Neuroendocrine Correlations of Food Intake

Neuroendocrinology 2002;75:34–44

Neuropeptide Y, but Not Agouti-Related Peptideor Melanin-Concentrating Hormone, Is a TargetPeptide for Orexin-A Feeding Actions in the RatHypothalamus

Miguel Lopez Luisa Marıa Seoane Marıa del Carmen GarcıaCarlos Diéguez Rosa Señarıs

Department of Physiology, Faculty of Medicine, University of Santiago de Compostela,Santiago de Compostela, Spain

Received: June 11, 2001Accepted after revision: October 18, 2001

Rosa Señarıs, MD, PhDDepartment of Physiology Faculty of MedicineS. Francisco s/nE–15705 Santiago de Compostela (A Coruña) (Spain)Tel. +34 981 582658, Fax +34 981 574145, E-Mail fsrsr@usc.es

ABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com

© 2002 S. Karger AG, Basel0028–3835/02/0751–0034$18.50/0

Accessible online at:www.karger.com/journals/nen

Key WordsOrexins W Neuropeptide Y W Agouti-related peptide W

Melanin-concentrating hormone W Orexin receptors W

Feeding behavior

AbstractWe examined the effects of orexin A on the mRNA levelsof neuropeptide Y, agouti-related peptide, melanin-con-centrating hormone, prepro-orexin and orexin receptorsin the rat hypothalamus. Adult male rats were treatedcentrally (i.c.v.) with a single dose of orexin A (3 nmol).After 2, 6 and 12 h, neuropeptide Y, agouti-related pep-tide, melanin-concentrating hormone, and prepro-orexinmRNA levels were measured by semiquantitative RT-PCR and in situ hybridization; orexin receptors mRNAcontent was quantified by semiquantitative RT-PCR. Wefound that orexin A increased neuropeptide Y expres-sion in the arcuate nucleus of the rat hypothalamus. Thisstimulatory effect was transient, being observed 2 h afterthe treatment, and disappearing after longer periods (6and 12 h). In contrast, no change was demonstrated inhypothalamic agouti-related peptide, melanin-concen-trating hormone, prepro-orexin or orexin receptorsmRNA levels at any time evaluated. Our results suggestthat neuropeptide Y synthesized in the arcuate nucleus

of the hypothalamus, but not agouti-related peptide andmelanin-concentrating hormone pathways, is likely in-volved in orexin-induced feeding behavior, and raise thepossibility that this functional linkage may also be in-volved in other actions mediated by orexins such aslocomotor activity and sympathetic function.

Copyright © 2002 S. Karger AG, Basel

Introduction

The hypothalamus plays a major role in the regulationof food intake. Among other factors, feeding is regulatedby hypothalamic neuropeptides that promote (orexigenic)or inhibit (anorexigenic) food intake [1, 2]. Of these, neu-ropeptide Y (NPY) and agouti-related peptide (AgRP) inthe arcuate nucleus, and melanin-concentrating hormone(MCH) and orexins/hypocretins, in the lateral hypothala-mus, appear to have prominent roles in these orexigenicpathways [1, 2]. The expression of these four neuropep-tides is increased following food deprivation [3–6] andtheir central exogenous administration increase feeding[4, 5, 7, 8].

The orexins (OX-A and OX-B) are two recently discov-ered neuropeptides derived from a common precursor,the prepro-orexin (prepro-OX, also called prepro-hypo-

Orexin-A and Orexigenic Pathways Neuroendocrinology 2002;75:34–44 35

cretin) [5, 9]. These peptides have been involved in theregulation of different biological processes such as feeding[5, 8] sleep [10–13], arousal, locomotor and behaviouralactivities [14–17], blood pressure, heart rate [18, 19], pain[20], sympathetic function [19, 21] and neuroendocrinefunction [14, 22, 23]. Prepro-OX gene expression isknown to be regulated by several nutritional states or sig-nals, such as fasting [5, 24], glucose [25–27] and leptin[24] but not by thyroid hormones [28]. These peptidesbind and activate two closely related G-protein coupledreceptors (OXRs), called orexin receptor-1 (OX1R), andorexin receptor-2 (OX2R) [5]. Orexin receptors show awidespread distribution in the rat central nervous system[29, 30] and in the periphery [31, 32].

The orexin-containing neurones project to multiplesites of the central nervous system [22, 33–36]. Interest-ingly, orexin-containing fibers synapse with perikarya anddendrites of NPY/AgRP-containing neurons in the ar-cuate nucleus, and also terminate in close apposition withNPY containing nerve terminals in the paraventricularnucleus of the hypothalamus [36]. These anatomical find-ings suggest a possible interaction between orexin andNPY/AgRP signalling in the rat hypothalamus. In thissense, it has recently been demonstrated that the blockadeof NPY Y1 and NPY Y5 receptors using selective antago-nists suppressed orexin-induced feeding [37–40]. More-over, these orexigenic actions, the role of orexins andNPY in other physiological actions related with foodintake, as locomotor activity and sympathetic function[15, 41, 42] suggest possible interactions of these neuro-peptides.

Melanin-concentrating hormone (MCH) is a peptidethat is also expressed in the lateral hypothalamic area, butorexin and melanin concentrating hormone-expressingcells constitute distinct populations in this hypothalamicnucleus [33, 34]. No functional relationship between orex-ins and MCH has been described, but the presence of bothOXRs in the lateral hypothalamic area [29] suggested thatorexins might regulate MCH gene expression.

In order to fully understand the possible functionallinkage between orexins and these orexigenic peptides, wedetermined the effects of OX-A administration (i.c.v.) onhypothalamic NPY, MCH and prepro-OX mRNA levels.Moreover, we examined whether OXRs expression in thehypothalamus could be altered by OX-A treatment, sincewe and others demonstrated that OX1R mRNA content isregulated by the nutritional status of the animal, beingupregulated in fasting conditions [24, 43].

Material and Methods

AnimalsAdult male Sprague-Dawley rats (250–300 g) were housed on a

12-hour light (08:00 to 20:00 h), 12-hour dark cycle, in a temperatureand humidity controlled room. Animals were allowed free access tostandard laboratory pellets of rat chow and tap water. The protocolswere approved by the Ethics Committee of the University of Santia-go de Compostela, and experiments performed in agreement with therules of laboratory animal care and international law on animalexperimentation.

Implantation of Intracerebroventricular Cannulae and OX-ATreatmentChronic intracerebroventricular (i.c.v.) cannulae were implanted

under ketamine-xylacine anesthesia (50 mg/kg i.p.), as described pre-viously [44], and demonstrated to be located in the lateral ventricleby methylene blue staining. Animals were caged individually andused for the experiments 1 week later, during this postoperativerecovery period the rats became accustomed to the handling proce-dure under nonstressful conditions. Rats received either a singleadministration of OX-A (Bachem; Switzerland) (3 nmol per rat, [5, 8,38, 39] dissolved in 5 Ìl of distilled water saturated with argon) orvehicle (control rats) 2, 6, or 12 h after the treatment, vehicle andOX-A-treated animals were quickly decapitated, in an independentroom, and their brains removed rapidly. For the RT-PCR analysis,the hypothalamus, defined by the posterior margin of the opticchiasm and the anterior margin of the mamillary bodies to the depthof approximately 2 mm, was dissected out and immediately frozen indried ice. For in situ hybridization the whole brains were maintainedat –80°C until processed.

All treatments started in the lights-on phase (09:00 h). Imme-diately after the i.c.v. injection, a pre-weighed amount of food wasprovided to the animals. The food consumed during the next 2, 3, 6and 12 h was determined by weighing the remaining food and thespillage. 8–23 animals were used in each experimental group, 4–11 ofthem were used for RT-PCR analysis and 4–12 were used for in situhybridization analysis.

RNA Isolation and RT-PCR AnalysisTotal RNA was isolated using Trizol® reagent (Gibco-BRL, Life

Technologies, USA). The hypothalamic content of NPY, AgRP,MCH, prepro-OX, OX1R and OX2R mRNA was analyzed in eachhypothalamus individually.

The RT reaction was carried out in a volume of 30 Ìl containing1 Ìg of total RNA, and incubated at 37°C for 50 min and at 42°C for15 min. The enzyme was inactivated by heating at 95 °C for 5 min.We amplified 3 Ìl of the RT product, using the specific sets of prim-ers (table 1). The temperature and times used were: AgRP: 94°C(1 min), 49°C (1 min), 72 °C (1 min); MCH: 94°C (1 min), 58°C(1 min), 72°C (1 min); NPY: 94°C (1 min), 59°C (1 min), 72°C(30 s); prepro-OX, OXRs and HPRT (hypoxanthine-guanine phos-phoribosyltransferase), used as a housekeeping gene: 94°C (1 min),63°C (1 min), 72°C (1 min); in all cases amplification was carriedout at 28 cycles with a final extension step at 72°C for 10 min.

In order to assure that the PCR reaction was performed within alinear range, samples were amplified for 15, 20, 25, 30, 35 and 40cycles. As can be seen in figure 1, the reactions for AgRP, MCH andNPY were linear under the conditions used in the experiments. Thekinetics for HPRT, prepro-OX, OX1R and OX2R have been pub-

AgRP

36 Neuroendocrinology 2002;75:34–44 Lopez/Seoane/Garcıa/Diéguez/Señarıs

Fig. 1. PCR-amplification plots for AgRP, MCH and NPY. Samples were amplified for 15, 20, 25, 30, 35 and 40cycles.

Table 1. PCR primers for rat AgRP, rat MCH, rat NPY, rat Prepro-OX, rat OXRs and rat HPRT

mRNA GenBankaccessionnumber

Primer Sequence 5) position PCRproductbp

AF206017 sense 5)-GTTCCCAGGTCTAAGTCTGAA-3) 14 205antisense 5)-TGAAGAAGCGGCAGTAGCAC -3) 219

MCH M29712 sense 5)-GACATAGTATTTAATACATTCAGG-3) 146 400antisense 5)-GCAGGTATCAGACTTGCCAACAGG-3) 545

NPY M20373 sense 5)-ATCACCAGACAGAGATATGGC-3) 226 108antisense 5)-AGGGTCTTCAAGCCTTGTTCT-3) 333

Prepro-OX AF041241 sense 5)-CGGATTGCCTCTCCCTGAGC-3) 61 397antisense 5)-CTAAAGCGGTGGCGGTTGC-3 457

OX1R AF041244 sense 5)-TGGGCTGTGTCGCTGGCTG-3) 1070 328antisense 5)-GTTGGGGCTCTGTACACAGG-3) 1397

OX2R AF041246 sense 5)-TTGGGGTTCATCGTCGTCAAG-3) 1207 226antisense 5)-AGCCAGGTGGACAGGAGTGA-3) 1432

HPRT X62085 sense 5)- CAGTCCCAGCGTCGTGATTA-3) 38 139antisense 5)-AGCAAGTCTTTCAGTCCTGTC-3) 176

lished elsewhere [24]. The amplified products were resolved in a 2%agarose gel and quantitated by densitometry using a digital imagingsystem (Molecular Analyst®, Biorad, Calif., USA) [24]. All PCRproducts were digested with specific restriction enzymes, and hybrid-ized with complementary RNA or DNA probes in order to determinetheir specificity (data not shown).

In situ HybridizationFor in situ hybridization, coronal hypothalamic sections (16 Ìm)

were cut on a cryostat, and immediately stored at –80°C untilhybridization.

For the determination of prepro-OX mRNA levels we used a pre-pro-OX antisense riboprobe labelled with 35S-·CTP. A sense riboprobe

was used as a negative control. The specificity of the probe has pre-viously been demonstrated [24]. Frozen sections were fixed with 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temper-ature for 30 min, dehydrated through 70, 80, 90, 95% and absoluteethanol (5 min each). Hybridization was carrried out overnight at55°C in a moist chamber. Hybridization solution contained 1! 106

cpm per slide of the labeled probe, 4! SSC, 50% deionized formam-ide, 1! Denhardt’s solution, 10% dextran sulfate, 10 Ìg/ml sheared,single-stranded salmon sperm DNA and 5 Ìg/ml yeast tRNA. Afterhybridization sections were sequentially washed in 2! SSC (20! SSC:3 M NaCl, 0.3 M sodium citrate, pH 7) at room temperature, twice in2! SSC /50% formamide at 55°C (15 min per wash); rinsed briefly in2! SSC at 37 °C, incubated in RNase buffer containing 20 Ìg RNase

AgRP

Orexin-A and Orexigenic Pathways Neuroendocrinology 2002;75:34–44 37

Table 2. Antisense oligonucleotides for rat AgRP, rat MCH and rat NPY

mRNA GenBankaccessionnumber

Antisense oligonucleotide sequence 5) position

AF206017 5)-CGACGCGGAGAACGAGACTCGCGGTTCTGTGGATCTAGCACCTCTGCC-3) 136MCH M29712 5)-CCAACAGGGTCGGTAGACTCGTCCCAGCAT-3) 529NPY M20373 5)-AGATGAGATGTGGGGGGAAACTAGGAAAAGTCAGGAGAGCAAGTTTCATT-3) 400

A/ml at 37°C (30 min), rinsed in 2! SSC at 37°C, washed threetimes in 2! SSC/50% formamide at 55°C (15 min per wash), twicein 2! SSC at room temperature (5 min per wash), rinsed in waterand ethanol. Finally, sections were air-dried and exposed to Hyper-film ß-Max (Amersham, UK) at room temperature for 7 days.

For NPY, AgRP and MCH mRNA detection we employed thespecific antisense oligodeoxynucleotides (table 2). These probes were3)-end labelled with 35S-·dATP using terminal deoxynucleotidyltransferase. The specificity of the probes was confirmed by incuba-tion of the sections with an excess of the unlabelled probes (data notshown). In situ hybridizations were performed as already reported[45]. Frozen sections were fixed with 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.4) at room temperature for 30 min, dehy-drated through 70, 80, 90, 95%, and absolute ethanol (5 min each).Hybridization was carrried out overnight at 37°C in a moist cham-ber. Hybridization solution contained 5 ! 105 (AgRP) or 1 ! 106

cpm (MCH, NPY) per slide of the labeled probe, 4! SSC, 50%deionized formamide, 1! Denhardt's solution, 10% dextran sulfate,10 Ìg/ml sheared, single-stranded salmon sperm DNA. After hybrid-ization sections were sequentially washed in 1! SSC at room tem-perature, four times in 1! SSC at 42°C (NPY and MCH) or 55°C(AgRP) (30 min per wash), one time in 1! SSC at room temperature(1 h), rinsed in water and ethanol. Finally, sections were air-dried andexposed to Hyperfilm ß-Max (Amersham; UK) at room temperaturefor 4–6 days for AgRP, MCH, and NPY in the arcuate nucleus of thehypothalamus, and 14 days for NPY in the dorsomedial nucleus.

To compare anatomically similar regions, slides were matchedaccording to the rat brain atlas of Paxinos and Watson [46]. Slidesfrom control and treated animals, at each treatment time, werealways apposed to the same autoradiographic film. All sections werescanned and the specific hybridization signal was quantitated bydensitometry using a digital imaging system (Molecular Analyst®,Biorad, Calif., USA) [24]. The optical density of the hybridizationsignal was determined and subsequently corrected by the optical den-sity of its adjacent background value. For that, a rectangle, with thesame dimensions in all cases, was drawn enclosing the hybridizationsignal over each nucleus and over adjacent brain areas of each section(background).

Statistical Analysis and Data PresentationFood intake, RT-PCR and in situ data were analyzed by non-

parametric Mann-Whitney test. We compared control with treatedanimals. In order to analyze the time-dependent effect of the treat-ment we performed an ordinary ANOVA, followed by post hoc Bon-ferroni test, among the treated groups of the different time points.For all tests, significance was set at the p ! 0.05 level. Because of thepresence of circadian changes in hypothalamic mRNA content of the

Fig. 2. Effect of OX-A on food intake when administered i.c.v.(3 nmol) to satiated rats in the light phase (09:00 h). Food intake wasexamined 2 h (11:00), 3 h (12:00), 6 h (15:00) and 12 h (21:00) afterthe treatment. Data (mean B SEM) were expressed in relation tovehicle-treated rats. * p ! 0.05 vs. vehicle. n = 8–23 animals perexperimental group.

neuropeptides evaluated in this study [47–49], the results were nor-malized by comparisons with control animals killed at the same time.RT-PCR results were corrected by the endogenous HPRT mRNAlevels and the two lanes presented in all RT-PCR agarose gels repre-sent two different animals.

In all cases data (mean B SEM) were expressed as percentage ofchange in relation to control (vehicle-treated animals = 100%). Allthe statistical analysis and graphical representations presented in thiswork has been performed using one data point per animal.

Results

Orexin-A Induces Feeding in the Light PhaseICV administration of OX-A significantly increased

food intake when administered to satiated rats during theday. This orexigenic effect was transient being significant

38 Neuroendocrinology 2002;75:34–44 Lopez/Seoane/Garcıa/Diéguez/Señarıs

Fig. 3. A Upper panel: Representative RT-PCR detection of HPRTand NPY levels in the hypothalamus of control rats and rats treatedwith OX-A at the described times. Lower panel: NPY mRNA levelsin the hypothalamus of the described experimental groups. * p ! 0.05vs. vehicle; ## p ! 0.01, OX-A 2 h vs. OX-A 6 h and OX-A 12 h.B Upper panel: Autoradiographic images of representative braincoronal sections, incubated with a 35S-labelled antisense oligonucleo-tide NPY probe, in the vehicle-treated group and OX-A-treatedgroup for 2 h. Areas delineated in both sections are shown at higher

magnification in the right side. Lower panel: NPY mRNA levels inthe arcuate nucleus of the hypothalamus of the described experimen-tal groups. 3V = Third ventricle; * p ! 0.05 vs. vehicle; *** p ! 0.001vs. vehicle; ## p ! 0.01, OX-A 2 h vs. OX-A 4h; ### p ! 0.001, OX-A2 h vs. OX-A 6 h and OX-A 12h. C Autoradiographic images of rep-resentative brain coronal sections, at the level of the dorsomedialnucleus of the hypothalamus, incubated with a 35S-labelled antisenseoligonucleotide NPY probe in control rats and rats treated with OX-A for 2 h.

2 and 3 h after the treatment and disappearing 6 h afterthe administration of the OX-A (fig. 2).

Treatment with OX-A Increases NPY mRNA Contentbut Not AgRP mRNA Levels in the Arcuate Nucleus ofthe HypothalamusUsing RT-PCR analysis a significant increase of NPY

mRNA content was observed in the hypothalamus afterthe treatment with OX-A. This increase was transientbeing evident 2 h after the treatment, and absent 6 and12 h after OX-A administration (fig. 3A).

In order to further confirm the anatomical localizationof OX-A effects on NPY mRNA within the hypothalamuswe performed an in situ hybridization analysis looking atthe expression of NPY in the arcuate and the dorsomedialnuclei. Moreover, we added to the experimental protocola new group of rats treated with OX-A for 4 h. As it can beseen in figure 3B, our data confirmed the transient stimu-latory effect of OX-A shown by RT-PCR. Furthermore,we demonstrated that the stimulatory effect on hypotha-lamic NPY mRNA levels was circumscribed to the ar-cuate nucleus of the hypothalamus. No effect was noted in

Orexin-A and Orexigenic Pathways Neuroendocrinology 2002;75:34–44 39

Fig. 4. A Upper panel: Representative RT-PCR detection of HPRT and AgRP levels in the hypothalamus of controlrats and rats treated with OX-A at the described times. Lower panel: AgRP mRNA levels in the hypothalamus of thedescribed experimental groups. B Upper panel: Autoradiographic images of representative brain coronal sections,incubated with a 35S-labelled antisense oligonucleotide AgRP probe in the vehicle-treated group and the OX-A-treated group for 2 h. Areas delineated in both sections are shown at higher magnification on the right side. Lowerpanel: AgRP mRNA levels in the arcuate nucleus of the hypothalamus of the described experimental groups. 3V =Third ventricle.

the dorsomedial nucleus of the hypothalamus 2 h after thetreatment (fig. 3C)

On the other hand, we did not find any change onAgRP mRNA content in the arcuate nucleus of the hypo-thalamus at any time evaluated (2, 6 and 12 h) as assessedby RT-PCR and in situ hybridization (fig. 4).

OX-A Administration Does Not Change either MCHmRNA Content or Prepro-OX mRNA Content in theLateral HypothalamusUsing both RT-PCR analysis and in situ hybridization,

we were unable to demonstrate any effect of OX-A treat-ment on MCH mRNA content or prepro-OX mRNA con-

tent in the lateral hypothalamus at any time evaluated inthis study (fig. 5, 6).

OX-A Treatment Does Not Change HypothalamicOXRs mRNA LevelsOXRs mRNA content in the hypothalamus was not

modified after treatment with OX-A as assessed by RT-PCR (fig. 7).

40 Neuroendocrinology 2002;75:34–44 Lopez/Seoane/Garcıa/Diéguez/Señarıs

Fig. 5. A Upper panel: Representative RT-PCR detection of HPRT and MCH levels in the hypothalamus of controlrats and rats treated with OX-A at the described times. Lower panel: MCH mRNA levels in the hypothalamus of thedescribed experimental groups. B Upper panel: Autoradiographic images of representative brain coronal sections,incubated with a 35S-labelled antisense oligonucleotide MCH probe in vehicle-treated group and OX-A-treated groupfor 2 h. Lower panel: MCH mRNA levels in the lateral hypothalamic area of the described experimental groups.

Discussion

The role of orexins in the control of food intake hasrecently been demonstrated. The expression of their pre-cursor prepro-OX in the hypothalamus is upregulated byfasting, and the central administration of these peptidesto rats stimulates feeding [5, 8, 24]. Moreover, the i.c.v.treatment of an anti-orexin antibody to fasted rats sup-pressed feeding in a dose-dependent manner [50].

In spite of all this evidence the physiology of orexinsand their role within the orexigenic pathways are not com-pletely clear. Immunohistochemical studies have revealedthat axon terminals containing orexin make synaptic con-

tacts with arcuate nucleus NPY/AgRP cells and alsoexhibit close apposition with NPY containing nerve ter-minals in the paraventricular nucleus of the hypothala-mus [36]. Moreover, it has recently been published thatcentral administration of selective antagonists of NPY Y1and NPY Y5 receptors blocked feeding stimulation in-duced by orexins [37–40]. These findings suggested thatthe orexin system might interact with the NPY systemand serve as a stimulator of NPY containing cells in theregulation of hypothalamic mechanisms involved in foodintake. In order to confirm this hypothesis, we examinedthe effect of a single administration of OX-A (i.c.v.) onhypothalamic NPY mRNA levels, 2, 4, 6 and 12 h after

Orexin-A and Orexigenic Pathways Neuroendocrinology 2002;75:34–44 41

Fig. 6. A Upper panel: Representative RT-PCR detection of HPRT and prepro-OX levels in the hypothalamus ofcontrol rats and rats treated with OX-A at the described times. Lower panel: Prepro-OX mRNA levels in the hypo-thalamus of the described experimental groups. B Upper panel: Autoradiographic images of representative braincoronal sections, incubated with a 35S-labelled antisense prepro-OX mRNA probe in vehicle-treated group and OX-A-treated group for 2 h. Lower panel: Prepro-OX mRNA levels in the lateral hypothalamic area of the describedexperimental groups.

the treatment. Our results showed that central adminis-tration of OX-A induced a significant fast and transientincrease on hypothalamic NPY mRNA content. Withinthe hypothalamus these changes were clearly observedonly in NPY mRNA content of the arcuate nucleus; noeffect was seen in the dorsomedial nucleus. The functionof NPY in this latter nucleus is still unknown, and it hasonly been described to be upregulated by lactation in therat [51]. The transient effect of OX-A on NPY is in accor-dance with our food intake data showing a rapid increaseof feeding with a peak at about 3 h after the treatmentwhich disappears further on.

On the other hand, the rapid stimulation of NPYexpression in the arcuate nucleus after OX-A treatment,and the already described lack of effect of NPY on prepro-OX expression [52] suggest an upstream position for orex-ins, in relation to NPY, in the orexigenic pathways. More-over, orexin neurons in the lateral hypothalamus are sen-sitive to glucose and leptin [24-27]. Thus, it is quite possi-ble that peripheral information arriving at the hypothala-mus through circulating glucose and leptin levels maymodulate the orexins-NPY signalling network, regulatingfood intake and other neuroendocrine functions. Further-more, taking into in account that orexins are involved inthe regulation of locomotor and behavioral activities and

42 Neuroendocrinology 2002;75:34–44 Lopez/Seoane/Garcıa/Diéguez/Señarıs

Fig. 7. A Upper panel: Representative RT-PCR detection of HPRT and OX1R levels in the hypothalamus of controlrats and rats treated with OX-A at the described times. Lower panel: OX1R mRNA levels in the hypothalamus of thedescribed experimental groups. B Upper panel: Representative RT-PCR detection of HPRT and OX2R levels in thehypothalamus of control rats and rats treated with OX-A at the described times. Lower panel: OX2R mRNA levels inthe hypothalamus of the described experimental groups.

the sympathetic nervous system [14–17,19, 21], it is pos-sible that these peptides might stimulate some of theseactions via NPY neurons, suggesting that this functionallinkage acts as a general activating system and not only asa specifically feeding system.

The hypothalamic melanocortin system plays an im-portant role in the regulation of feeding [1, 2]. ·-Melano-cyte-stimulating hormone (·-MSH) synthesized in the hy-pothalamic arcuate nucleus, binds and activates the mela-nocortin-4 receptor (MC4-R), leading to reduced foodintake [7]. Agouti-related peptide (AgRP), which is alsosynthesized exclusively in the arcuate nucleus of the hypo-thalamus, where it colocalizes with NPY [34, 53], com-

petes with ·-MSH for the binding to MC4-R, therebystimulating food intake [7, 54, 55]. Then NPY/AgRP neu-rones, when they are activated, exert a dual effect to pro-mote hyperphagia: stimulation of NPY pathway andblockage of MC4-R by AgRP. The existence of an impor-tant orexin innervating network on NPY/AgRP neurones,suggests that orexins might regulate the melanocortin sys-tem through AgRP function. Using both, RT-PCR and insitu hybridization, we were unable to detect any change inAgRP mRNA content in the arcuate nucleus of the hypo-thalamus following OX-A administration, at any timeevaluated. These findings demonstrate a specific effect ofOX-A on NPY in NPY/AgRP expressing cells in the

Orexin-A and Orexigenic Pathways Neuroendocrinology 2002;75:34–44 43

arcuate nucleus of the hypothalamus. Our results suggestthat NPY synthesized at this level, but not AgRP system,is involved in the short-term orexin-induced feeding be-havior.

The hypothalamic melanin-concentrating hormonesystem is an essential regulator of food intake and energyhomeostasis. Central injections of MCH into the lateralventricle increases food intake in the rat [4, 8], and fooddeprivation increases hypothalamic mRNA content [4].The levels of the mRNA coding for this peptide are ele-vated in ob/ob mice [4], and MCH knockout mice exhibitsignificantly reduced food intake and body weight as com-pared to wild-type mice [56]. Taking into account thatOXRs are expressed in the lateral hypothalamus, we pos-tulated that MCH could be regulated by orexins. How-ever, we failed to find any meaningful change in MCHmRNA levels following OX-A administration at any timepoint studied. This indicates that the orexigenic action ofOX-A, at least in the fed state, is likely exerted through anMCH independent pathway.

Finally, it was not possible to demonstrate any effect ofOX-A treatment on either prepro-OX mRNA levels orOXRs mRNA content, suggesting a lack of effect of thispeptide at the level of its own gene expression, and that of

its receptors during the lengths of time evaluated in thisstudy. However, that the lack of effect of OX-A on prepro-OX, OXRs and MCH expression, could be due to anincomplete diffusion of OX-A from the third ventricle tothe lateral hypothalamus cannot be excluded.

In summary, in this paper we demonstrate a stimulato-ry effect of OX-A on NPY expression in the arcuatenucleus of the hypothalamus, without any change inAgRP and MCH mRNA levels, suggesting a rather spe-cific relationship between orexin and NPY. Taken togeth-er, these data can help to our understand further the inter-actions among the different systems involved in thecontrol of food intake, energy balance, as well as thepathophysiology of obesity.

Acknowledgements

We would like to thank Luz Casas for her excellent technicalassistance, Adolfo Figueiras (Department of Public Health, Facultyof Medicine, USC) for his help with the statistical analysis, andMasashi Yanagisawa for providing the prepro-OX, OX1R and OX2Rprobes.

Supported by grants from Fondo de Investigaciones Sanitarias,Spanish Ministry of Health, Xunta de Galicia and the DGICYT.

References

1 Kalra SP, Dube MG, Pu S, Xu B, Horvath TL,Kalra PS: Interacting appetite-regulating path-ways in the hypothalamic regulation of bodyweight. Endocr Rev 1999;20:68–100.

2 Meister B: Control of food intake via leptinreceptors in the hypothalamus. Vitam Horm2000;59:265–304.

3 Bray LS, Smith MA, Gold PW, Herkenham M:Altered expression of hypothalamic neuropep-tide mRNAs in food-restricted and food-de-prived rats. Neuroendocrinology 1990;52:441–447.

4 Qu D, Ludwig DS, Gammeltoft S, Piper M,Pelleymounter MA, Cullen MJ, Mathes WF,Przypek R, Kanarek R, Maratos-Flier E: A rolefor melanin-concentrating hormone in the cen-tral regulation of feeding behaviour. Nature1996;380:243–247.

5 Sakurai T, Amemiya A, Ishii M, Matsuzaki I,Chemelli R, Tanaka H, Willians S, RichardsonJ, Kozlowski G, Wilson S, Arch J, BuckinghamR, Haynes A, Carr S, Annan R, MacNutty D,Li W, Terrett J, Elshourbagy N, Bergsma D,Yanagisawa M: Orexins and orexins receptors:A family of hypothalamic neuropeptides and Gprotein-coupled receptors that regulate feedingbehaviour. Cell 1998;92:573–585.

6 Ziotopoulou M, Erani DM, Hileman SM, Bjor-baek C, Mantzoros CS: Unlike leptin, ciliaryneurotrophic factor does not reverse the starva-tion-induced changes of serum corticosterone

and hypothalamic neuropeptide levels but in-duces expression of hypothalamic inhibitors ofleptin signaling. Diabetes 2000;49:1890–1896.

7 Rossi M, Kim MS, Morgan DG, Small CJ,Edwards CM, Sunter D, Abusnana S, Gold-stone AP, Russell SH, Stanley SA, Smith DM,Yagaloff K, Ghatei MA, Bloom SR: A C-termi-nal fragment of Agouti-related protein in-creases feeding and antagonizes the effect ofalpha-melanocyte stimulating hormone invivo. Endocrinology 1998;139:4428–4431.

8 Edwards CM, Abusnana S, Sunter D, MurphyKG, Ghatei MA, Bloom SR: The effect of theorexins on food intake: Comparison with neu-ropeptide Y, melanin-concentrating hormoneand galanin. J Endocrinol 1999;160:R7–R12.

9 de Lecea L, Kilduff T, Peyron C, Gao X, FoyeP, Danielson P, Fukuhara C, Battenberg E,Gautvik V, Bartlett F, Frankel W, van den PolA, Bloom F, Gautvik K, Sutcliffe J: The hypo-cretins: Hypothalamus-specific peptides withneuroexcitatory activity. Proc Natl Acad SciUSA 1998;95:322–327.

10 Lin L, Faraco J, Li R, Kadotani H, Rogers W,Lin X, Qiu X, de Jong PJ, Nishino S, Mignot E:The sleep disorder canine narcolepsy is causedby a mutation in the hypocretin (orexin) recep-tor 2 gene. Cell 1999;98:365–376.

11 Chemelli R, Willie J, Sinton C, Elmquist J,Scammell T, Lee C, Richardson J, Williams S,Xiong Y, Kisanuki Y, Fitch T, Nakazato M,

Hammer R, Saper C, Yanagisawa M: Narco-lepsy in orexin knockout mice: Molecular ge-netics of sleep regulation. Cell 1999;98:437–451.

12 Nishino S, Ripley B, Overeem S, Lammers GJ,Mignot E: Hypocretin (orexin) deficiency inhuman narcolepsy. Lancet 2000;355:39–40.

13 Peyron C, Faraco J, Rogers W, Ripley B, Over-eem S, Charnay Y, Nevsimalova S, Aldrich M,Reynolds D, Albin R, Li R, Hungs M, Pedraz-zoli M, Padigaru M, Kucherlapati M, Fan J,Maki R, Lammers GJ, Bouras C, KucherlapatiR, Nishino S, Mignot E: A mutation in a case ofearly onset narcolepsy and a generalized ab-sence of hypocretin peptides in human narco-leptic brains. Nat Med 2000;6:991–997.

14 Hagan JJ, Leslie RA, Patel S, Evans ML, Wat-tam TA, Holmes S, Benham CD, Taylor SG,Routledge C, Hemmati P, Munton RP, Ash-meade TE, Shah AS, Hatcher JP, Hatcher PD,Jones DN, Smith MI, Piper DC, Hunter AJ,Porter RA, Upton N: Orexin A activates locuscoeruleus cell firing and increases arousal in therat. Proc Natl Acad Sci USA 1999;96:10911–10916.

15 Ida T, Nakahara K, Katayama T, Murakami N,Nakazato M: Effect of lateral cerebroventricu-lar injection of the appetite-stimulating neuro-peptide, orexin and neuropeptide Y, on thevarious behavioral activities of rats. Brain Res1999;821:526–529.

44 Neuroendocrinology 2002;75:34–44 Lopez/Seoane/Garcıa/Diéguez/Señarıs

16 Ida T, Nakahara K, Murakami T, Hanada R,Nakazato M, Murakami N: Possible involve-ment of orexin in the stress reaction in rats.Biochem Biophys Res Commun 2000;270:318–323.

17 Jones DN, Gartlon J, Parker F, Taylor SG,Routledge C, Hemmati P, Munton RP, Ash-meade TE, Hatcher JP, Johns A, Porter RA,Hagan JJ, Hunter AJ, Upton N: Effects of cen-trally administered orexin-B and orexin-A: Arole for orexin-1 receptors in orexin-B-inducedhyperactivity. Psychopharmacology 2001;153:210–218.

18 Samson WK, Gosnell B, Chang JK, Resch ZT,Murphy TC: Cardiovascular regulatory actionsof the hypocretins in brain. Brain Res 1999;831:248–253.

19 Shirasaka T, Nakazato M, Matsukura S, Takas-aki M, Kannan H: Sympathetic and cardiovas-cular actions of orexins in conscious rats. Am JPhysiol 1999;277:R1780–R1785.

20 Bingham S, Davey PT, Babbs AJ, Irving EA,Sammons MJ, Wyles M, Jeffrey P, Cutler L,Riba I, Johns A, Porter RA, Upton N, HunterAJ, Parsons AA: Orexin-A, an hypothalamicpeptide with analgesic properties. Pain 2001;92:81–90.

21 van den Pol AN: Hypothalamic hypocretin (or-exin): Robust innervation of the spinal cord. JNeurosci 1999;19:3171–3182.

22 Date Y, Ueta Y, Yamashita H, Yamaguchi H,Matsukura S, Kangawa K, Sakurai T, Yanagi-sawa M, Nakazato M: Orexins, orexigenic hy-pothalamic peptides, interact with autonomic,neuroendocrine and neuroregulatory systems.Proc Natl Acad Sci USA 1999;96:748–753.

23 Pu S, Jain MR, Kalra PS, Kalra SP: Orexins, anovel family of hypothalamic neuropeptides,modulate pituitary luteinizing hormone secre-tion in an ovarian steroid-dependent manner.Regul Pept 1998;78:133–136.

24 Lopez M, Seoane L, Garcıa MC, Lago F, Cas-anueva FF, Senarıs R, Diéguez C: Leptin regu-lation of prepro-orexin and orexin receptormRNA levels in the hypothalamus. BiochemBiophys Res Commun 2000;269:41–45.

25 Moriguchi T, Sakurai T, Nambu T, Yanagisa-wa M, Goto K: Neurons containing orexin inthe lateral hypothalamic area of the adult ratbrain are activated by insulin-induced acutehypoglycemia. Neurosci Lett 1999;264:101–104.

26 Griffond B, Risold PY, Jacquemard C, ColardC, Fellmann D: Insulin-induced hypoglycemiaincreases preprohypocretin (orexin) mRNA inthe rat lateral hypothalamic area. Neurosci Lett1999;262:77–80.

27 Cai XJ, Widdowson PS, Harrold J, Wilson S,Buckingham RE, Arch JR, Tadayyon M, Cla-pham JC, Wilding J, Williams G: Hypothalam-ic orexin expression: Modulation by blood glu-cose and feeding. Diabetes 1999;48:2132–2137.

28 Lopez M, Seoane L, Señarıs R, Diéguez C: Pre-pro-orexin mRNA levels in the rat hypothala-mus, and orexin mRNA levels in the rat hypo-thalamus and adrenal gland are not influenceby thyroid status. Neurosci Lett 2001;300:171–175.

29 Trivedi P, Yu H, MacNeil D, Van der Ploeg L,Guan X: Distribution of orexin receptormRNA in the rat brain. FEBS Lett 1999;438:71–75.

30 Hervieu GJ, Cluderay JE, Harrison DC, Ro-berts JC, Leslie RA: Gene expression and pro-tein distribution of the orexin-1 receptor in therat brain and spinal cord. Neuroscience 2001;103:777–797.

31 Lopez M, Señarıs R, Gallego R, Garcıa-Cabal-lero T, Lago F, Seoane L, Casanueva F, Dié-guez C: Orexin receptors are expressed in theadrenal medulla of the rat. Endocrinology1999;140:5991–5994.

32 Kirchgessner AL, Liu M: Orexin synthesis andresponse in the gut. Neuron 1999;24:941–951.

33 Elias C, Saper C, Marathos-Flier E, Tritos N,Lee C, Kelly J, Tatro J, Hoffman G, OllmannM, Barsh G, Sakurai T, Yanagisawa M, Elm-quist J: Chemically defined projections linkingthe mediobasal hypothalamus and the lateralhypothalamic area. J Comp Neurol 1998;402:442–459.

34 Broberger C, de Lecea L, Sutcliffe J, Hökfelt T:Hypocretin/orexin and melanin concentratinghormone-expressing cells form distinct popula-tions in the rodent lateral hypothalamus: Rela-tionship to the neuropeptide Y and agouti generelated protein systems. J Comp Neurol 1998;402:460–474.

35 Peyron C, Tighe D, van den Pol A, de Lecea L,Heller H, Sutcliffe J, Kilduff T: Neurons con-taining hypocretin (orexin) project to multipleneuronal systems. J Neurosci 1998;18:9996–10015.

36 Horvath T, Diano S, van den Pol A: Synapticinteraction between hypocretin (orexin) andneuropeptide Y cells in the rodent and primatehypothalamus: A novel circuit implicated inmetabolic and endocrine regulations. J Neuro-sci 1999;19:1072–1087.

37 Yamanaka A, Kunii K, Nambu T, Tsujino N,Sakai A, Matsuzaki I, Miwa Y, Goto K, Saku-rai T: Orexin-induced food intake involvesneuropeptide Y pathway. Brain Res 1999;859:404–409.

38 Jain MR, Horvath TL, Kalra PS, Kalra SP:Evidence that NPY Y1 receptors are involvedin stimulation of feeding by orexins (hypocre-tins) in sated rats. Regul Pept 2000;87:19–24.

39 Ida T, Nakahara K, Kuroiwa T, Fukui K, Na-kazato M, Murakami T, Murakami N: Bothcorticotropin releasing factor and neuropeptideY are involved in the effect of orexin (hypocre-tin) on the food intake in rats. Neurosci Lett2000;293:119–122.

40 Dube MG, Horvath TL, Kalra PS, Kalra SP:Evidence of NPY Y5 receptor involvement infood intake elicited by orexin A in sated rats.Peptides 2000;21:1557–1560.

41 Bannon AW, Seda J, Carmouche M, FrancisJM, Norman MH, Karbon B, McCaleb ML:Behavioral characterization of neuropeptide Yknockout mice. Brain Res 2000;868:79–87.

42 Egawa M, Yoshimatsu H, Bray GA: Neuropep-tide Y suppresses sympathetic activity to inter-scapular brown adipose tissue in rats. Am JPhys 1991;260:R1314–R1320.

43 Lu XY, Bagnol D, Burke S, Akil H, Watson SJ:Differential distribution and regulation of OX1and OX2 orexin/hypocretin receptor messen-ger RNA in the brain upon fasting. HormBehav 2000;37:335–344.

44 Carro E, Señarıs R, Seoane L, Frohman L, Ari-mura A, Casanueva F, Diéguez C: Role ofgrowth hormone (GH)-releasing hormone andsomatostatin on leptin-induced GH Secretion.Neuroendocrinology 1999;69:3–10.

45 Señarıs RM, Lago F, Coya R, Pineda J, Dié-guez C: Regulation of hypothalamic somatosta-tin, growth hormone-releasing hormone, andgrowth hormone receptor messenger ribonucle-ic acid by glucocorticoids. Endocrinology 1996;137:5236–5241.

46 Paxinos G, Watson C: The Rat Brain in Stereo-taxic Coordinates. Sydney, Academic Press, 1986.

47 Akabayashi A, Levin N, Paez X, Alexander JT,Leibowitz F: Hypothalamic neuropeptide Yand its gene expression: Relation to light/darkcycle and circulating corticosterone. Mol CellNeurosci 1994;5:210–218.

48 Bluet-Pajot MT, Presse F, Voko Z, Hoeger C,Mounier F, Epelbaum J, Nahon JL: Neuropep-tide-E-I antagonizes the action of melanin-con-centrating hormone on stress-induced releaseof adrenocorticotropin in the rat. J Neuroendo-crinol 1995;7:297–303.

49 Taheri S, Sunter D, Dakin C, Moyes S, Seal L,Gardiner J, Rossi M, Ghatei M, Bloom S: Diur-nal variation in orexin A immunoreactivity andprepro-orexin mRNA in the rat central nervoussystem. Neurosci Lett 2000;279:109–112.

50 Yamada H, Okumura T, Motomura W, Ko-bayashi Y, Kohgo Y: Inhibition of food intakeby central injection of anti-orexin antibody infasted rats. Biochem Biophys Res Commun2000;267:527–531.

51 Li C, Chen P, Smith MS: The acute sucklingstimulus induces expression of neuropeptide Y(NPY) in cells in the dorsomedial hypothala-mus and increases NPY expression in the ar-cuate nucleus. Endocrinology 1998;139:1645–1652.

52 Pu S, Dhillon H, Moldawer LL, Kalra PS, Kal-ra SP: Neuropeptide Y counteracts the anorec-tic and weight reducing effects of ciliary neuro-tropic factor. J Neuroendocrinol 2000;12:827–832.

53 Hahn TM, Breininger JF, Baskin DG,Schwartz MW: Coexpression of AgRP andNPY in fasting-activated hypothalamic neu-rons. Nat Neurosci 1998;1:271–272.

54 Ollmann MM, Wilson BD, Yang YK, KernsJA, Chen Y, Gantz I, Barsh G: Antagonism ofcentral melanocortin receptors in vitro and invivo by agouti-related protein. Science 1997;278:135–138.

55 Hagan MM, Rushing PA, Pritchard LM,Schwartz MW, Strack AM, Van Der Ploeg LH,Woods SC, Seeley RJ: Long-term orexigeniceffects of AgRP-(83–132) involve mechanismsother than melanocortin receptor blockade.Am J Physiol 2000;279:R47–R52.

56 Shimada M, Tritos NA, Lowell BB, Flier JS,Maratos-Flier E: Mice lacking melanin-concen-trating hormone are hypophagic and lean. Na-ture 1998;396:670–674.