The effects of galanin-like peptide on energy balance, body temperature and brain activity in the mouse and rat are independent of the GALR2/3 receptor

Galanin-like peptide (GALP) is a neuropeptide that is thought to act on the galanin receptors GALR1, GALR2 and GALR3. In rats, i.c.v. injection of GALP has dichotomous actions on energy balance, stimulating feeding over the first hour, but reducing food intake and body weight at 24 h, as well as causing an increase in core body temperature. In mice, GALP only induces an anorexic action, and its effects on core body temperature are unknown. One aim of the present study was to determine the effects of GALP on core body temperature in mice. Intracerebroventricular injection of GALP into conscious mice had no effect on feeding over 1 h, but caused a significant reduction in food intake and body weight at 24 h. It also caused an immediate drop in core body temperature, which was followed by an increase in body temperature. To understand these different effects of GALP on energy balance in mice compared to rats, and to determine the involvement of GALR2 and GALR3, immunohistochemistry was performed to localise c-Fos, a marker of cell activation. Intracerebroventricular injection of GALP induced c-Fos expression in the parenchyma surrounding the ventricles, the ventricular ependymal cells and the meninges in mice and rats. GALP also induced c-Fos expression in the supraoptic nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamus and nucleus tractus solitarius in rats but not in mice. Central administration of a GALR2/3 agonist in rats did not induce c-Fos in any of the brain regions that expressed this protein after GALP injection, and had no effect on food intake, body weight and body temperature in rats or mice. These data suggest that GALP induces differential effects on energy balance and brain activity in mice compared to rats, which are unlikely to be due to activation of the GALR2 or GALR3 receptor.     

Microinjection of galanin-like peptide into the medial preoptic area stimulates food intake in adult male rats

Galanin-like peptide (GALP) is a neuropeptide implicated in the regulation of feeding behaviour, metabolism and reproduction. GALP is an endogenous ligand of the galanin receptors, which are widely expressed in the hypothalamus. GALP is predominantly expressed in arcuate nucleus (ARC) neurones, which project to the paraventricular nucleus (PVN) and medial preoptic area (mPOA). Intracerebroventricular or intraparaventricular (iPVN) injection of GALP acutely increases food intake in rats. The effect of GALP injection into the mPOA on feeding behaviour has not previously been studied. In the present study, intra-mPOA (imPOA) injection of GALP potently increased 0-1-h food intake in rats. The dose-response effect of imPOA GALP administration on food intake was similar to that previously observed following iPVN administration. The effects of GALP (1 nmol) or galanin (1 nmol) on food intake were then compared following injection into the PVN, mPOA, ARC, dorsal medial nucleus (DMN), lateral hypothalamus and rostral preoptic area (rPOA). GALP (1 nmol) increased food intake to a similar degree when injected into the imPOA or iPVN, but produced no significant effect when injected into the ARC, DMN, lateral hypothalamus or rPOA. Similarly, galanin (1 nmol) significantly increased food intake following injection imPOA and iPVN. However, the effect was significantly smaller than that following administration of GALP (1 nmol). Galanin also had no significant effect on food intake when administered into the ARC, DMN, lateral hypothalamus and rPOA. These data suggest that the mPOA and the PVN may have specific roles in mediating the orexigenic effect of GALP and galanin.     

Exaggerated feeding response to central galanin-like peptide administration in diet-induced obese rats

Galanin-like peptide (GALP) is a newly identified neuropeptide implicated in the regulation of metabolism and reproduction. GALP gene expression is decreased in the hypothalamus of genetically obese rodents, such as fa/fa rats and ob/ob mice, and central administration of GALP increases feeding in satiated rats. The effect of dietary obesity on GALP-induced feeding is unknown, so this study characterized the effects of central administration of GALP on feeding in a rat model of diet-induced obesity. Male Sprague-Dawley rats (n = 21) were randomly assigned to receive standard laboratory chow (12% fat as kcal) or high-fat cafeteria diet (35% fat) for 12 weeks before intracerebroventricular (icv) cannulae were implanted. Seven days later, rats received 0,0.2 or 0.3 nmol doses of GALP in randomized order at least 48 h apart. Food intake was measured at 0.5,1,2, 4 and 24 h post administration and body weight was measured at 24 h. Rats were maintained on their respective diets throughout the entire feeding experiment. Implementation of the high-fat diet led to significantly greater caloric intake (230%) and body weight (28%) compared to chow-fed control rats. GALP-induced feeding was rapid and maximal in both dietary groups at 30 min post injection. The 0.3 nmol dose of GALP led to significantly larger increases in caloric intake in high-fat fed rats than in chow-fed controls (35.4 +/- 3.7 and 22.1 +/- 1.3 kcal, respectively, at 30 min). It is not known if diet-induced obesity alters endogenous GALP levels, but our data suggest that adaptive responses in GALP signaling might occur during chronic overfeeding. One possible explanation is an increased sensitivity and/or number of specific GALP receptors, although actions of exogenous GALP may also represent pharmacological actions at galanin receptors.     

Galanin-Like Peptide (GALP) Facilitates Thermogenesis via Synthesis of Prostaglandin E2 by Astrocytes in the Periventricular Zone of the Third Ventricle

Administration of galanin-like peptide (GALP) leads to a decrease in both total food intake and body weight 24 h after injection, compared to controls. Moreover, GALP induces an increase in core body temperature. To elucidate the mechanism by which GALP exerts its effect on energy homeostasis, urethane-anesthetized rats were intracerebroventricularly injected with GALP or saline, after which oxygen consumption, heart rate, and body temperature were monitored for 4 h. In some cases, animals were also pretreated with the cyclooxygenase (COX) inhibitor, diclofenac, via intracerebroventricular (i.c.v.) or intravenous (i.v.) injection. c-Fos expression in the brain was also examined after injection of GALP, and the levels of COX and prostaglandin E₂ synthetase (PGES) mRNA in primary cultured astrocytes treated with GALP were analyzed by using qPCR. The i.c.v. injection of GALP caused biphasic thermogenesis, an effect which could be blocked by pretreatment with centrally (i.c.v.), but not peripherally (i.v.) administered diclofenac. c-Fos immunoreactivity was observed in astrocytes in the periventricular zone of the third ventricle. GALP treatment also increased COX-2 and cytosolic PGES, but not COX-1, microsomal PGES-1, or microsomal PGES-2 mRNA levels in cultured astrocytes. We, therefore, suggest that GALP elicits thermogenesis via a prostaglandin E₂-mediated pathway in astrocytes of the central nervous system.     

Pharmacological and functional characterization of galanin-like peptide fragments as potent galanin receptor agonists

The hypothalamic galanin-like peptide (GALP) was isolated by its ability to activate galanin receptors. The mature porcine GALP is a 60-amino acid neuropeptide proteolytically processed from a 120-amino acid precursor protein. It contains a region identical to the N-terminal 13-amino acids of the neuropeptide galanin. Within the sequence of human GALP (1-60) a potential proteolytic cleavage site between two basic amino acids is present at position 33, which might lead to a shorter C-terminally amidated peptide. In addition, the first two amino acids could be potentially removed via the action of dipeptidase IV. Ligand binding assays using the human neuroblastoma cell line SH-SY5Y transfected with the respective galanin receptors revealed that human GALP (1-60) displayed the highest affinity for the galanin receptor subtype GalR3 (IC50 = 10 nM) followed by GalR2 (IC50 = 28 nM) and GalR1 (IC50 = 77 nM). Ligand binding assays and functional studies showed that the human GALP (3-32) fragment was at least as potent as full length GALP (1-60). Other studies have shown that shorter fragments like human GALP (1-21) and GALP (22-60) were not effective on feeding responses in mice as compared to the full length peptide. Taken together these data suggest that the putative fragment GALP (3-32) might represent the strongest mediator of biological GALP activity. Furthermore it might be a useful tool to study the affinity of GALP to galanin receptors and to search for specific GALP receptors.     

Weight loss in rats treated with intracerebroventricular cobalt protoporphyrin is not specific to the neuropeptide Y system

Cobalt protoporphyrin (CoPP) reduces food intake and body weight following intracerebroventricular (i.c.v.) administration in rats. We injected 0.2 μmol CoPP per kg body weight i.c.v. and monitored body weight and daily food intake for 7 days. The body weight and 24 h food intake of CoPP-treated animals was significantly lower than that of vehicle-treated animals in all studies (P < 0.01) from day 2 to day 7. The 2 h feeding response (CoPP vs. vehicle-treated) to 10 μg neuropeptide Y (NPY) (4.0 vs. 7.1 g;P < 0.05), the 1 h feeding response to 10 μg galanin (1.3 vs. 3.2 g;P < 0.05) and 30 μg norepinephrine (0.6 vs. 1.9 g;P < 0.05) in CoPP-treated animals were all reduced compared to the vehicle-treated group. In addition there was no change in hypothalamic NPY mRNA in CoPP-treated animals. I.c.v. COP decreases sensitivity to exogenous NPY, galanin and norepinephrine. The effect of COP is not specific to NPY as previously described.     

Evidence that neurotensin mediates the central effect of leptin on food intake in rat

Recent evidence suggests that leptin's action on food intake and body weight regulation is mediated by a number of orexigenic and anorectic neuronal systems in the hypothalamus. Our previous demonstration that central injections of leptin induce hypothalamic neurotensin (NT) gene expression in association with a reduced food intake and decreased body weight in rats indicates that NT, an anorectic peptide, is involved in mediating leptin's action on feeding and body weight regulation. To begin to examine the relative role of NT in this regard we evaluated the effects of NT antiserum (NT-AS) or NT receptor antagonist, SR 48692, on the satiety action of leptin in rats. In the first experiment, 3rd cerebroventricular (i.c.v.) administration of either 1 or 5 microl of NT-AS, 30 min prior to leptin (4 microg) injection, completely blocked the effects of leptin on food deprivation (FD)-induced feeding. In the second experiment, intraperitoneal (i.p.) administration of SR 48692 (40 microg/kg) also completely prevented leptin's satiety action on FD-induced feeding. These results showing the reversal of leptin's satiety action by either NT immunoneutralization or NT-receptor antagonism support our hypothesis that NT is involved in mediating leptin's action on feeding and further suggest that this neuropeptide is a quantitatively important component of the leptin sensitive neural circuitry.     

Acute, subacute and chronic effects of central neuropeptide Y on energy balance in rats

Central neuropeptide Y (NPY) injection has been reported to cause hyperphagia and in some cases also hypometabolism or hypothermia. Chronic central administration induced a moderate rise of short duration in body weight, without consistent metabolic/thermal changes. In the present studies the acute and subsequent subacute ingestive and metabolic/thermal changes were studied following intracerebroventricular (i.c.v.) injections of NPY in cold-adapted and non-adapted rats, or the corresponding chronic changes following i.c.v. NPY infusion. Besides confirming basic earlier data, we demonstrated novel findings: a temporal relationship for the orexigenic and metabolic/thermal effects, and differences of coordination in acute/subacute/chronic phases or states. The acute phase (30-60 min after injection) was anabolic: coordinated hyperphagia and hypometabolism/hypothermia. NPY evoked a hypothermia by suppressing any (hyper)metabolism in excess of basal metabolic rate, without enhancing heat loss. Thus, acute hypothermia was observed in sub-thermoneutral but not thermoneutral environments. The subsequent subacute catabolic phase exhibited opposite effects: slight increase in metabolic rate, rise in body temperature, reaching a plateau within 3-4 h after injection -- this was maintained for at least 24 h; meanwhile the food intake decreased and the normal daily weight gain stopped. This rebound is only indirectly related to NPY. Chronic (7-day long) i.c.v. NPY infusion induced an anabolic phase for 2-3 days, followed by a catabolic phase and fever, despite continued infusion. In cold-adaptation environment the primary metabolic effect of the infusion induced a moderate hypothermia with lower daytime nadirs and nocturnal peaks of the circadian temperature rhythm, while at near-thermoneutral environments in non-adapted rats the infusion attenuated only the nocturnal temperature rise by suppressing night-time hypermetabolism. Further finding is that in cold-adapted animals, the early feeding effect of NPY-infusion was enhanced, whereas the early hypothermic effect in cold was limited by interference with competing thermoregulatory mechanisms.     

Galanin/GALP receptors and CNS homeostatic processes

Galanin is a 29/30 amino acid peptide neurotransmitter that is widely distributed throughout the central nervous system and periphery. There are three well-characterized G-protein coupled galanin receptors subtypes (GalR1-3). A more recently discovered 60 amino acid galanin-like peptide (GALP) shares amino acid sequence homology with galanin (1-13) in position 9-21 and has high binding affinity for GalR1-3, with highest affinity for GalR3. Considerable evidence has accumulated that implicates both galanin and GALP as playing important roles in regulating food and water intake behavior and related neuroendocrine functions. Pharmacological tools are emerging that will allow dissociation of specific roles for the peptides and their associated receptor subtypes in mediating the homeostatic processes of energy and fluid balance.     

The role of galanin in feeding behavior

Galanin inhibits food consumption in satiated rats. Discovered relatively recently, galanin is a 29-amino-acid neuropeptide, not homologous with any other known peptide. Three G-protein-linked galanin receptor subtypes have been cloned. This review summarizes the mechanisms by which exogenously administered galanin may stimulate ingestion, discusses pharmacological and genetic investigations of the role of endogenous galanin on feeding and body weight, and speculates on the therapeutic potential of non-peptide galanin receptor antagonists for the treatment of appetite disorders.     

Regulation by orexin of feeding behaviour and locomotor activity in the goldfish

Orexin is a hypothalamic neuropeptide that is implicated in the regulation of feeding behaviour and the sleep-wakefulness cycle in mammals. However, in spite of a growing body of knowledge concerning orexin in mammals, the orexin system and its function have not been well studied in lower vertebrates. In the present study, we first examined the effect of feeding status on the orexin-like immunoreactivity (orexin-LI) and the expression of orexin mRNA in the goldfish brain. The number of cells showing orexin-LI in the hypothalamus of goldfish brain showed a significant increase in fasted fish and a significant decrease in glucose-injected fish. The expression level of orexin mRNA in the brains of fasted fish increased compared to that of fed fish. We also examined the effect of an i.c.v. injection of orexin or an anti-orexin serum on food intake and locomotor activity in the goldfish. Administration of orexin by i.c.v. injection induced a significant increase of food intake and locomotor activity, whereas i.p. injection of glucose or i.c.v. injection of anti-orexin serum decreased food consumption. These results indicate that the orexin functions as an orexigenic factor in the goldfish brain.     

Injections of Galanin-Like Peptide directly into the nucleus of the tractus solitarius (NTS) reduces food intake and body weight but increases metabolic rate and plasma leptin

Galanin-Like Peptide (GALP) is a hypothalamic neuromediator of metabolism and reproduction. GALP is known to stimulate reproduction and alter food intake and body weight in multiple species. The regulation of body weight involves control of both energy intake and energy expenditure. Since GALP is known to alter food intake - possibly via the autonomic nervous system - we first hypothesized that GALP would increase metabolic rate. First, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannulae and abdominal radiotelemetry temperature transmitters. Following ICV injection with either 5 nmol GALP or vehicle, the oxygen consumption of each rat was monitored for 8 h. Food intake, core temperature, and general motor activity were monitored for 24 h. GALP significantly increased oxygen consumption, an indirect estimator of metabolic rate, without having any significant effect on motor activity. Compared to controls, GALP increased core body temperature during the photophase and reduced food intake over the 24 h period following injection. ICV GALP also increased plasma levels of luteinizing hormone (LH). A second group of male Sprague-Dawley rats were implanted with abdominal transmitters and given injections of GALP directly into the nucleus of the tractus solitarius (NTS). These injections resulted in a significant reduction in food intake, and a significant increase in both oxygen consumption and core body temperature compared to vehicle injections. Direct injections of GALP into the NTS compared to vehicle also resulted in a significant increase in plasma leptin levels, but not LH levels. GALP appears to increase energy expenditure in addition to decreasing energy input by actions within the NTS and thus may play an important role in the hypothalamic regulation of body weight.     

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.     

Galanin-like peptide promotes feeding behaviour via activation of orexinergic neurones in the rat lateral hypothalamus

Galanin-like peptide (GALP) is produced in neurones in the hypothalamic arcuate nucleus and is implicated in the neural control of feeding behaviour. Previously, we have reported that GALP immunoreactive fibres were in direct contact with orexin/hypocretin immunoreactive neurones in the rat lateral hypothalamus using double-immunofluorescence. Centrally administered GALP is known to stimulate feeding behaviour. However, the target neurones of this action have not been clarified. The present study aimed to determine features of the GALP-mediated neuronal feeding pathway in rat. Accordingly, at the ultrastructural level, GALP-immunoreactive axon terminals were found to make synapses on orexin/hypocretin immunoreactive cell bodies and dendritic processes in the lateral hypothalamus. c-Fos immunoreactivity was expressed in orexin/hypocretin-immunoreactive neurones but not in melanin concentrating hormone-immunoreactive neurones in the lateral hypothalamus at 90 min after the application of GALP by i.c.v. infusion. Furthermore, to determine whether GALP regulates feeding behaviour via orexin/hypocretin neurones, the feeding behaviour of rats was studied following GALP i.c.v. injection with or without anti-orexin A and B immunoglobulin (IgG) pretreatment. The anti-orexin IgGs markedly inhibited GALP-induced hyperphagia. These results suggest that orexin/hypocretin-containing neurones in the lateral hypothalamus are targeted by GALP, and that GALP-induced hyperphagia is mediated via orexin/hypocretin neurones in the rat hypothalamus.     

Anorexia is Associated with Stress‐Dependent Orexigenic Responses to Exogenous Neuropeptide Y

Chicken lines that have been divergently selected for either low (LWS) or high (HWS) body weight at 56 days of age for more than 57 generations have different feeding behaviours in response to a range of i.c.v. injected neurotransmitters. The LWS have different severities of anorexia, whereas the HWS become obese. Previously, we demonstrated that LWS chicks did not respond, whereas HWS chicks increased food intake, after central injection of neuropeptide Y (NPY). The present study aimed to determine the molecular mechanisms underlying the loss of orexigenic function of NPY in LWS. Chicks were divided into four groups: stressed LWS and HWS on day of hatch, and control LWS and HWS. The stressor was a combination of food deprivation and cold exposure. On day 5 post‐hatch, each chick received an i.c.v. injection of vehicle or 0.2 nmol of NPY. Only the LWS stressed group did not increase food intake in response to i.c.v. NPY. Hypothalamic mRNA abundance of appetite‐associated factors was measured at 1 h post‐injection. Interactions of genetic line, stress and NPY treatment were observed for the mRNA abundance of agouti‐related peptide (AgRP) and synaptotagmin 1 (SYT1). Intracerebroventricular injection of NPY decreased and increased AgRP and SYT1 mRNA, respectively, in the stressed LWS and increased AgRP mRNA in stressed HWS chicks. Stress was associated with increased NPY, orexin receptor 2, corticotrophin‐releasing factor receptor 1, melanocortin receptor 3 (MC3R) and growth hormone secretagogue receptor expression. In conclusion, the loss of responsiveness to exogenous NPY in stressed LWS chicks may be a result of the decreased and increased hypothalamic expression of AgRP and MC3R, respectively. This may induce an intensification of anorexigenic melanocortin signalling pathways in LWS chicks that block the orexigenic effect of exogenous NPY. These results provide insights onto the anorexic condition across species, and especially for forms of inducible anorexia such as human anorexia nervosa.     

Targeted inactivation of the neurotensin type 1 receptor reveals its role in body temperature control and feeding behavior but not in analgesia

Three subtypes of neurotensin receptor have been described, two members of the heptahelical transmembrane domain G protein-coupled receptor superfamily NT-1R and NT-2R, and NT-3R unrelated to this family. We have generated NT-1R deficient (NT-1R(-/-)) mice. NT-1R(-/-) mice were born at the expected Mendelian frequency without obvious abnormalities and they were fertile. The NT-induced analgesia on the writhing induced by phenyl-p-benzoquinone administration remained at wild-type levels in the NT-1R(-/-) mice demonstrating that the NT-1R is not implicated in the analgesic effect of NT in this test. The NT-1R(-/-) mice were hyperthermic; their body temperature was not affected by intracerebroventricular (i.c.v.) administration of NT, contrasting with the hypothermia induced in NT-1R(+/+) mice. NT-1R(-/-) mice showed a small significant increase in body weight compared to the NT-1R(+/+) congeners as early as 10 weeks after birth, correlated with a higher food intake. NT-1R(-/-) mice showed similar spontaneous locomotion to the control littermates, but did not respond to i.c.v. NT-induced hypolocomotion. I.c.v. injection of NT inhibited feeding in fasted wild-type mice, but had no effect on feeding of the NT-1R(-/-) mice. I.c.v. administration of the orexigenic neuropeptide Y (NPY) stimulated feeding to the same extent in both wild-type and NT-1R(-/-) mice. This analysis of NT-1R-deficient mice shows that the NT-1R does not play a role in NT-induced analgesia, but that it is clearly implicated in thermal and feeding regulation, weight control, and NT-induced hypolocomotion.     

Galanin-like peptide (GALP) is a hypothalamic regulator of energy homeostasis and reproduction

Galanin-like peptide (GALP) was discovered in 1999 in the porcine hypothalamus and was found to be a 60 amino acid neuropeptide. GALP shares sequence homology to galanin (1-13) in position 9-21 and can bind to, as well as activate, the three galanin receptor subtypes (GalR1-3). GALP-expressing cells are limited, and are mainly found in the arcuate nucleus of the hypothalamus (ARC) and the posterior pituitary. GALP-positive neurons in the ARC project to several brain regions where they appear to make contact with multiple neuromodulators. These neuromodulators are involved in the regulation of energy homeostasis and reproduction, anatomical evidence that suggests a role for GALP in these physiological functions. In support of this idea, GALP gene expression is regulated by several factors that reflect metabolic state including the metabolic hormones leptin and insulin, thyroid hormones, and blood glucose. Considerable evidence now exists to support the hypothesis that GALP has a role in the regulation of energy homeostasis and reproduction; and, that GALP’s role may be independent of the known galanin receptors. In this review, we (1) provide an overview of the distribution of GALP, and discuss the potential relationship between GALP and other neuromodulators of energy homeostasis and reproduction, (2) discuss the metabolic factors that regulate GALP expression, (3) review the evidence for the role of GALP in energy homeostasis and reproduction, (4) discuss the potential downstream mediators and mechanisms underlying GALP’s effects, and (5) discuss the possibility that GALP may mediate its effects via an as yet unidentified GALP-specific receptor.