Expression of neuropeptide W in rat stomach mucosa: regulation by nutritional status, glucocorticoids and thyroid hormones.

Abstract

Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain, NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis, namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next, we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.

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Expression of neuropeptide W in rat stomach mucosa: Regulation by
nutritional status, glucocorticoids and thyroid hormones
Jorge E. Caminosa,b, Susana B. Bravoa, María E.R. García-Renduelesa, C. Ruth Gonzáleza,
Maria F. Garcésb, Libia A. Cepedab, Ricardo Lagea, Miguel A. Suárezb, Miguel Lópeza,c,
Carlos Diégueza,c,⁎
aDepartment of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
bDepartment of Physiology and Genetic Institute, Faculty of Medicine, National University of Colombia, Bogotá, Colombia
cCIBER of Obesity and Nutrition, Instituto Salud Carlos III, Santiago de Compostela, Spain
Received 9 May 2007; received in revised form 16 July 2007; accepted 28 August 2007
Abstract
Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain,
NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in
peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis,
namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we
examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and
protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next,
we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW
expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in
the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Neuropeptide W; Body weight; Nutritional status; Glucocorticoids; Thyroid hormones
1. Introduction
Neuropeptide W (NPW) is a recently discovered peptide that
activates the previously described orphan G-protein-coupled
receptors, GPR7 and GPR8 [1,2]. Two endogenous molecular
forms of NPW that consist of 23- and 30-amino acid residues
have been identified [1,2]. NPW mRNA in rodent brain is
confined to specific nuclei in hypothalamus, midbrain and
brainstem [1,3,4]. This pattern of expression, alongside the
localization of GPR7 and GPR8 in some hypothalamic nuclei,
such as the paraventricular, supraoptic, ventromedial, dorsome-
dial, suprachiasmatic and arcuate [5,6], initially suggested that
NPW may be implicated in feeding regulation. However, the
reported results are contradictory. Firstly, Mondal et al.
observed a mild anorectic effect (associated to increased body
temperature and heat production) after NPW central adminis-
tration [7]. On the other hand, Levine et al. have demonstrated
an orexigenic action for NPW [8]. Whatever the case, this
evidence suggests the possibility that NPW may act as an
endogenous feeding–regulating molecule in the brain.
Besides this role in body weight homeostasis, NPW may
play a role in the regulation of hypothalamic–pituitary–adrenal
(HPA) and thyroid (HPT) axes. It has been reported that central
Available online at www.sciencedirect.com
Regulatory Peptides xx (2007) xxx–xxx
+ MODEL
REGPEP-03744; No of Pages 6
www.elsevier.com/locate/regpep
Abbreviations: AMT, aminotriazole; BAT, brown adipose tissue; BBS,
bombesin; CCK, cholecystokinin; CNS, Central Nervous System; HPA axis,
hypothalamic–pituitary–adrenal axis; HPT axis, hypothalamic–pituitary–thy-
roid axis; GLP-1, glucagon-like peptide-1; GLP-2, glucagon-like peptide-2;
GPR7, G-protein-coupled receptor 7; GPR8, G-protein-coupled receptor 8;
NPW, neuropeptide W; OXM, oxyntomodulin; PYY, Peptide YY; T4, L-
thyroxine; WAT, white adipose tissue.
⁎Corresponding author. Department of Physiology, School of Medicine and
CIBER of Obesity and Nutrition, Instituto Salud Carlos III, University of
Santiago de Compostela, S. Francisco s/n, 15705, Santiago de Compostela (A
Coruña), Spain. Tel.: +34 981582658; fax: +34 981574145.
E-mail address: fscadigo@usc.es (C. Diéguez).
0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.regpep.2007.08.021
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NPW administration increases plasmatic levels of corticoste-
rone [9,10]. Moreover, a direct effect of NPWon adrenocortical
cells has been also described in rodents [11,12] and humans
[13]. On addition, both NPW receptors, GPR7 and GPR8, are
highly expressed in the thyroid gland [14], which suggest the
existence of direct effect of NPW on thyroid function.
In addition to the central nervous system (CNS), NPW
expression has been also detected in peripheral tissues. Recent
reports have demonstrated the presence of NPW in multiple
endocrine glands of the rat, including pituitary, thyroid, para-
thyroid, pancreas, adrenal gland (cortex and medulla), ovary
andtestis[3,12,14,15],aswellasinantralgastrin(G)cellsofrat,
mouse, and human stomach [16]. However, data are lacking
about the specific regulation of NPW expression in stomach.
This issue is particularly relevant considering that other gut-
derivedmoleculesregulatingfoodintake,suchasleptin,ghrelin,
bombesin (BBS), cholecystokinin (CCK), glucagon-like pep-
tide-1 (GLP-1) and -2 (GLP-2), oxyntomodulin (OXM) and
peptide YY (PYY) have been shown to be sensitive to the
nutritional state, being rapidly mobilized in response to food
intake following fasting, or after the administration of satiety
factors[17–21].Thissuggestsarole forgastric-derived peptides
in the short-term regulation offeeding, giving information to the
brainontheavailabilityofexternal(food)energyresources,thus
participating in short- and long-term satiation [18–21].
In this study we aim to characterize the effects of nutritional
status on NPW expression in rat stomach mucosa. In addition,
given the possible involvement of NPW on the regulation of
HPA and HPT axes, we studied the effect on glucocorticoids
and thyroid hormone status on stomach NPW expression.
2. Materials and methods
2.1. Animals
Adult Sprague–Dawley rats (Animalario General USC,
Spain) were housed in a temperature-controlled room, with a
12-hour light/dark cycle (light: 8:00–20:00). Food and water
were available ad libitum prior to the initiation of the ex-
periment. All experiments were conducted in accordance with
the Ethics Committee of the University of Santiago de
Compostela. The experiments were performed in agreement
with the Rules of Laboratory Animal Care and International
Law on Animal Experimentation.
2.2. Fasting–refeeding experiments
Male rats (n=8–10 per experimental group) were deprived
of food for 24 or 48 h and refed ad libitum during 24 h [22,23].
Body weights, food intake (refeeding), leptin, insulin and
glucose levels were measured in all the animals to check the
effects of fasting–refeeding (Table 1).
2.3. Chronic food restriction
Food restriction was performed as described previously
[24,25]. Briefly, male rats (n=8–10 per experimental group)
were fed 30% of the daily ad libitum intake during 12, 16 or
21 days. Control rats were fed ad libitum. To determine the
pattern of rat NPW mRNA expression throughout pregnancy
(n=8–10 animals per experimental group) and analyze the food
restriction effect on stomach tissue at different times during
pregnancy, rats on gestational days 12, 16 and 21 were obtained
[24,25]. All animals were fed every day at 18:00 h.
2.4. Treatments with dexamethasone
Male rats (n=8–10 animals per experimental group)
received a daily subcutaneous (SC) administration of dexa-
methasone (DX) (Fortecortin®, Merck; Barcelona, Spain) in a
dose of 40 μg/day, dissolved in 200 μl of saline for 8 and
15 days. Control animals were treated with the same SC volume
of vehicle. All the treatments began at 9:00 h.
2.5. Induction of hypo- and hyperthyroidism
Hypothyroidismwasinducedaspreviouslydescribed[26,27]
by administration of 0.1% aminotriazole (Sigma, Poole, UK) in
drinking water for a period of 8 days. Hyperthyroidism was
induced by chronic subcutaneous administration of L-thyroxine
(100 μg/day, dissolved in 200 μl of saline; Sigma, Poole, UK).
Control animals and hypothyroid animals were treated with the
same subcutaneous volume of vehicle. Ten male rats per
experimental group were used. To confirm the hyperthyroid
status we studied plasma T3, T4 and TSH levels (Table 2).
Table 1
Corporal and plasma parameters in fed, fasted and refed rats
Fed Fast 24 hFast 48 h Refed
2.2±1.3⁎⁎⁎(vs. fed)
Body weight
change (g)
Food intake (g)
Glucose
(mmol/l)
Insulin (ng/ml)
20.0±1.5
−31.6±
1.1⁎⁎⁎
NFI
5.3±0.5⁎⁎⁎
−41.6±
1.8⁎⁎⁎
NFI
5.3±0.8⁎⁎⁎
29.3±0.5
7.8±0.3
38.5±0.6⁎⁎⁎(vs. fed)
8.0±0.4
2.2±0.4 0.4
±0.1⁎⁎⁎
0.9
±0.2⁎⁎⁎
0.2
±0.08⁎⁎⁎
0.7±0.1⁎⁎⁎
2.0±0.3
Leptin (ng/ml) 5.5±0.4
5.1±0.8
⁎⁎⁎: Pb0.001 vs. fed and refed. In order to simplify the table, comparisons
between Fast 24 hours vs. Fast 48 hours and refed vs. fast (24/48) have been
omitted.
NFI: non-food intake.
Table 2
Corporalandplasmaparametersin euthyroid,hypothyroid andhyperthyroidrats
EuthyroidHypothyroid
27.2±1.8⁎⁎⁎
ND⁎⁎⁎
0.9±0.1⁎⁎⁎
Hyperthyroid
0.4±0.1⁎⁎⁎
321.6±33.3⁎⁎⁎
5.1±0.6⁎⁎⁎
TSH (ng/ml)
T4 (nmol/l)
T3 (nmol/l)
⁎⁎⁎: Pb0.001 vs. euthyroid. In order to simplify the table, comparisons
between hypothyroid vs. hyperthyroid have been omitted.
ND: non-detected.
3.4±0.2
80.2±7.2
3.2±0.2
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2.6. RNA preparation and RT-PCR
Stomach mucosa was dissected as previously described
[24,28,29]. Total RNA was extracted from the tissues using
Trizol (Invitrogen, Inc., Carlsbad, CA, USA) according to the
manufacturer's instructions. For amplification of the targets, RT
and PCR were run in two separate steps as previously described
[22–24,29]. Briefly, equal amounts of total RNA (2 μg) were
used as a template to generate cDNA by RT with random
hexamer primers. For PCR, 3 μl of the RT products were
amplifiedinseparatereactionsusingprimersforratNPWcDNA
andhypoxanthinephosphoribosyltransferase(HPRT).ThePCR
consistedofavariablenumberofcyclesofamplificationdefined
bydenaturationat95°Cfor15s,annealingat64°Cfor30s,and
extensionat72°Cfor1min.Afinalextensioncycle of72°Cfor
10 min was included. We used the same set of primers as those
used for real-time PCR (see below). Amplification of the NPW
signal from rat hypothalamus was conducted as a positive
control. PCR products were analyzed by agarose gel-electro-
phoresis, and visualized by image analysis software (Molecular
Analyst, Biorad; Hercules, CA, USA).
2.7. Real-time semiquantitative RT-PCR analysis
Two micrograms of total RNA were used for each RT
reaction as previously described [22–24,29]. Real-time reverse-
transcription polymerase chain reaction (RT-PCR) analyses
were performed in a fluorescent temperature cycler (Light-
Cycler, Roche Molecular Biochemicals; Mannheim, Germany)
[30,31]. We used the following primer sequences (5′–3′): NPW
(GenBank accession number: NM_153294): NPW-forward:
GAG TCT GCG GGA CCT GGA; NPW-reverse: CTG ACA
GGATCG GCA AAG AT; HPRT (GenBank accession number:
NM_012583): HPRT-forward CAG TCC CAG CGT CGT GAT
TA; HPRT-reverse AGC AAG TCT TTC AGT CCT GTC. The
amount of PCR products formed in each cycle was evaluated on
the basis of SYBR Green I fluorescence. Data were normalized
by using the delta–delta Ct method [30,31].
2.8. Western blotting
Stomach protein lysates were subjected to SDS-PAGE,
electrotransferred on a PVDF membrane and probed with the
indicated antibodies [23]: NPW (Phoenix; Burlingame, CA,
USA) and α-Tubulin (Sigma; Poole, UK). For protein detection
we used HRP-conjugated secondary antibodies and chemilu-
minescence (Tropix, Bedford; MA, USA).
2.9. Blood biochemistry
Plasma thyroid-stimulating hormone (TSH) levels were
measured by radioimmunoassay as previously described
[26,27] by using reagents provided by the National Hormone
and Pituitary Program. Plasma T3, T4, leptin and insulin levels
were measured by ELISA kits (Crystal Chem Inc; Downers
Grove, IL, USA). Kits for the measurement of glucose were
obtained from Roche (Basel, Switzerland).
2.10. Statistical analysis
Data were expressed as mean±SEM and analyzed by using
StatView 4.57 (Abacus Concepts). Statistical significance was
determined by t-Student (when 2 groups were compared) or
Fig. 1. Expression of NPW gene in rat stomach mucosa. Representative RT-PCR
assay of the expression levels of NPW mRNA in the described tissues. Positive
(rat hypothalamus) and negative (water) controls are shown. A 100-bp marker
(MW) was used. Stomach: stomach mucosa; hypotha: hypothalamus; WAT:
white adipose tissue; BAT: brown adipose tissue.
Fig. 2. NPW mRNA levels in stomach mucosa of fed, fasted (24 and 48 h) and
refed rats (A). Western blot analysis of stomach mucosa NPW protein levels in
fed, fasted 24 (F24), fasted 48 (F48) and refed rats (B). NPW mRNA levels in
stomach mucosa of fed and food restricted rats, at the described times (C).⁎⁎:
Pb0.01;⁎⁎⁎: Pb0.001 between the labeled groups.
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ANOVA and post-hoc Bonferroni test (when more than 2
groups were compared). Pb0.05 was considered significant.
3. Results
3.1. NPW is expressed in stomach mucosa of the rat
Assessment of NPW mRNA expression by RT-PCR
analysis, using specific primers, demonstrated expression of
this gene in rat stomach mucosa. As anticipated, a weaker signal
of similar size was amplified from the hypothalamus, the
positive control. For each sample, parallel amplification using a
specific primer set of rat HPRTserved as an internal control. No
products were detected either from other examined tissues, such
as white adipose tissue (WAT), brown adipose tissue (BAT),
ileum and blood, or the negative controls (water) (Fig. 1).
3.2. Nutritional status markedly decreases NPW expression in
stomach mucosa
Twenty four- and 48-hour fasting induced a marked de-
creased in the mRNA expression of NPW in rat stomach
mucosa and this effect was reverted by refeeding (Fig. 2A).
Similar data were obtained by assessing NPW protein levels by
Western blot (Fig. 2B). In addition, our real-time RT-PCR
analysis showed that NPW mRNA levels in stomach mucosa
were decreased after food restriction (Fig. 2C). These data
suggest that NPW expression in the stomach is positively
regulated by nutritional status.
3.3. Gestation and food restriction decreased NPW expression
in stomach mucosa
Next, we studied the effect of food restriction on other
models, such as virgin females and pregnant rats. Interestingly,
we detected that, independently of nutritional status, pregnancy
induced a marked decrease in the expression of NPW in the
stomach mucosa, at any evaluated time point (12, 16 and
21 days of gestation) (Fig. 3A). In addition, food restriction
further decreased NPW mRNA levels in the stomach of preg-
nant rats, an effect also observed in virgin females (Fig. 3B).
3.4. Dexamethasone treatment decreased NPW expression in
stomach mucosa
Chronic dexamethasone administration to male rats induced
a marked decrease in NPW mRNA levels in stomach mucosa
(Fig. 4A).
3.5. Thyroid status negatively regulates NPW expression in
stomach mucosa
Hypothyroid rats showed a marked increase in NPW mRNA
levels in stomach mucosa (Fig. 4B). On the other hand,
Fig. 3. NPW mRNA levels in stomach mucosa of female rats at different
pregnancy stages (12, 16 and 21 days) (A) and female virgin and pregnant rats
food restricted at the described times (B).⁎: Pb0.05;⁎⁎: Pb0.01;⁎⁎⁎:
Pb0.001 between the labeled groups.
Fig. 4. NPW mRNA levels in stomach mucosa of male rats receiving chronic
treatments with dexamethasone for 8 and 15 days (A). NPW mRNA levels in
stomach mucosa of male hypothyroid and hyperthyroid rats (B).⁎: Pb0.05;
⁎⁎⁎: Pb0.001 between the labeled groups.
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hyperthyroid rats displayed a significant decrease in the NPW
mRNA expression in stomach mucosa.
4. Discussion
The gastrointestinal tract is the body's largest endocrine
organ and releases about thirty different regulatory peptide
hormones that modulate multiple physiological processes and
act on a range of tissues [18–21]. Despite their wide phy-
siological roles, nearly all these hormones have two common
characteristics; firstly they are sensitive to gut nutrient content
and secondly, as a consequence of this, short-term regulation of
feeding is partly mediated by coordinated changes in circulating
gut hormone levels [18–21].
NPW is a recently discovered peptide, originally detected in
the CNS [1,2] but also recently identified in peripheral tissues,
such as antral gastrin (G) cells of rat, mouse, and human stom-
ach [16]. Since brain NPW has been implicated in the regulation
of food intake [7,8], we hypothesized that stomach NPW may
also be involved in feeding control and therefore NPW gene
expression in this tissue may be regulated by changes in nu-
tritional status. Our data demonstrates that stomach NPW gene
expression is markedly decreased by fasting/food restriction and
reverted by refeeding in all animal models we studied: male,
female and pregnant rats. This suggests an anorectic role for
stomach NPW [7]. This idea is further supported by the down-
regulation of stomach NPW in pregnant rats. Pregnancy is a
hypermetabolic state in which a great increase in maternal body
fat and weight occurs. This positive energy balance in preg-
nancy occurs mostly due to an increase in food intake [32,33].
We found that NPW mRNA levels were decreased both during
pregnancy and this was further pronounced in pregnant food
restricted rats, which suggests that the increased feeding or
hunger of these models may be mediated by downregulation of
this anorectic signal. The fact that a negative energy balance
during fasting-food restriction and a positive energy balance
during pregnancy are both able to downregulate NPW ex-
pression, is apparently paradoxical. However, similar patterns
of expression have been described for other signals involved in
feeding control. For example, recent evidence has shown that
anorectic peripheral signals, such as peptide YY (PYY) are
decreased in fasting [19,20] and pregnancy [34]. In addition,
orexigenic neuropeptide systems are highly stimulated during
pregnancy [32] and fasting [21] and leptin signaling is markedly
reduced in both states [21,35]. The reasons of this paradoxical
effect are still not fully understand, but it may be related to
specific changes in pregnancy hormones, such as prolactin [32].
Further work will clarify these issues.
Finally, it is interesting to note that besides their effects on
feeding, current data have also demonstrated that NPW may play
a role in regulation of HPA and HPT axes [11–14]. To further
explore this inter-relationship, we examined the effect of
glucocorticoid and thyroid status on NPWexpression in stomach
mucosa. Our data showed that chronic dexamethasone or thyroid
hormone administration decreased mRNA expression of NPWin
thistissue,whereasthyroidhormonedeficiencyinducedamarked
increase in NPW expression in the stomach mucosa. Thus, it is
reasonable to speculate that NPW is negatively regulated by
glucocorticoids and thyroid hormones as a compensatory
mechanismattemptingtomodulateadrenalandthyroidfunctions.
Further work is necessary to confirm this hypothesis.
In summary, NPW is regulated by nutritional, glucocorticoid
and thyroid status in stomach mucosa. Overall these data
suggest that stomach NPW may play a role in food intake
control and body weight homeostasis.
Acknowledgements
We are very grateful to Dr. Chris Lelliott (AstraZeneca R&D;
Mölndal, Sweden) for his comments and criticisms. This work
has been supported by grants from Instituto de Salud Carlos III
(PI061700), Spanish Ministry of Education (BFU-2005), Xunta
de Galicia and the National Direction of Investigations of the
National University of Colombia.
References
[1] Tanaka H, Yoshida T, Miyamoto N, Motoike T, Kurosu H, Shibata K,
Yamanaka A, Williams SC, Richardson JA, Tsujino N, Garry MG, Lerner
MR, King DS, O'Dowd BF, Sakurai T, Yanagisawa M. Characterization of
a family of endogenous neuropeptide ligands for the G protein-coupled
receptors GPR7 and GPR8. Proc Natl Acad Sci U S A 2003;100:6251–6.
[2] Singh G, Davenport AP. Neuropeptide B and W: neurotransmitters in an
emerging G-protein-coupled receptor system. Br J Pharmacol 2006;148:
1033–41.
[3] Dun SL, Brailoiu GC, Yang J, Chang JK, Dun NJ. Neuropeptide W-
immunoreactivity in the hypothalamus and pituitary of the rat. Neurosci
Lett 2003;349:71–4.
[4] Kitamura Y, Tanaka H, Motoike T, Ishii M, Williams SC, Yanagisawa M,
SakuraiT. Distributionof neuropeptide Wimmunoreactivity andmRNA in
adult rat brain. Brain Res 2006;1093:123–34.
[5] Lee DK, Nguyen T, Porter CA, Cheng R, George SR, O'Dowd BF. Two
related G protein-coupled receptors: the distribution of GPR7 in rat brain
and the absence of GPR8 in rodents. Brain Res Mol Brain Res
1999;71:96–103.
[6] Jackson VR, Lin SH, Wang Z, Nothacker HP, Civelli O. A study of the rat
neuropeptide B/neuropeptide W system using in situ techniques. J Comp
Neurol 2006;497:367–83.
[7] Mondal MS, Yamaguchi H, Date Y, Shimbara T, Toshinai K, Shimomura
Y, Mori M, Nakazato M. A role for neuropeptide W in the regulation of
feeding behavior. Endocrinology 2003;144:4729–33.
[8] Levine AS, Winsky-Sommerer R, Huitron-Resendiz S, Grace MK, de
Lecea L. Injection of neuropeptide W into paraventricular nucleus of
hypothalamus increases food intake. Am J Physiol Regul Integr Comp
Physiol 2005;288:R1727–32.
[9] Baker JR, Cardinal K, Bober C, Taylor MM, Samson WK. Neuropeptide
W acts in brain to control prolactin, corticosterone, and growth hormone
release. Endocrinology 2003;144:2816–21.
[10] Taylor MM, Yuill EA, Baker JR, Ferri CC, Ferguson AV, Samson WK.
Actions of neuropeptide W in paraventricular hypothalamus: implications
for the control of stress hormone secretion. Am J Physiol Regul Integr
Comp Physiol 2005;288:R270–5.
[11] Hochol A, Albertin G, Nussdorfer GG, Spinazzi R, Ziolkowska A, Rucinski
M, Malendowicz LK. Effects of neuropeptidesB andWon the secretion and
growth of rat adrenocortical cells. Int J Mol Med 2004;14:843–7.
[12] Hochol A, Tortorella C, Ricinski M, Ziolkowska A, Nussdorfer GG,
Malendowicz LK. Effects of neuropeptides B and W on the rat pituitary–
adrenocortical axis: in vivo and in vitro studies. Int J Mol Med
2007;19:207–11.
[13] Mazzocchi G, Rebuffat P, Ziolkowska A, Rossi GP, Malendowicz LK,
Nussdorfer GG. G protein receptors 7 and 8 are expressed in human
5 J.E. Caminos et al. / Regulatory Peptides xx (2007) xxx–xxx
ARTICLE IN PRESS
Please cite this article as: Caminos JE et al. Expression of neuropeptide W in rat stomach mucosa: Regulation by nutritional status, glucocorticoids and thyroid
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Page 6

adrenocortical cells, and their endogenous ligands neuropeptides B and
w enhance cortisol secretion by activating adenylate cyclase- and
phospholipase C-dependent signaling cascades. J Clin Endocrinol Metab
2005;90:3466–71.
[14] Hochol A, Belloni AS, Rucinski M, Ziolkowska A, Di Liddo R,
Nussdorfer GG, Malendowicz LK. Expression of neuropeptides B and
W and their receptors in endocrine glands of the rat. Int J Mol Med
2006;18:1101–6.
[15] Rucinski M, Nowak KW, Chmielewska J, Ziolkowska A, Malendowicz
LK. Neuropeptide Wexerts a potent suppressive effect on blood leptin and
insulin concentrations in the rat. Int J Mol Med 2007;19:401–5.
[16] Mondal MS, Yamaguchi H, Date Y, Toshinai K, Kawagoe T, Tsuruta T,
Kageyama H, Kawamura Y, Shioda S, Shimomura Y, Mori M, Nakazato
M. Neuropeptide W is present in antral G cells of rat, mouse, and human
stomach. J Endocrinol 2006;188:49–57.
[17] Horvath TL, Castaneda T, Tang-Christensen M, Pagotto U, Tschop MH.
Ghrelinasapotentialanti-obesitytarget.CurrPharmDes2003;9:1383–95.
[18] Pico C, Oliver P, Sanchez J, Palou A. Gastric leptin: a putative role in the
short-term regulation of food intake. Br J Nutr 2003;90:735–41.
[19] Perez-Tilve D, Nogueiras R, Mallo F, Benoit SC, Tschoep M. Gut
hormones ghrelin, PYY, and GLP-1 in the regulation of energy, balance,
and metabolism. Endocrine 2006;29:61–72.
[20] Murphy KG, Bloom SR. Gut hormones and the regulation of energy
homeostasis. Nature 2006;444:854–9.
[21] López M, Tovar S, Vázquez MJ, Williams LM, Diéguez C. Peripheral
tissue–brain interactions in the regulation of food intake. Proc Nutr Soc
2007;66:131–55.
[22] LópezM,SeoaneL,GarcíaMC,LagoF,CasanuevaFF,SenarísR,Diéguez
C. Leptin regulation of prepro-orexin and orexin receptor mRNA levels in
the hypothalamus. Biochem Biophys Res Commun 2000;269:41–5.
[23] López M, Lelliott CJ, Tovar S, Kimber W, Gallego R, Virtue S, Blount M,
Vázquez MJ, Finer N, Powles T, O'Rahilly S, Saha AK, Diéguez C, Vidal-
Puig AJ. Tamoxifen-induced anorexia is associated with fatty acid
synthase inhibition in the ventromedial nucleus of the hypothalamus and
accumulation of malonyl-CoA. Diabetes 2006;55:1327–36.
[24] Gualillo O, Caminos JE, Nogueiras R, Seoane LM, Arvat E, Ghigo E,
Casanueva FF, Dieguez C. Effect of food restriction on ghrelin in normal-
cycling female rats and in pregnancy. Obes Res 2002;10:682–7.
[25] Nogueiras R, Gualillo O, Caminos JE, Casanueva FF, Dieguez C.
Regulation of resistin by gonadal, thyroid hormone, and nutritional status.
Obes Res 2003;11:408–14.
[26] López M, Seoane L, Señarís R, Diéguez C. Prepro-orexin mRNA levels in
the rat hypothalamus, and orexin receptors mRNA levels in the rat
hypothalamus and adrenal gland are not influenced by the thyroid status.
Neurosci Lett 2001;300:171–5.
[27] López M, Seoane LM, Tovar S, Señarís R, Diéguez C. Thyroid status
regulates cocaine and amphetamine-regulated transcript but not agouti-
related peptide mRNA levels in the rat hypothalamus. Neuroreport
2002;13:1775–9.
[28] Gualillo O, Caminos JE, Kojima M, Kangawa K, Arvat E, Ghigo E,
Casanueva FF, Dieguez C. Gender and gonadal influences on ghrelin
mRNA levels in rat stomach. Eur J Endocrinol 2001;144:687–90.
[29] Caminos JE, Seoane LM, Tovar SA, Casanueva FF, Dieguez C. Influence
of thyroid status and growth hormone deficiency on ghrelin. Eur
J Endocrinol 2002;147:159–63.
[30] NogueirasR, CaminosJE, GallegoR, RaghayK,BravoS, TovarS, Pombo
C, López M, Tena-Sempere M, Garcia-Caballero T, Dieguez C. Regulation
of peroxisome proliferator activated receptor-gamma in rat pituitary.
J Neuroendocrinol 2005;17:292–7.
[31] Lage R, Diéguez C, López M. Caffeine treatment regulates neuropeptide S
system expression in the rat brain. Neurosci Lett 2006;410:47–51.
[32] García MC, López M, Gualillo O, Seoane L, Diéguez C, Señarís R.
Hypothalamic levels of NPY, MCH, and prepro-orexin mRNA during preg-
nancy and lactation in the rat: role of prolactin. FASEB J 2003;17:1392–400.
[33] Brailoiu GC, Dun SL, Ohsawa M, Yin D, Yang J, Chang JK, Brailoiu E,
Dun NJ. KiSS-1 expression and metastin-like immunoreactivity in the rat
brain. J Comp Neurol 2005;481:314–29.
[34] Tovar SA, Seoane LM, Caminos JE, Nogueiras R, Casanueva FF, Dieguez
C. Regulation of peptide YY levels by age, hormonal, and nutritional
status. Obes Res 2004;12:1944–50.
[35] García MC, Casanueva FF, Diéguez C, Señarís R. Gestational profile of
leptin messenger ribonucleic acid (mRNA) content in the placenta and
adipose tissue in the rat, and regulation of the mRNA levels of the leptin
receptor subtypes in the hypothalamus during pregnancy and lactation.
Biol Reprod 2000;62:698–703.
6 J.E. Caminos et al. / Regulatory Peptides xx (2007) xxx–xxx
ARTICLE IN PRESS
Please cite this article as: Caminos JE et al. Expression of neuropeptide W in rat stomach mucosa: Regulation by nutritional status, glucocorticoids and thyroid
hormones. Regulatory Pept (2007), doi:10.1016/j.regpep.2007.08.021