Expression Of Galanin-like Peptide mRNA Research Articles

Galanin-like peptide (GALP) neurone-specific phosphoinositide 3-kinase signalling regulates GALP mRNA levels in the hypothalamus of males and luteinising hormone levels in both sexes.

Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110β via conditional gene targeting (cKO) in mice (GALP-p110α/β cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/β cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/β cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/β cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/β cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP neurones are direct targets of steroid hormones and that PI3K signalling regulates hypothalamic GALP mRNA expression and LH levels in a sex-specific fashion.

Postnatal development of galanin-like peptide mRNA expression in rat hypothalamus

We examined the developmental change of GALP mRNA in male and female rat hypothalamus during postnatal day 1 to 60, using in situ hybridization histochemistry. Neuropeptide Y (NPY) and proopiomelanocortin (POMC) mRNA in the hypothalamus were also examined because they are important in the regulation of food intake. GALP mRNA was first detected in the arcuate nucleus (ARC) on day 8. GALP mRNA was gradually increased between day 8 and 14 and markedly increased between day 14 and 40, which is the weaning and pubertal period in rats. After day 40, there were no significant differences in GALP mRNA. In contrast to GALP, NPY and POMC mRNAs were detected in the ARC from day 1 and lasted to day 60. There was no sexual dimorphism in GALP, NPY and POMC mRNAs during postnatal development. Next, we examined the effect of the milk deprivation for 24 h on GALP, NPY and POMC mRNA in pups. GALP mRNA did not change by milk deprivation on day 9 and 15, while milk deprivation had a significant effect on NPY and POMC mRNA on day 15. These results suggest that the development of GALP may be associated with developmental changes such as weaning, feeding and maturation of reproductive functions. The regulatory mechanism of GALP mRNA is different from that of the NPY and POMC genes during postnatal development.

Various Dietary Fats Differentially Change the Gene Expression of Neuropeptides Involved in Body Weight Regulation in Rats

Various high-fat diets are obesogenic but not to the same extent. The aim of the present study was to investigate the effects of saturated fat n-6 and n-3 polyunsaturated fatty acids (PUFAs) on the central neuropeptidergic system in adult rats. Using reverse transcriptase-polymerase chain reaction and in situ hybridisation, we evaluated the net effect of feeding in these fats, comparing the effects of a high- to low-fat diet, and the diversity of the effects of these fats in the same amount within the diet. We also determined plasma lipids, glucose, insulin and leptin concentrations. Six-week feeding with high-saturated fat evoked hyperpahagia and the largest weight gain compared to both high-PUFA diets. Rats fed high-saturated fat were found to have decreased neuropeptide Y (NPY) mRNA expression in the arcuate nucleus (ARC) and the compact zone of the dorsomedial nucleus (DMHc), unchanged pro-opiomelanocortin (POMC), galanin-like peptide (GALP) mRNA expression in the ARC, as well as melanin-concentrating hormone (MCH) and prepro-orexin (preORX) mRNA expression in the lateral hypothalamus, compared to low-saturated fed rats. By contrast, feeding with both high-PUFA diets increased POMC and GALP mRNA expression in the ARC compared to the corresponding low-fat diet and the high-saturated fat diet. Furthermore, feeding with both low-PUFA diets reduced NPY mRNA expression compared to the low-saturated fat diet exclusively in the DMHc. Uniquely, the high n-3 PUFA feeding halved MCH and preORX mRNA expression in the lateral hypothalamus compared to the other high-fat and low n-3 PUFA diets. In rats fed three high-fat diets, plasma insulin and leptin concentrations were significantly increased and the type of fat had no effect on these hormone levels. Rats fed high-saturated fat had both hyperglycaemia and hypertriacylglycerolemia and rats fed high n-3 PUFA only had hyperglycaemia. The present study demonstrates that various forms of dietary fat differentially change the expression of neuropeptide genes involved in energy homeostasis.

Neuronal interactions between galanin-like-peptide- and orexin- or melanin-concentrating hormone-containing neurons

Galanin-like peptide (GALP) is a novel orexigenic neuropeptide that is recently isolated from the porcine hypothalamus. GALP-containing neurons predominantly locate in the hypothalamic arcuate nucleus (ARC). The expression of GALP mRNA within the ARC is increased after the administration of leptin. GALP-containing neurons express leptin receptor and contain α-melanocyte-stimulating hormone. We have recently reported that neuropeptide Y (NPY)- and orexin-containing axon terminals are in close apposition with GALP-containing neurons in the ARC. In addition, GALP-containing neurons express orexin-1 receptor (OX 1-R). Thus, GALP may function under the influence of leptin and orexin. However, the target neurons of GALP have not yet been clarified. To clarify the neuronal interaction between GALP-containing and other feeding regulating neurons, double-immunostaining method using antibodies against GALP- and orexin- or melanin-concentrating hormone (MCH) was performed in the rat lateral hypothalamus (LH). GALP-immunoreactive fibers appeared to project to the LH around the fornix. They were also found from the rostral to the caudal part of the ARC, paraventricular nucleus (PVH), stria terminalis (BST), medial preoptic area (MPA), and lateral septal nucleus (LSV). Moreover, GALP-like immunoreactive nerve fibers were directly contacted with orexin- and melanin-concentrating hormone (MCH)-like immunoreactive neurons in the LH. Our findings strongly suggest that GALP-containing neurons interact with orexin- and/or MCH-containing neurons in the lateral hypothalamus and that it participates in the regulation of feeding behavior in harmony with other feeding-regulating neurons in the hypothalamus.

Effects of diabetes and insulin on the expression of galanin-like peptide in the hypothalamus of the rat.

Galanin-like peptide (GALP) is produced in a small population of neurons in the arcuate nucleus of the hypothalamus, and leptin stimulates the hypothalamic expression of GALP mRNA. Because insulin and leptin share common signaling pathways in the brain, we reasoned that GALP neurons might also be responsive to changes in circulating concentrations of insulin. To test this hypothesis, we first studied the effect of insulin deficiency on the expression of GALP by comparing levels of GALP mRNA between normal and diabetic animals. Streptozotocin-induced diabetes was associated with a significant reduction in the expression of GALP mRNA, which was reversed by treatment with either insulin or leptin. Second, we examined the effect of insulin administered directly into the brain on the expression of GALP mRNA in fasted rats. Hypothalamic levels of GALP mRNA were lower in animals after a 48-h fast, and central treatment with insulin reversed this effect. These results suggest that GALP neurons are direct targets for regulation by insulin and implicate these cells for a role in the metabolic and behavioral sequelae of type 1 diabetes.

Galanin-like peptide as a possible link between metabolism and reproduction in the macaque.

Galanin-like peptide (GALP) is a hypothalamic neuropeptide that has been implicated in the control of feeding, metabolism, and reproduction. The goal of this study was to examine the effects of central infusions of GALP on GnRH and LH secretion and to identify physiological factors that influence the expression of GALP mRNA in the brain of a primate species. Infusions of GALP into the lateral cerebroventricle of the macaque caused a significant increase in LH secretion, which was blocked by administration of the GnRH receptor antagonist acyline. However, the expression of GALP mRNA in the arcuate nucleus, as determined by in situ hybridization, was not regulated by either estradiol or progesterone. Compared with feeding ad libitum, fasting for 48 h produced a significant reduction in the hypothalamic expression of GALP mRNA. GALP neurons were found to express both neuropeptide Y Y1 receptor and serotonin 2C receptor by double-label in situ hybridization. Taken together, these results suggest that GALP neurons play a role of integrating metabolic signals, which are relayed to circuits controlling GnRH release in the macaque.

Novel G-protein coupled receptor ligands and neurohypophysial hormones.

Recently, novel peptides have been identified as unknown ligands of orphan G-protein coupled receptors. In the paraventicular (PVN) and supraoptic nuclei (SON), the expression of the G-protein genes are abundant. In this review, we focus on the physiological role of neuromedin U and galanin-like peptide, which were recently identified as ligands of G-protein coupled receptors, in the regulation of neurohypophysial hormones. Intracerebroventricular (i.c.v) administration of neuromedin U induced the expression of c-fos mRNA in both the magnocellular and parvocellular division of the PVN and throughout the SON. Administration of i.c.v. neuromedin U caused a significant increase in plasma concentrations of vasopressin and oxytocin as well as adrenocorticotropic hormone. The expression of galanin-like peptide mRNA was observed in the pituicytes of rat posterior pituitary gland. The expression of galanin-like peptide mRNA in the posterior pituitary gland was markedly increased after dehydration, salt loading and intraperitoneal administration of lipopolysaccaride, challenges that stimulate the secretion of vasopressin and oxytocin, and which activate the hypothalamic-pituitary adrenal axis. These results suggest that neuromedin U and galanin-like peptide may have an important role in the regulation of both the hypothalamic-neurohypophysial system and the hypothalamic-adrenohypophysial system.

Changes in hypothalamic expression levels of galanin-like peptide in rat and mouse models support that it is a leptin-target peptide.

Galanin-like peptide (GALP) is a novel peptide that has been isolated from the porcine hypothalamus. The expression of GALP mRNA is localized to the hypothalamic arcuate nucleus and is thought to be under the regulation of leptin. First, we confirmed by real-time PCR analysis that sc administration of leptin to Wistar rats under food-deprived conditions resulted in a 1.5-fold increase in hypothalamic GALP mRNA levels. Next, GALP mRNA levels were found to be reduced by 50% in 11-wk-old male Zucker obese rats compared with age-matched Zucker lean rats, whereas neuropeptide Y mRNA levels were increased by 55% and proopiomelanocortin mRNA levels were reduced by 53% in Zucker obese rats. Analysis using a two-site enzyme immunoassay revealed a lower level of hypothalamic GALP immunoreactivity in 11-wk-old Zucker obese rats (5.9 fmol/mg protein) than in age-matched Zucker lean rats (19.6 fmol/mg protein). Immunohistochemical studies demonstrated that Zucker obese rats (11 wk old) had a reduced number of GALP immunoreactivity-positive cells (29.4 cells/3 slices) in the arcuate nucleus compared with age-matched Zucker lean rats (115 cells/3 slices). Furthermore, Zucker obese rats showed increased sensitivity to intracerebroventricularly administered GALP compared with Zucker lean rats, in that a lower dose of GALP increased plasma LH levels in male Zucker obese rats, but not in male Zucker lean rats. In addition, a reduction in the level of hypothalamic GALP mRNA was found in db/db and ob/ob mice. The result supports the hypothesis that the hypothalamic GALP gene expression is controlled by leptin signals and suggests possible involvement of GALP in the reproductive abnormalities of the Zucker obese rat.

A role for galanin-like peptide in the integration of feeding, body weight regulation, and reproduction in the mouse.

Galanin-like peptide (GALP) shares sequence homology with galanin and binds to galanin receptors in vitro. GALP neurons in the arcuate nucleus coexpress leptin receptors, and GALP mRNA expression is up-regulated by leptin. Based on these observations, we postulated that GALP plays a role in mediating leptin's inhibitory effects on food intake (FI) and body weight (BW), as well as its stimulatory effect on the reproductive axis. To test these hypotheses, we performed several studies in which mice received intracerebroventricular injections of either GALP or vehicle. Acute GALP treatment elicited a dose-dependent suppression of FI and BW. Long-term treatment with GALP caused only transient reductions in FI and BW, demonstrating that the mice became refractory to continued exposure to GALP. GALP inhibited FI as early as 1 h post injection. Central injection of GALP suppressed locomotor activity and elicited the formation of a conditioned taste aversion. In male mice, serum levels of LH and testosterone were increased by GALP administration. Although we cannot rule out possible nonspecific effects of GALP on FI, the present observations are consistent with the argument that GALP is a downstream effector of leptin's actions within the central nervous system.

Cloning and distribution of galanin-like peptide mRNA in the hypothalamus and pituitary of the macaque.

Galanin-like peptide (GALP) is a newly discovered hypothalamic neuropeptide, which is regulated by leptin and implicated in the regulation of GnRH secretion in the rodent. We searched the human genome database and determined that the human GALP gene comprises six exons, as has been shown for human galanin. We used rapid amplification of cDNA ends to clone a full-length cDNA (802 bp) of the macaque homologue of GALP and found it to be highly conserved between human and macaque at both the nucleotide (93%) and peptide (94%) levels. Mature GALP is predicted to be 60 amino acids in the macaque as in other species, and the region of GALP (9-21) that shows homology to the N-terminal 13 amino acids of galanin is perfectly conserved. We mapped the distribution of GALP mRNA in the hypothalamus and pituitary of the macaque by in situ hybridization and observed that, as in rodent species, the expression of GALP mRNA is confined to the arcuate nucleus, median eminence, and neurohypophysis. Using double-label in situ hybridization, we found that nearly all (98%) GALP mRNA-expressing cells in the arcuate nucleus also express mRNA for the long form of the leptin receptor. These findings suggest that a leptin-GALP signaling pathway exists in a primate species.

Distribution and Regulation of Galanin-Like Peptide (GALP) in the Hypothalamus of the Mouse

Galanin-like peptide (GALP) is a newly discovered molecule whose expression in the brain is confined to the arcuate nucleus and median eminence. In the rat, cellular levels of GALP mRNA are reduced by fasting and reversed by peripheral administration of leptin. The purpose of this investigation was 1) to clone and map the distribution of GALP mRNA in the brain of the mouse; 2) to compare the pattern and magnitude of GALP mRNA expression in the leptin-deficient obese (ob/ob) mouse with that of wild-type controls; and 3) to examine the effects of leptin delivered into the brain on the expression of GALP mRNA in the ob/ob mouse. We report the sequence of a mouse GALP cDNA and show that GALP mRNA is expressed in the arcuate nucleus, median eminence, infundibular stalk, and the neurohypophysis of this species. The expression of GALP mRNA in the brain was markedly reduced in the ob/ob mice, compared with wild-type animals. Intracerebroventricular infusion of leptin to ob/ob mice increased both the number of GALP mRNA-expressing neurons and their content of GALP mRNA, compared with vehicle-treated controls. These observations demonstrate that GALP mRNA is induced by leptin through a direct action on the brain.

Galanin-Like Peptide mRNA in Neural Lobe of Rat Pituitary

Galanin-like peptide (GALP) was recently identified in the porcine hypothalamus, pituitary gland and gut, and has reported selectivity for the GalR2, c.f. the GalR1 receptor. GALP cDNAs have been cloned from pig, rat and human, and GALP mRNA expression is restricted to the arcuate nucleus in normal rat brain. This study examined the regional and cellular distribution of GALP mRNA in the rat pituitary gland, and subsequently determined the effect of osmotic stimulation on GALP transcript levels. GALP mRNA was not detected in the anterior or intermediate lobes, but moderate levels of GALP mRNA were present in the neural (posterior) lobe, in presumed pituicytes, of normal male and female rats. Osmotic stimulation by dehydration or salt loading produced a time-dependent increase in GALP mRNA levels in the neural lobe. Thus, dehydration for 4 days increased GALP mRNA 40-fold, while salt loading for 4, 7 or 10 days increased GALP levels 14-, 21- and 25-fold, respectively (p ≤ 0.001). Levels of vasopressin (VP) mRNA in the neural lobe were also increased by these treatments, consistent with previous reports. Galanin (GAL) and GalR2 receptor mRNAs were not detected in the neural lobe, under normal or osmotic stimulation conditions. In addition, GALP mRNA levels in the arcuate nucleus were not altered in dehydrated or salt-loaded rats; and GALP mRNA was not detected in magnocellular neurons of the supraoptic or paraventricular nucleus, despite the characteristic up-regulation of VP and GAL mRNA in these cells. In view of the established anatomy and function of VP/oxytocin neurons in the hypothalamo-neurohypophysial system and the role played by pituicytes in their regulation, the likely synthesis/release of GALP by these specialized astrocytes strongly suggests a role for this novel peptide in regulation of pituicyte morphology/function and/or neurohormone release.

Galanin-like peptide (GALP) is expressed in rat hypothalamus and pituitary, but not in DRG.

Galanin-like peptide (GALP) was recently purified on the basis of its preferential activation of galanin receptor subtype 2 (GALR2) compared with galanin receptor subtype 1 (GALR1). Using in situ hybridization of adult rat brain, pituitary and dorsal root ganglia (DRG) we demonstrate that GALP mRNA expression is restricted to the arcuate nucleus and median eminence of the hypothalamus, and to the posterior lobe of the pituitary. No expression was detected elsewhere in brain, or in the DRG. In adult mouse, no expression was detected in brain or in DRG either before or after axotomy, suggesting that GALP has no apparent role in the axotomy response of DRG.

Galanin-Like Peptide (GALP) mRNA Expression Is Restricted to Arcuate Nucleus of Hypothalamus in Adult Male Rat Brain

Galanin-like peptide (GALP) is a novel 60-amino acid neuropeptide, isolated from porcine hypothalamus and subsequently identified in rats and humans, which has reported selectivity for the Gal-R2 galanin receptor [Ohtaki T et al: J Biol Chem 1999; 274: 37041–37045]. In the current study, the regional and cellular distribution of GALP mRNA in rat brain has been investigated by in situ hybridization of [³⁵S]-labelled oligonucleotide probes. In a thorough screening of adult male rat brain, GALP mRNA expression was detected only throughout the rostrocaudal extent of the arcuate nucleus (ARC) with the most abundant hybridization signal in the posterior, periventricular zones. GALP mRNA-positive neurons were mostly localized in the ventromedial division of the ARC, with many closely adjacent to the wall of the third ventricle. Smaller numbers of labelled neurons were also found in ventrolateral areas. The distribution of GALP mRNA was somewhat complementary to that for galanin (GAL) mRNA in the ARC, but contrasted with the broad distribution of this transcript throughout the hypothalamus. GAL mRNA was also distributed along the rostrocaudal extent of the ARC, but was most abundant in the anterior to middle levels and in ventrolateral regions. Interestingly, somatostatin mRNA expression appeared to overlap the distribution of GALP mRNA in posterior, ventromedial regions of the ARC. Thus, in adult rat brain GALP mRNA expression was restricted to a discrete subpopulation of neurons in the ARC, with a unique localization pattern unlike GAL or many other known peptide- and transmitter-containing cells in this region. GALP could, however, be co-expressed in sub-populations of other neuronal phenotypes (e.g. somatostatin neurons) or within cells that express Gal-R2 receptors. In view of the established anatomy and function of the ARC and the restricted localization of GALP mRNA, this novel peptide is likely to play a role in regulation of anterior pituitary hormone secretion, or in regulation of other hypothalamic peptide and transmitter systems.