MINIREVIEW Galanin-like peptide and the regulation of feeding behavior and energy metabolism Kanako Shiba 1 , Haruaki Kageyama 1 , Fumiko Takenoya 1,2 and Seiji Shioda 1 1 Department of Anatomy, Showa University School of Medicine, Tokyo, Japan 2 Department of Physical Education, Hoshi University School of Pharmacy and Pharmaceutical Science, Tokyo, Japan Introduction Neuropeptides of G protein-coupled receptor (GPCR) ligands are shown to perform a range of physiological functions. Subsequent to the discovery of leptin [1] and ghrelin [2], a number of studies have demonstrated structural and functional characters of appetite-regu- lating neuropeptides, such as orexin, melanin-concen- trating hormone (MCH), neuropeptide Y (NPY), a-melanocyte stimulating hormone (a-MSH) derived from pro-opiomelanocortin (POMC) [3], neuropeptide W [4], relaxin-3 [5] and prolactin-releasing peptide [6]. Galanin is a 29 amino acid peptide that was dis- covered by the detection of its C-terminal amide sequence in porcine intestinal extract in 1983 [7]. The galanin receptors (GALRs) belong to one of the GPCR families and have three known subtypes: GALR1, GALR2 and GALR3. Sixteen years after the discovery of galanin, a galanin-like peptide (GALP) that consists of 60 amino acids was isolated from porcine hypothalamus using a binding assay for GALRs [8]. The 9–21 amino acid sequence of GALP is identical to that of the first 13 amino acids of gala- nin (Fig. 1). However, galanin and GALP are encoded by separate genes that are typically located on separate chromosomes: the GALP gene is located Keywords clinical implication; feeding regulation; galanin; GPCRs leptin; mouse; neuronal network; obesity; rat; thermogenesis Correspondence S. Shioda, Department of Anatomy, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142- 8555, Japan Fax: +81 3 3784 6815 Tel: +81 3 3784 8103 E-mail: shioda@med.showa-u.ac.jp (Received 14 June 2010, revised 5 September 2010, accepted 12 October 2010) doi:10.1111/j.1742-4658.2010.07933.x The hypothalamic neuropeptides modulate physiological activity via G pro- tein-coupled receptors (GPCRs). Galanin-like peptide (GALP) is a 60 amino acid neuropeptide that was originally isolated from porcine hypo- thalamus using a binding assay for galanin receptors, which belong to the GPCR family. GALP is mainly produced in neurons in the hypothalamic arcuate nucleus. GALP-containing neurons form neuronal networks with several other types of peptide-containing neurons and then regulate feeding behavior and energy metabolism. In rats, the central injection of GALP produces a dichotomous action that involves transient hyperphasia fol- lowed by hypophasia and a reduction in body weight, whereas, in mice, it has only one action that reduces both food intake and body weight. In the present minireview, we discuss current evidence regarding the function of GALP, particularly in relation to feeding and energy metabolism. We also examine the effects of GALP activity on food intake, body weight and locomotor activity after intranasal infusion, a clinically viable mode of delivery. We conclude that GALP may be of therapeutic value for obesity and life-style-related diseases in the near future. Abbreviations ARC, arcuate nuclei; a-MSH, a-melanocyte stimulating hormone; DMH, dorsomedial hypothalamus; GALP, galanin-like peptide; GALR, galanin receptor; GPCR, G protein-coupled receptors; IL-1, interleukin-1; LH, lateral hypothalamus; MCH, melanin-concentrating hormone; MPA, medial preoptic area; NPY, neuropeptide Y; NTS, nucleus tractus solitarii; POA, preoptic area; POMC, pro-opiomelanocortin; PVN, paraventricular nuclei; SON, supraoptic nuclei; VMH, ventromedial hypothalamic nuclei. 5006 FEBS Journal 277 (2010) 5006–5013 ª 2010 The Authors Journal compilation ª 2010 FEBS on chromosome 7, whereas the galanin gene is on chromosome 19 in mice. The primary structures of both rat and human GALP have been deduced from the corresponding cDNA. Mature GALP is cleaved from the precursor protein preproGALP, which consists of 115–120 amino acids depending on the species. The 1–24 and 41–53 amino acid sequences of GALP are highly conserved between mice [9], rats [8], pigs [8], monkeys [10] and humans [8]. Ligand binding assays and functional studies show that the human GALP (3–32) fragment is at least as potent as mature GALP [11], whereas neither GALP (1–21), nor GALP (22–60) has any discernible effect on the feeding response in mice [12]. This suggests that the putative fragment GALP (3–32) might represent the strongest mediator of the peptide’s biological activity. GALP is involved in feeding behavior and energy metabolism via neuronal circuits formed with sev- eral types of appetite-regulating peptide-containing neurons. The present minireview summarizes the neu- ronal network involving GALP in the hypothalamus where the appetite regulation centers are located, and discusses the physiological actions of this peptide, par- ticularly in relation to feeding and energy metabolism. We also consider the therapeutic value of the intrana- sal administration of GALP. In addition, this review will provide an overview of a novel peptide, alarin, generated by alternative splicing of the GALP gene. GALP receptors Receptor binding studies using membranes from the Chinese hamster ovary cells transfectants expressing rat GALR1 and rat GALR2 initially reveal that the binding affinity of galanin for GALR1 is IC 50 = 0.097 nm and, for GALR2, is IC 50 = 0.48 nm [8]. By contrast, porcine mature GALP has a higher affinity for the receptor GALR2 (IC 50 = 0.24 nm) than for GALR1 (IC 50 = 4.3 nm ) [8]. The latest studies on the Fig. 1. The primary structure and gene structure of galanin and GALP in several species. Black shaded characters indicate the amino acid sequences that are common to galanin and GALP. Galanin and GALP are encoded by separate genes that are typically located on separate chromosomes: the GALP gene is located on chromosome 7, whereas the galanin gene is on chromosome 19 in mice. Galanin: the first exon encodes the 5¢-untranslated region of preprogalanin. Cording region of galanin is present on exons 2–4. Galanin message-associated peptide is encoded on exons 4–6 [48]. GALP: the first exon is untranslated region. The preproGALP is encoded by exons 2–6. Amino acid is repre- sented by one letter code. EX, exon. K. Shiba et al. GALP in feeding and energy metabolism FEBS Journal 277 (2010) 5006–5013 ª 2010 The Authors Journal compilation ª 2010 FEBS 5007 binding affinity of GALP for GALRs have demon- strated, using human neuroblastoma cells expressing all three human GALRs, that GALR3 binds GALP with the highest affinity, with the order of binding potency of the GALRs for GALP being GALR3 (IC 50 =10nm), GALR2 (IC 50 =28nm) and GALR1 (IC 50 =77nm) [11]. In situ hybridization mapping studies have shown that the three galanin receptor transcripts are present throughout the hypothalamus. High levels of expression of GALR1 are found in the medial preoptic area (MPA), paraventricular nuclei (PVN) and supraoptic nuclei (SON) [13]. GALR2 is expressed in the preoptic area (POA), arcuate nuclei (ARC), dorsomedial hypothalamus (DMH), PVN, periventricular suprachiasmatic and mammillary nuclei [14]. GALR3 expression is confined to the PVN, DMH and ventromedial hypothalamic nuclei (VMH) [15]. GALP reduces food intake and body weight in both GALR1 and GALR2 knockout mice, similar to the situation in wild-type mice [12]. It is therefore pos- sible that GALR3 mediates feeding behavior. How- ever, the central administration of a GALR2 ⁄ 3 agonist had no effect on food intake, body weight and body temperature in rodents [16]. In addition, other studies have used quantitative analysis of c-Fos immunoreac- tivity to show that, although galanin induces a signifi- cantly greater number of c-Fos-positive nuclei in the PVN compared to GALP, GALP induces significantly more c-Fos-positive cells in the horizontal limb of the diagonal band of Broca, caudal POA, ARC and med- ian eminence [17]. These results suggest that GALP and galanin act through different receptor-mediated pathways to exert their effects on the regulation of feeding. In other words, it is possible that GALP mediates its effect via a yet-to-be-identified GALP receptor. In 2006, the novel 25 amino acid peptide, alarin, was discovered as an alternate transcript of the GALP gene [18–20]. Recently, it was shown that intracerebro- ventricular injection of alarin increased food intake and body weight [21]. Alarin immunoreactive cell bodies are detected within the locus coeruleus and locus subcoeruleus of the midbrain [21]. Alarin stimu- lates Fos induction in the hypothalamic nuclei, such as the PVN and nucleus tractus solitarii (NTS) [21]. Because alarin does not share any homology to gala- nin, alarin is most unlikely to activate GALR [19,21]. In alarin, the signal sequence of the GALP precursor peptide and the first five amino acids of the mature GALP are followed by 20 amino acids without homol- ogy to any other murine protein [19]. These studies suggest that alarin is a neuromediator of food intake and body weight via a specific receptor for alarin. Regulation of GALP mRNA expression GALP mRNA gradually increases between postnatal days 8 and 14, and markedly increases between days 14 and 40, which represent the weaning and pubertal periods in rats [22]. These findings suggest that GALP may be associated with developmental changes such as weaning, feeding and maturation of reproductive function. Fasting decreases both the number of GALP- expressing neurons [23] and the expression of GALP mRNA [24]. Leptin administration restores the number of GALP-expressing cells in fasted rats [23] and leptin- deficient ob ⁄ ob mice [9], with the expression levels of GALP mRNA being reduced in the hypothalamus of leptin receptor-deficient Zucker obese rats, and db ⁄ db and ob ⁄ ob obese mice [25]. These findings clearly show that leptin positively regulates activity of GALP neurons in the hypothalamus. Furthermore, streptozo- tocin-induced diabetic rats are associated with a signifi- cant reduction in the expression of GALP mRNA, which is reversed by treatment with either insulin or leptin [26]. This suggests that GALP-expressing neu- rons are direct regulatory targets not only for leptin, but also for insulin. Neuronal networks involving GALP- containing neurons Galanin is broadly distributed in the brain [27], whereas GALP-immunoreactive neuronal cell bodies are located in the hypothalamic ARC, being particu- larly dense in the medial posterior section of the nucleus [28]. In the rat brain, GALP mRNA is expressed only in the ARC [23,29,30], with GALP- positive fibers projecting from this nucleus to several other hypothalamic nuclei, including the PVN, lateral septal nucleus, bed nucleus of the stria terminalis and MPA [28], as well as to the lateral hypothalamus (LH) around the fornix [31]. On the basis of these results, at least two major neural pathways involving GALP have been proposed: one in which GALP- containing neurons project from the ARC to the PVN, and the other in which they project to the MPA, bed nucleus of the stria terminalis and lateral septal nucleus. Central administration of GALP activates neurons in various regions of the rat brain. Injection of GALP into the third ventricle induces c-Fos expression, a marker of cell activation, in the horizontal limb of the diagonal band of Broca, POA, ARC and median emi- nence [17], whereas injection into the lateral ventricle activates several brain regions, including the DMH, GALP in feeding and energy metabolism K. Shiba et al. 5008 FEBS Journal 277 (2010) 5006–5013 ª 2010 The Authors Journal compilation ª 2010 FEBS LH, NTS of the brainstem, PVN and SON [32]. In mice, intracerebroventricular injection of GALP into the lateral ventricle induces c-Fos expression in the parenchyma surrounding the ventricles, the ventric- ular ependymal cells and the meninges, but not in the SON, DMH, LH and NTS [33], highlighting the exis- tence of species-specific differences between rats and mice. Additional work is therefore required to clarify the link between GALP-induced c-Fos expression and the neural circuitry involving GALP-containing neurons. Neuropeptides are divided into two groups: orexi- genic peptides, including orexin, MCH and NPY, and anorexigenic peptides, including a-MSH derived from POMC [3]. GALP neurons in the ARC are innervated by orex- inergic neurons in the LH and NPY-expressing neu- rons in the ARC. Nine percent of GALP-positive neurons express orexin-1 receptor [34]. GALP-positive neurons have also been shown to express NPY Y1 receptor by double-label in situ hybridization [35], with NPY- and orexin-containing fibers lying in close appo- sition with GALP-containing neurons in the ARC [34,36]. In addition, more than 85% of GALP-contain- ing neurons express the leptin receptor [28]. However, the GALP-containing neurons in the ARC are reported to be different from the leptin receptor- expressing neurons that express NPY ⁄ agouti-related protein and galanin [30,34,36,37]. Taken together, these morphological studies suggest that GALP-con- taining neurons are regulated by both orexigenic and anorexigenic signals. With regard to the targets of GALP-containing neu- rons in rats, morphological studies have shown that GALP-like-immunoreactive nerve fibers make direct contact with orexin- and MCH-like-immunoreactive neurons in the LH [31]. At the ultrastructural level, GALP-immunoreactive axon terminals have been found to make synapses on orexin-immunoreactive cell bodies and dendritic processes in the LH [38]. We have previously reported that 3–12% of GALP-positive neu- rons in the ARC also express a-MSH derived from POMC [36]. These observations suggest that GALP- containing neurons introduce feeding and ⁄ or satiety signals. In addition, we have found that GALP-posi- tive nerve fibers appear to make direct contact with tyrosine hydroxylase-containing neurons in the ARC [39], suggesting that GALP may interact with dopami- nergic neurons in this region. GALP-positive neurons have been shown to form circuits involving many neu- rons. Although galanin is co-expressed with a number of transmitters (monoamines and amino acids) and dif- ferent peptides in neurons in various brain regions [40], it is yet to be reported that GALP-neurons express other neuropeptides or transmitters except a- MSH in the ARC, indicating that GALP-expressing neurons are unique. A schematic diagram summarizing the hypothalamic neuronal networks involved in feeding regulation is presented in Fig. 2. GALP-positive neurons are affected by leptin, which conveys satiety signals from the peripheral tissues, NPY and orexin. GALP regu- lates both orexigenic (NPY and ⁄ or orexin) and anorex- igenic (POMC) pathways in the central nervous system. POMC MCH NPY Orexin Leptin Leptin adipose tissue adipose tissue 3V DA LH VMH ARC NPY DMH GALP Fig. 2. Distribution of GALP-producing neurons in the hypothala- mus. GALP-induced hyperphagia is mediated via activation of orexin neurons in the LH and NPY neurons in the DMH. GALP nerve fibers make direct contact with MCH neurons in the LH and tyro- sine hydroxylase-containing neurons in the ARC, although their physiological actions are uncertain. GALP neurons in the ARC are innervated by orexin neurons in the LH and NPY neurons in the ARC, although their physiological actions are uncertain. More than 85% of GALP neurons express the leptin receptor. Leptin positively regulates the activity of GALP neurons in the hypothalamus. GALP neurons in the ARC also express a-MSH derived from POMC. 3V, third cerebroventricle; DA, dopamine. Red arrows indicate stimula- tory effects. Blue arrows indicate an uncertain function. K. Shiba et al. GALP in feeding and energy metabolism FEBS Journal 277 (2010) 5006–5013 ª 2010 The Authors Journal compilation ª 2010 FEBS 5009 Effect of GALP on feeding behavior and energy metabolism Galanin and biologically active fragments such as gala- nin (1–16) stimulate food intake after acute microinjec- tion into the PVN, LH, VMH and the central nucleus of the amigdala, producing a rapid increase in the feeding response and total caloric intake without alter- ing feeding-associated behaviors such as drinking, grooming and motor activity [20], whereas GALP has complex actions on feeding behavior and energy bal- ance. Intracerebroventricular injection of GALP signif- icantly stimulates feeding during the first hour in rats [32,41], whereas it inhibits food intake in mice [42]. The physiological significance of this behavioral differ- ence between the rats and mice remains unclear, although it may be a result of species differences in neuronal circuitry. In rats, three pathways have been demonstrated to mediate the orexigenic effect of GALP: one via orexin- ergic neurons in the LH; one via NPY-expressing neu- rons in the DMH; and the third via POMC-expressing neurons in the ARC. c-Fos immunoreactivity is increased in orexin-immunoreactive neurons but not in MCH-immunoreactive neurons in the LH after intra- cerebroventricular injection of GALP [38]. Further- more, anti-orexin IgG markedly inhibits GALP- induced hyperphagia [38]. These results suggest that orexin-containing neurons in the LH are targeted by GALP, and that GALP-induced hyperphagia is medi- ated via orexinergic neurons in the rat hypothalamus. In addition, GALP focally injected into the DMH stimulates food intake for 2 h after injection [43]. Intracerebroventricular injection of GALP induces c-Fos expression in NPY-containing neurons in the DMH. GALP also increases the cytosolic calcium con- centration in NPY-immunoreactive neurons isolated from the DMN. Furthermore, both anti-NPY IgG and NPY antagonists, when preinjected, counteract the feeding induced by GALP administration. In an in vi- tro study of GALP-treated rat hypothalamic explants, it was suggested that GALP-induced hyperphagica could be mediated by an increase in NPY release [44]. These results reveal that GALP mediates a potent short-term stimulation of food intake via activation of NPY-containing neurons in the DMN. Moreover, in vivo, the number of POMC mRNA-expressing cells in the ARC of the ob ⁄ ob mouse is reduced after chronic GALP injection [45]. These findings suggest that GALP also promotes feeding behavior through suppression of the anorexigenic POMC system. GALP also increases food intake when injected into the POA or PVN [46]. Although it is possible that the POA and the PVN have specific roles in mediating the orexigenic effect of GALP, the subpopulations of neu- rons in these regions that mediate GALP-induced overeating remain unknown. Long-term continuous treatment with GALP causes only transient reductions in both food intake and body weight in wild-type mice, leading to the conclu- sion that these animals become insensitive to contin- ued exposure to GALP [17,42]. However, in the ob ⁄ ob mouse, chronic GALP administration results in a sus- tained decrease in body weight, despite a significant recovery in food intake [42,45]. This suggests that GALP promotes ongoing energy expenditure under leptin-deficient conditions. Indeed, GALP promotes thermogenesis, with intracerebroventricular injection of GALP being shown to cause a dose-dependent increase in core body temperature, which lasts for 6–8 h after injection. GALP-induced thermogenesis is attenuated by peripheral administration of the cyclo- oxygenase inhibitor, flurbiprofen, suggesting a depen- dence on the actions of prostaglandins [47]. Astrocytes produce prostaglandins and have been implicated in thermogenesis in the brain, with an immunohistochem- ical study revealing that GALP induces c-Fos expres- sion in astrocytes but not in microglia [32]. These findings suggest that GALP mediates the production of fever via the prostaglandin pathway in the brain. Recent data also suggest that GALP induces the expression of interleukin-1 (IL-1) in the brain, and that its anorexic and febrile actions are mediated by this cytokine acting via the IL-1 type I receptor [48]. This indicates that IL-1 is a key mediator of inflam- mation that acts to induce fever via the release of prostaglandins in response to GALP in the hypothala- mus. Brown adipose tissue innervated and activated by the sympathetic nervous system plays an important role in the regulation of thermogenesis. Repeated treatment with GALP has been shown to increase both mRNA and protein expression of uncoupling protein-1, a key thermogenic molecule, in the brown adipose tissue of the ob ⁄ ob mouse [45]. These findings suggest that GALP may partly mediate energy metab- olism through thermogenesis by long-term activation of the sympathetic nervous system. Therefore, both prostaglandins in the brain and uncoupling protein-1 in peripheral tissue are involved in GALP-induced thermogenesis. Although GALP is also present in blood [49], the production of GALP in the peripheral organs remains to be elucidated. Further studies are required to deter- mine the link between the brain and peripheral tissues involved in the regulation of feeding and energy metabolism by GALP. GALP in feeding and energy metabolism K. Shiba et al. 5010 FEBS Journal 277 (2010) 5006–5013 ª 2010 The Authors Journal compilation ª 2010 FEBS Overall, these findings suggest that acute hyperpha- gia mediated by GALP occurs via the activation of orexin- and ⁄ or NPY-expressing neurons, and that long-term body weight loss is a result of the promotion of energy expenditure. Clinical implications To determine the potential clinical efficacy of GALP, we investigated its intranasal delivery into the brain. Recently, we have reported that the uptake by the whole brain, olfactory bulb and cerebrospinal fluid after intranasal administration is greater than that after intravenous injection [50]. These findings indicate that intranasal administration is an effective route of delivery of GALP to the brain. We also studied the effect of intranasal infusion of GALP on feeding behavior in mice (K. Shiba, H. Kageyama, N. Non- aka, F. Takenoya and S. Shioda, unpublished data). Intranasal infusion of GALP significantly reduced body weight over the course of 1 week. These results suggest that intranasal administration of GALP repre- sents a viable option for obese people who seek to combat obesity and similar life-style-related diseases. Conclusions GALP is mainly produced in the hypothalamic ARC, and plays important roles in the regulation of feeding behavior and energy metabolism through complicated neuronal networks. The central administration of GALP produces a short-term increase (followed by a subsequent decrease) in food intake in rats, whereas it produces only a decrease in mice. GALP also reduces body weight and stimulates thermogenesis in rodents. The short-term orexigenic actions of GALP are mediated via NPY and the orexinergic pathway in the rat. The long-term ano- rectic and thermogenic actions of GALP are mediated via the pro-inflammatory pathway in rodents. The iden- tification of a specific receptor for GALP is of consider- able importance if the physiological functions and mechanism of action of GALP are to be fully under- stood. Little is known about the role of alarin, which was discovered as an alternate transcript of the GALP gene. Further elucidation of the function of GALP and alarin will provide the necessary basis for the treatment and prevention of obesity and related disorders. Acknowledgements The authors thank Dr Tetsuya Ohtaki from Takeda Pharmaceutical Company. The present work was sup- ported in part by the High-Technology Research Cen- ter Project from the Ministry of Education, Sports, Science and Technology and by grant-in-Aid for Exploratory Research (#21659059). References 1 Friedman JM & Halaas JL (1998) Leptin and the regula- tion of body weight in mammals. 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Neuroendocrinology 74, 423–432. 50 Nonaka N, Farr SA, Kageyama H, Shioda S & Banks WA (2008) Delivery of galanin-like peptide to the brain: targeting with intranasal delivery and cyclodextrins. J Pharmacol Exp Ther 325, 513–519. K. Shiba et al. GALP in feeding and energy metabolism FEBS Journal 277 (2010) 5006–5013 ª 2010 The Authors Journal compilation ª 2010 FEBS 5013 . MINIREVIEW Galanin-like peptide and the regulation of feeding behavior and energy metabolism Kanako Shiba 1 , Haruaki Kageyama 1 , Fumiko Takenoya 1,2 and Seiji Shioda 1 1 Department of Anatomy,. in feeding and energy metabolism FEBS Journal 277 (2010) 5006–5013 ª 2010 The Authors Journal compilation ª 2010 FEBS 5009 Effect of GALP on feeding behavior and energy metabolism Galanin and. neurons project from the ARC to the PVN, and the other in which they project to the MPA, bed nucleus of the stria terminalis and lateral septal nucleus. Central administration of GALP activates