GALR) subtypes in the hypothalamus and the amygdala using quantitative receptor autoradiography to analyze the binding characteristics of NPY-Y1 and Y2 receptor subtypes in the presence and absence of GAL. Food intake in satiated animals was evaluated after intraventricular co-injections of GAL and NPY-Y1 or Y2 agonists. The expression of c-Fos IR in both regions was also investigated. GAL decreases NPY-Y1 agonist binding in the arcuate nucleus by about 15% (p<0.01), but increases NPY-Y1 agonist binding in amygdala (18%) (p<0.01). These effects were blocked with the GAL antagonist M35. Y2-agonist binding was not modified by GAL. GAL blocked the food intake induced by the Y1 agonist (p<0.01). Co-injections of Y1 agonist and GAL also reduced the c-Fos expression induced by the Y1 agonist in the arcuate nucleus and the dorsomedial hypothalamic nucleus but increased c-Fos expression in amygdala. These results indicate the existence of antagonistic interactions between GALR and NPY-Y1 receptors in the hypothalamus and their functional relevance for food intake. In contrast, a facilitatory interaction between GALR and Y1 receptors exists in the amygdala which may be of relevance for fear related behaviour.

Metastin and its G protein-coupled receptor, GPR54: critical pathway modulating GnRH secretion.

Photoperiod, food availability, temperature, stress, and hormonal cues are some of the varied signals used by mammalian species to activate or suppress their timing of sexual maturation. All ultimately converge upon gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. Through its stimulation of LH and FSH from the pituitary, GnRH represents a critical step in the reproductive cascade. While few dispute this central role of GnRH, little is understood of the mechanisms influencing the developmental fate and physiologic controls of GnRH neurons. Identification of the signals which modulate pulsatile GnRH secretion is critical to advancing understanding of normal puberty and reproductive competency. The recent identification of loss-of-function mutations in GPR54, a receptor for kisspeptin-1, has highlighted a new pathway for the timing of puberty and reproductive control.

G protein-coupled galanin receptor distribution in the rat central nervous system.

The action of galanin in the central nervous system is mediated by at least three galanin receptor subtypes (GalR1, GalR2 and GalR3) which belong to the family of G protein-coupled receptors. GalR1 and GalR2 are coupled to G(i/o) proteins, although the latter may also be coupled to G(q/11) proteins. The aim of the present study was to identify the anatomical distribution and quantify the density of GalRs coupled to G proteins. The galanin (10(-6) M) stimulated guanosine 5'-(gamma-[35S] thio)triphosphate binding assay was used in tissue sections from the rat brain. Maximal percentages of stimulation over basal levels were found in the anterior olfactory nucleus and in the lateral olfactory tract nucleus ( approximately 54%). High levels of stimulation were recorded in diverse hypothalamic nuclei (16-28%), in the amygdala (central amygdaloid nucleus, 40%), in the spinal trigeminal tract (23%) and in layers 1-2 of the spinal cord (26%). Moderate binding stimulation (5-13%) was observed in thalamus, substantia nigra pars compacta, parabrachial nucleus, locus coeruleus and dorsal raphe nucleus. The lowest stimulation induced by galanin was recorded in diverse areas of the cortex, striatum, hippocampus and substantia nigra pars reticulata. The results show an anatomical distribution similar to that described for GalR1. However, in diverse brain areas, in which a high density of these receptors has previously been reported, only a moderate coupling to G proteins was found. These findings would suggest that the efficacy of galanin to induce an effective coupling of its receptors to G proteins could be different depending on the brain area.

Galanin-like peptide as a link between metabolism and reproduction.

Galanin-like peptide (GALP) is a hypothalamic neuropeptide that binds and activates galanin receptors in vitro. Following the discovery of GALP, researchers have attempted to properly place it in the context of galanin receptor physiology. Central injections of GALP have revealed some common actions with galanin, such as acutely increased food intake and suppression of the thyroid axis. Other actions are unique to GALP, such as long-term inhibition of food intake and stimulation of luteinizing hormone (LH) secretion in male rats. GALP and galanin also produce differential effects on expression of the immediate early gene product Fos in the brain. Determining which of these actions are dependent on galanin receptors (versus a putative GALP-specific receptor), as well as which actions represent the authentic physiology of endogenous GALP will require continued experimentation. GALP gene expression is positively regulated by several hormones involved in the control of energy balance and metabolism, namely leptin, insulin and thyroid hormone. Based on current evidence, GALP neurones may serve as a hypothalamic relay, transmitting information from the periphery to circuits within the brain involved in the physiological control of metabolism and reproduction.