Báo cáo khoa học: Galanin-like peptide and the regulation of feeding behavior and energy metabolism pptx

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].. How

Galanin-like peptide and the regulation of feeding

behavior and energy metabolism

Kanako Shiba1, Haruaki Kageyama1, Fumiko Takenoya1,2and Seiji Shioda1

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.

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 IC50= 0.097 nm and, for GALR2, is IC50= 0.48 nm [8] By contrast, porcine mature GALP has a higher affinity for the receptor GALR2 (IC50= 0.24 nm) than for GALR1 (IC50= 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.

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

(IC50= 10 nm), GALR2 (IC50= 28 nm) and GALR1

(IC50= 77 nm) [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,

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 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.

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 feedalter-ing-associated behaviors such as drinkalter-ing,

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

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)

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