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Roles of prolactin-releasing peptide and RFamide related peptides in the control of stress and food intake Yuki Takayanagi and Tatsushi Onaka Division of Brain and Neurophysiology, Depar

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Roles of prolactin-releasing peptide and RFamide related peptides in the control of stress and food intake

Yuki Takayanagi and Tatsushi Onaka

Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Japan

Introduction

RFamide peptides, defined by their carboxy-terminal

arginine (R) and amidated phenylalanine (F) residues

(hence RFamide), were originally discovered in

inbrates [1] and have recently been identified in

verte-brates [2] The first reported RFamide peptides in

mammals were neuropeptide FF (NPFF) and

neuro-peptide AF, which were later confirmed to be encoded

on a single gene [3] By applying a reverse

pharmaco-logical approach, in which orphan G protein-coupled

receptor ligands were identified by detecting signal

transduction induced in cells expressing a targeted

orphan G protein-coupled receptor after stimulation,

prolactin-releasing peptide (PrRP) was identified to be

a ligand of an orphan G protein-coupled receptor,

GPR10 (hGR3⁄ UHR-1 ⁄ PRLHR) and to belong to the RFamide peptide [4] Subsequently, by utilizing DNA databases, another gene for RFamide peptides was identified in mammals [5,6] The RFamide peptides encoded by the gene were named RFamide related peptide (RFRP)-1⁄ NPSF and RFRP-3 ⁄ NPVF, which were found to be orthologs of avian peptide LPLRFa-mide Thus, RFRPs are allocated into the LPXRFa-mide peptide family (X = L or Q) Several other RFamide peptides also have been discovered by a reverse pharmacological method and by DNA sequence database searching, and there are now five families of RFamide peptides known to exist in mammals: NPFF, PrRP, LPXRFamide, kisspeptin

Keywords

dorsomedial hypothalamus; energy

consumption; energy metabolism; food

intake; nucleus tractus solitarii; oxytocin;

prolactin-releasing peptide; RFamide

peptide; RFamide-related peptide; stress

Correspondence

T Onaka, Division of Brain and

Neurophysiology, Department of Physiology,

Jichi Medical University, Shimotsuke-shi,

Tochigi-ken 329-0498, Japan

Fax: +81 285 44 8147

Tel: +81 285 58 7318

E-mail: tonaka@jichi.ac.jp

(Received 13 June 2010, revised 2

September 2010, accepted 13 October

2010)

doi:10.1111/j.1742-4658.2010.07932.x

Subsequent to the isolation of the first recognized RFamide neuropeptide, FMRFamide, from the clam, a large number of these peptides have been identified There are now five groups of RFamide peptides identified in mammals RFamide peptides show diversity with respect to their N-termi-nal sequence and biological activity RFamide peptides have been impli-cated in a variety of roles, including energy metabolism, stress and pain modulation, as well as effects in the neuroendocrine and cardiovascular systems In the present minireview, we focus on prolactin-releasing peptide (PrRP) and RFamide related peptide (RFRP) with respect to their roles in the control of energy metabolism and stress responses Both food intake and stressful stimuli activate PrRP neurons The administration of PrRP affects energy metabolism and neuroendocrine systems PrRP-deficient or PrRP receptor-deficient mice show abnormal energy metabolism and⁄ or stress responses On the other hand, RFRP neurons are activated by stress-ful stimuli and the administration of RFRP induces neuroendocrine and behavioral stress responses Taken together, these data suggests that PrRP and RFRP neurons play a role in the control of energy metabolism and/or stress responses

Abbreviations

ACTH, adrenocorticotropic hormone; CCK, cholecystokinin-8; CRH, corticotropin-releasing hormone; NPFF, neuropeptide FF;

NTS, nucleus tractus solitarii; PrRP, prolactin-releasing peptide; QFRP, pyroglutamylated RFamide peptide; RFRP, RFamide related peptide.

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(previously known as metastin) and pyroglutamylated

RFamide peptide (QFRP) (Table 1) RFamide peptides

show diversity in their N-terminal sequence and, as a

result, a broad pattern of biological activities,

includ-ing the control of energy metabolism and stress, as

well as effects in the neuroendocrine and

cardiovascu-lar systems In the present minireview, we focus on

PrRP and RFRP (Table 2) and review recent progress

in research investigating the roles of these two peptides

in the control of energy metabolism and stress

PrRP

PrRP was considered to serve as a

hypothalamic-releasing factor and to act on the anterior pituitary

to stimulate prolactin release from the pituitary

However, no PrRP immunoreactivity was found in the external layer of the median eminence, from where classic hypothalamic hormones are released into the portal blood to control anterior pituitary hormone release Thus, PrRP is not a classic hypothalamic hor-mone in mammals Instead, PrRP appears to play an important role in the control of energy metabolism and stress [7]

Localization of PrRP and its receptors PrRP neurons are localized mostly in the nucleus trac-tus solitarii (NTS), with modest expression in the ven-trolateral medulla regions of the brainstem, and slight expression in the dorsomedial hypothalamus In the medulla oblongata, PrRP expression is restricted to the

Table 1 Summary of mammalian RFamide peptides and their receptors The effects of administration of RFamide peptides upon stress responses (hormone release) and energy metabolism are also described.

Family

name

Peptide in

Energy metabolism

Food intake

Energy consumption

NPAF

GPR74 (NPFF-2, NPGPR, HLWAR77) Decrease in vasopressin

release

Decrease [51] Increase PrRP PrRP GPR10 (hGR3, UHR-1, PRLHR) Increase in the release

of ACTH, oxytocin, vasopressin and prolactin

Decrease Increase

LPXRF RFRP-1 (NPSF)

RFRP-3 (NPVF)

GPR147 (NPFF-1, OT7T022, RFRPR) Increase in the release

of ACTH, oxytocin and prolactin

Increase

?

?

Table 2 Amino acid sequences of PrRP and RFRP in human, rats and mice are shown *Deduced from the cDNA sequence.

Number of

31

TPDINPAWYASRGIRPVGRF-NH2 SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH 2

[4]* [4]*

31

TPDINPAWYTGRGIRPVGRF-NH2 SRAHQHSMETRTPDINPAWYTGRGIRPVGRF-NH2

[4]* [4]*

31

TPDINPAWYTGRGIRPVGRF-NH 2

SRAHQHSMETRTPDINPAWYTGRGIRPVGRF-NH2

*

*

12

SLNFEELKDWGPKNVIKMSTPAVNKMPHSFANLPLRF-NH2 MPHSFANLPLRF-NH 2

[5]* [5]*, [54]

12

SVTFQELKDWGAKKDIKMSPAPANKVPHSAANLPLRF-NH2 VPHSAANLPLRF-NH2

[55] [5]*

12

SVSFQELKDWGAKKDIKMSPAPANKVPHSAANLPLRF-NH 2

VPHSAANLPLRF-NH 2

[5]* [5]*

17

VPNLPQRF-NH2 NMEVSLVRRVPNLPQRF-NH2

[5]*, [54] [5]*

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caudal A2 and A1 noradrenergic neurons

Immunohis-tochemical studies have shown that

PrRP-immunoreac-tive fibers are widely distributed in the brain [8] The

main receptor for PrRP, GPR10, is also widely

expressed in the brain (especially in the reticular

nucleus of the thalamus, bed nucleus of the stria

termi-nalis, preoptic areas, hypothalamic paraventricular

nucleus, periventricular nucleus, dorsomedial

hypothal-amus, NTS and area postrema) [9] In addition to

GPR10, PrRP has a high affinity for NPFF receptor 2

(also known as GPR74⁄ NPGPR ⁄ HLWAR77) NPFF

receptor 2 is expressed in the dorsal horn of the spinal

cord, thalamus, hypothalamus and hippocampus [10]

Although the physiological functions of NPFF

recep-tor 2 activated by PrRP remain to be clarified, a recent

study has proposed that central cardiovascular effects

of PrRP are mediated via NPFF receptor 2 [11]

Role of PrRP in the control of energy metabolism:

food intake

Intracerebroventricular injection of PrRP reduces food

intake [12] Both PrRP-deficient mice [13] and

GPR10-deficient mice [14] show hyperphagia Acute inhibition

of endogenous PrRP signaling by injections of

neutral-izing monoclonal antibodies against PrRP also induces

hyperphagia [13] From experiments with

PrRP-defi-cient mice or PrRP-neutralizing antibodies, PrRP has

been shown to regulate meal size rather than meal

fre-quency [13] Meal size is regulated by satiety signals

that terminate each meal, and one important satiety

sig-nal is cholecystokinin-8 (CCK) CCK is released from

the intestine in response to meals, and acts via the

CCKAreceptor on afferent vagal fibers that project into

the medulla oblongata, which relays information into

the hypothalamus Food intake [13] or the

administra-tion of CCK [15] activates PrRP neurons in the NTS

The anorectic effects of CCK are impaired in both

PrRP-deficient mice [13] and GPR10-deficient mice [16],

suggesting that PrRP relays satiety signaling of CCK

The downstream actions of PrRP neurons remain to

be clarified However, neurons expressing

corticotro-pin-releasing hormone (CRH) or oxytocin may relay

PrRP signaling to reduce food intake Anatomical

studies have shown that oxytocin neurons in the

hypo-thalamus receive direct projections from PrRP neurons

in the medulla oblongata The administration of PrRP

activates neurons expressing CRH or oxytocin in the

hypothalamus, both of which are anorexic peptides

PrRP-induced anorexia is attenuated by a CRH

recep-tor antagonist or oxytocin receprecep-tor antagonist [16]

Furthermore, an oxytocin receptor antagonist reduces

the anorexic actions of CCK [17,18], and increases

meal size [19] Oxytocin receptor-deficient mice show

an increased meal size [20] Taken together, these results suggest that the PrRP-oxytocin system plays a pivotal role in relaying the satiety signaling of CCK PrRP neurons in the brainstem and hypothalamus express leptin receptors [21] Leptin regulates long-term energy metabolism Leptin induces the expression of phosphorylated signal transducer and activator of transcription protein 3 in PrRP neurons, especially in the dorsomedial hypothalamus [13] (Fig 1) and the anorectic effects of leptin are impaired in PrRP-defi-cient mice [13] These data suggest that the anorectic effects of leptin signaling are mediated, at least in part,

by PrRP

Role of PrRP in the control of energy metabolism: energy expenditure

PrRP has also been associated with energy expendi-ture An intracerebroventricular injection of PrRP

Fig 1 Activation of PrRP and RFRP neurons in the dorsomedial hypothalamus after stressful stimuli and leptin administration The number of PrRP-immunoreactive neurons expressing Fos protein (the protein product of the immediate early gene, c-fos) or phos-phorylated signal transducer and activator of transcription 3 (i.e a transcription factor downstream of leptin) after conditioned fear stimuli or leptin is shown (top) Both conditioned fear stimuli and leptin administration activate PrRP neurons in the dorsomedial hypothalamus The number of RFRP-immunoreactive neurons expressing Fos protein after footshocks is shown (bottom) Foot-shocks activate RFRP neurons in the dorsomedial hypothalamus Data are obtained from previous studies [13,27,48] *P < 0.05,

**P < 0.01, ***P < 0.001.

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increases body temperature and oxygen consumption

[12,22], although neither PrRP-deficient [13], nor

GPR10-deficient male mice [23] show significant difference in

oxygen consumption under basal conditions

PrRP-deficient mice also show no significant difference in

core body temperature either at room temperature or

after cold exposure [13] Pair-fed PrRP-deficient mice

show no obese phenotype and no significant difference

in oxygen consumption [13], suggesting that

endoge-nous PrRP is not important for regulating energy

expenditure under resting conditions On the other

hand, obese GPR10-deficient female mice show slightly

reduced oxygen consumption [23], suggesting that

GPR10 might be important for regulating energy

expenditure in females Obesity has been reported to

be more pronounced in female than in male

GPR10-deficient mice It is interesting to note that PrRP

neu-rons in the brainstem express estrogen receptors and

that PrRP expression in the brainstem is higher in

female than in male rats [24] Thus, the function of

PrRP-GPR10 system in the control of energy

con-sumption might differ between sexes

PrRP has been suggested to be involved in energy

consumption under stressful conditions Stressful

stim-uli increase oxygen consumption This increase in

oxy-gen consumption after stressful stimuli is lower in

PrRP-deficient mice [25] Stressful stimuli activate

PrRP neurons, and thus it is possible that PrRP

increases energy consumption under the conditions in

which PrRP neurons are activated

Roles of PrRP in the control of stress responses

PrRP neurons in the medulla oblongata and⁄ or in the

dorsomedial hypothalamus are activated by a variety

of stressful stimuli [26], including restraint of body

movement, conditioned fear [27] (Fig 1), footshocks,

hemorrhage [28], exercise [29] and inflammatory stress

(e.g lipopolysaccharide injection) [30] PrRP neurons

have been suggested to be involved in neuroendocrine

responses to stress PrRP neurons project directly to

CRH neurons and oxytocin neurons in the

hypothala-mus [31] An intracerebroventricular injection of PrRP

activates CRH neurons and oxytocin neurons in the

hypothalamus, and facilitates adrenocorticotropic

hor-mone (ACTH) and oxytocin release into the systemic

circulation Blockade of endogenous PrRP signaling by

the administration of PrRP-neutralizing antibodies

reduces the activation of hypothalamic paraventricular

neurons after noxious stimuli (formalin injection) [30]

or reduces oxytocin release in response to conditioned

fear [27], suggesting that endogenous PrRP has

facilita-tive roles in neuroendocrine stress responses On the

other hand, the administration of PrRP-neutralizing antibodies facilitates ACTH release in response to exercise, suggesting that PrRP inhibits ACTH release

in response to exercise [29] Corticosterone release in response to restraint stress has been reported to be enhanced in PrRP-deficient mice [32] These data sug-gest that PrRP has inhibitory effects on neuroendo-crine responses to stress At present, the mechanisms underlying these apparent discrepant data remain to

be clarified However, the roles of PrRP in the control

of stress responses may depend upon the nature of stressful stimuli used

Stressful stimuli affect food intake and increase energy expenditure On the other hand, food intake affects stress responses [33] PrRP-deficient mice show

a lower increase in oxygen consumption after stressful stimuli [25], although the effects of stress on food intake and the effects of food intake on stress responses have not yet been examined in PrRP-defi-cient mice PrRP might be involved in the integration

in the control of energy metabolism and stress responses, whereas the underlying detailed mechanisms need further investigation

RFRP

The mammalian members of the LPXRFamide peptide family are 1 and 3 1 and

RFRP-3 are derived from a single precursor protein Immu-nohistochemical studies have shown that single cells contain both RFRP-1 and RFRP-3 RFRP-1 and RFRP-3 bind with high affinity to a G protein-coupled receptor, GPR147 (also known as OT7T022, NPFF receptor 1 or RFRP receptor)

In birds, peptides of the LPXRFamide family are termed gonadotropin inhibitory hormones RFRPs have also been reported to serve a similar function in mammals [34] The administration of RFRP-3 sup-presses plasma luteinizing hormone or follicle-stimulat-ing hormone concentrations in mammals However, the mechanisms are not fully understood RFRP-3 inhibits the activity of gonadotropin-releasing hormone neurons in the hypothalamus [35–37] RFRP-3 has been also reported to act on pituitary gonadotrophs to inhibit luteinizing hormone or follicle-stimulating hor-mone release [38–40], although it is currently a matter

of controversy as to whether RFRP-3 exerts a hypo-physiotropic role in mammals [37,41] RFRP mRNA expression is not affected either by estrogen [42] or tes-tosterone [43], whereas RFRP neurons have been reported to express estrogen receptors [35] The precise physiological roles of RFRP in the mammalian repro-ductive system need further investigation

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Localization of RFRPs and their receptors

The RFRP gene is expressed in the caudal

hypothala-mus, including the dorsomedial hypothalamus and the

periventricular nucleus Immunohistochemical studies

have shown that RFRP-immunoreactive fibers are

widely distributed within the brain [44] Expression of

the main receptor for RFRP, GPR147, is broadly

dis-tributed within the brain, including the septal areas,

amygdala, bed nucleus of the stria terminalis,

hypotha-lamic paraventricular nucleus, dorsomedial

hypothala-mus, ventromedial hypothalamus and the anterodorsal

thalamic nucleus

Roles of RFRP in the control of food intake and

stress responses

The dorsomedial hypothalamus plays an important

role in the control of energy metabolism [45] Indeed,

RFRPs have been suggested to contribute not only to

the control of the reproductive system, but also to the

control of energy balance RFRP neurons project

directly to cells expressing neuropeptide Y or

pro-opio-melanocortin in the arcuate nucleus of the

hypothala-mus, both of which are key molecules in the control of

energy balance [46] The administration of RFRPs

induces Fos protein in the arcuate nucleus, which is a

center for food intake, and stimulates food intake in

rats [36,38] However, food restriction does not change

the expression of RFRP in Siberian hamsters [47] The

effects of RFRP upon energy consumption or oxygen consumption are not known The downstream and physiological significance of orexigenic actions by RFRP remain to be determined

The involvement of RFRP in the control of stress responses has also been reported The dorsomedial hypothalamus where RFRP neurons exist plays an important role in the control of stress responses as well

as food intake [45] Subsequent to stressful stimuli, the percentage of RFRP neurons expressing Fos protein [48] (Fig 1) and the expression of RFRP mRNA in the hypothalamus are increased [49] RFRP fibers are observed in the hypothalamic paraventricular nucleus and appear to project directly to cells containing CRH

or oxytocin [46] in the hypothalamus The administra-tion of RFRP increases Fos expression in the hypotha-lamic paraventricular nucleus and in hypothahypotha-lamic oxytocin neurons, and facilitates the release of ACTH and oxytocin into the peripheral circulation Similar patterns of Fos expression and hormone release are observed after stressful stimuli Furthermore, the cen-tral application of RFRP-1 or RFRP-3 induces anxi-ety-related behavior [48] Taken together, these data are consistent with the view that stressful stimuli acti-vate RFRP neurons and that RFRP-1 and RFRP-3 are involved in neuroendocrine and behavioral responses to stressful stimuli

RFRP neurons express glucocorticoid receptors and the administration of glucocorticoid increases the expression of RFRP mRNA in the hypothalamus [49]

Fig 2 Possible neural pathways controlling stress and food intake by PrRP and RFRP The PrRP and RFRP systems influence energy homeostasis and stress responses The dorsomedial hypothalamus has direct connections to and from the limbic areas, other hypothalamic nuclei and the brainstem, which are involved in stress and energy balance AMY, amygdala; ARC, arcuate nucleus; BST, bed nucleus of the stria terminalis; DMH, dorsomedial hypothalamus; LHA, lateral hypothalamic area; NTS, nucleus tractus solitarii (A2 noradrenergic region); PBN, parabrachial nucleus; POA, preoptic area; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; VLM, ventrolateral medulla (A1 noradrenergic region); VMH, ventromedial hypothalamus.

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RFRP neurons also express serotonin receptors and

the number of RFRP neurons is increased after

chronic administration of a selective serotonin

reup-take inhibitor, citalopram [50] Glucocorticoid and

serotonin play major roles in the control of food

intake and stress responses The physiological

func-tions of these receptors expressed in RFRP neurons

remain to be determined

Conclusions

Epidemiological studies have shown that both stress

and obesity cause deleterious effects on human health

Obesity is caused by a positive energy balance

Stress-ful stimuli affect neurons in the brainstem and

hypo-thalamus, and induce neuroendocrine and behavioral

responses Food intake also activates the brainstem

and hypothalamus, resulting in the termination of

meals and the induction of energy consumption

Energy homeostasis and stress interact with each other

Stress affects food intake and energy expenditure On

the other hand, energy balance conditions affect stress

responses As described in the present minireview,

neu-rons expressing RFamide peptides receive information

concerning both internal metabolic states and

environ-mental conditions, and play a role in energy

homeosta-sis and stress responses (Fig 2) Thus, it is interesting

to speculate that RFamide peptides are pivotal in

interactions between stress and energy metabolism

Other neuropeptides, including GALP [57–59] and

relaxin-3 [60], also may play a role in these

tions The detailed mechanisms underlying the

interac-tions between stress and energy metabolism remain to

be clarified

Acknowledgements

The present work was supported by KAKENHI

(20590237, 20020023, 20790194, 22120512, 22659050)

from MEXT and JSPS

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