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Peptides 47 (2013) 20–28

Contents lists available at ScienceDirect

Peptides

j ourna l ho me pa g e: www.elsev ier .com/ locate /pept ides

YY(3-36) into the arcuate nucleus inhibits food deprivation-induced increases inood hoarding and intake�

rett J.W. Teubner, Timothy J. Bartness ∗

epartment of Biology and Obesity Reversal Center, Georgia State University, Atlanta, GA 30302-4010, USA

r t i c l e i n f o

rticle history:eceived 24 January 2012eceived in revised form 11 May 2013ccepted 13 May 2013vailable online 29 June 2013

eywords:ppetitive behavioriberian hamstereptide YYIIE0246

a b s t r a c t

Central administration of neuropeptide Y (NPY) increases food intake in laboratory rats and mice, as wellas food foraging and hoarding in Siberian hamsters. The NPY-Y1 and Y5 receptors (Rs) within the hypo-thalamus appear sufficient to account for these increases in ingestive behaviors. Stimulation of NPY-Y2Rsin the Arcuate nucleus (Arc) has an anorexigenic effect as shown by central or peripheral administrationof its natural ligand peptide YY (3-36) and pharmacological NPY-Y2R antagonism by BIIE0246 increasesfood intake. Both effects on food intake by NPY-Y2R agonism and antagonism are relatively short-livedlasting ∼4 h. The role of NPY-Y2Rs in appetitive ingestive behaviors (food foraging/hoarding) is untested,however. Therefore, Siberians hamsters, a natural food hoarder, were housed in a semi-natural bur-row/foraging system that had (a) foraging requirement (10 revolutions/pellet), no free food (true foraginggroup), (b) no running wheel access, free food (general malaise control) or (c) running wheel access, free

ood foragingood deprivation

food (exercise control). We microinjected BIIE0246 (antagonist) and PYY(3-36) (agonist) into the Arc to testthe role of NPY-Y2Rs there on ingestive behaviors. Food foraging, hoarding, and intake were not affectedby Arc BIIE0246 microinjection in fed hamsters 1, 2, 4, and 24 h post injection. Stimulation of NPY-Y2Rsby PYY(3-36) inhibited food intake at 0–1 and 1–2 h and food hoarding at 1–2 h without causing generalmalaise or affecting foraging. Collectively, these results implicate a sufficiency, but not necessity, of theArc NPY-Y2R in the inhibition of food intake and food hoarding by Siberian hamsters.

. Introduction

In modern industrialized nations, the incidence of obesity hasncreased markedly over the last few decades and has led to a risen severe secondary health consequences. Given that most animalsorage for food, including humans [for reviews see: [7,31]], we pos-ulated recently that a largely ignored set of related factors leads toizeable food hoards and has helped propel the obesity crisis: (a)ize of refrigerators, freezers and pantries, (b) processes that extendhe shelf lives of food well beyond that of 25–50 years ago, andc) ample and inexpensive calorically dense food stuffs [7]. There-ore, a deepened understanding of food foraging and hoarding mayead to behavioral and/or pharmacological treatments for over-

eight/obese humans, as we have suggested previously [5,7,31].Using Wallace Craig’s [14] division of animal behavior into appe-

itive (behavior leading to the goal) and consummatory (realization

� This work was supported by the NIH R01 DK078358 to TJB and NIH F32K091984 to BJWT.∗ Corresponding author at: Department of Biology and Obesity Reversal Center,4 Peachtree Center Avenue NE, Georgia State University, Atlanta, GA 30302-4010,SA. Tel.: +1 404 413 5334; fax: +1 404 413 5301.

E-mail address: bartness@gsu.edu (T.J. Bartness).

196-9781 Published by Elsevier Inc.ttp://dx.doi.org/10.1016/j.peptides.2013.05.005

Open access under CC BY-NC-ND license.

Published by Elsevier Inc.

of the goal) phases, ingestive behavior is dichotomized as foodforaging/hoarding (appetitive phase) and food intake (consumma-tory phase). We know considerably more about consummatoryingestive behaviors than appetitive behaviors because the mostcommonly studied animals in ingestive behavior research are lab-oratory rats and mice. They are not natural hoarders [for review:[7]] and are typically housed in standard cages that do not permit asignificant effort to obtain food. We are able to measure food forag-ing, hoarding, and intake using our simulated burrow system [17]and Siberian hamsters (Phodopus sungorus), as they hoard food innature [49] and in the laboratory (for review see: [7,31]).

Unlike laboratory rats and mice that overeat after a fast [e.g.,[27,53]], food deprived Siberian hamsters do not overeat, nor dohumans, once access to food is restored but instead ‘overhoard’, asdo humans [for review see: [7]]. Therefore, we reasoned that otherstimuli that increase food intake by laboratory rats and mice maytrigger increases in food hoarding by these hamsters. Indeed, welaunched several studies of the peptidergic control of food hoardingguided by this premise. Some of these studies focused on the arcu-ate nucleus (Arc) and the neuropeptide Y (NPY) and agouti-related

Open access under CC BY-NC-ND license.

protein (AgRP) neurons found therein [15,16,19,20,28,29]. As inlaboratory rats [41,42,44], and mice [8], NPY and AgRP are nearlyexclusively co-localized in neurons within the medial portions ofthe Arc in Siberian hamsters and Arc NPY and AgRP synthesis is

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timulated by food deprivation in Siberian hamsters [22,25,34]aking them a possible mediator of food deprivation-induced

ncreases in foraging/hoarding.NPY is a powerful orexigenic peptide when applied centrally in

aboratory rats [e.g., [33,43]] and other species [for review see: [6]].oreover, NPY is not only a powerful orexigenic peptide in Siberian

amsters [10,15], but also is a powerful short-term (1–4 h, but upo 24 h) stimulator of food hoarding [15,16,20,28,29]. NPY has sev-ral receptor (R) sub-types (NPY-Y1-5) that are broadly distributednd their stimulation results in a diverse range of functions [foreview see: [48]]. The NPY Y1- and Y5-R have been implicated in theontrol of food intake in laboratory rats and mice [for review see:21]]. Microinjections of a Y1-R agonist into the PVH or PFA triggers

dose-dependent increase in food intake in laboratory rats [45]nd, conversely, prior or co-injection of a NPY Y1-R antagonist intohe PVH blocks the ability of PVH NPY injections to increase foodntake [50,51]. NPY Y1-R agonism primarily increases food hoard-ng, whereas NPY Y5-R agonism primarily increases food intake inur foraging/hoarding model using Siberian hamsters [20,29].

Another NPY receptor subtype that has been strongly implicatedn food intake, the NPY Y2-R, is located presynaptically and found in

number of CNS sites, including the Arc and appears to function asn autoreceptor on NPY/AgRP neurons to inhibit their activity andhereby inhibit food intake [11]. A naturally-occurring ligand forhe NPY Y2-R is peptide tyrosine–tyrosine (PYY), a gut-derived hor-

one released from L cells in the intestine after a meal primarily inhe form of PYY(3-36) [2]. PYY(3-36) is a selective agonist for the NPY-2R resulting in inhibition of food intake, both endogenously andxogenously [1,9]. Consistent with these effects, antagonism of thePY-Y2R using the Y2-R selective antagonist BIIE0246, increases

ood intake, adding further support for a role of NPY-Y2R in theessation of food intake [1]. Therefore, the purpose of the presentxperiments was to test the role of the NPY Y2-R in food forag-ng, food hoarding, and food intake in Siberian hamsters. To do so

e asked two questions: (1) Does antagonism of NPY Y2-R usingIIE0246 increase ingestive behaviors in fed animals and (2) doesgonism of NPY Y2-R using the naturally-occurring PYY(3-36) inhibithe food deprivation-induced increases in ingestive behaviors?

. Materials and methods

.1. Animals and housing

Two separate cohorts of 40 male Siberian hamsters 2.5–3onths of age and weighing 35–45 g were selected from our breed-

ng colony. After weaning animals were group housed accordingo sex and raised in a long day photoperiod (16L:8D, light off-et: 1900) with ad libitum access to rodent chow (LabDiet® 5001,urina, St. Louis, MO) and tap water unless otherwise indicated.oom temperature was maintained at 21 ± 2 ◦C. Each cohort wasreated identically. All procedures were approved by the Georgiatate University Institutional Animal Care and Use Committee andere in accordance with Public Health Service and United Statesepartment of Agriculture guidelines.

.2. Foraging and hoarding apparatus

Animals were transferred to the foraging and hoard-ng room where they were singly housed in shoebox cages90 mm × 180 mm × 130 mm (length × width × height), main-ained in a 16L:8D photoperiod (light offset: 1330), and with

d libitum access to the pelleted test diet (DPPs, Purified 75 mgellets; Bio-Serve, Frenchtown, NJ) and water. After two weekso acclimate to the new light offset, animals were placed intohe foraging and hoarding apparatus modified from Perrigio and

eptides 47 (2013) 20–28 21

Bronson [39] and previously described [19]. Briefly, a bottom, “bur-row”, cage 290 mm × 180 mm × 130 mm (length × width × height)containing Alpha-Dri bedding (Specialty Papers, Kalamazoo,MI) and one cotton nestlet (Anacare, Belmore, NY). The bottomcage was opaque and covered to simulate the darkness of aburrow. The top, “foraging”, cage 456 mm × 234 mm × 200 mm(length × width × height) was equipped with a pellet dispenser,running wheel (525 cm circumference), and ad libitum access towater. The two cages were connected via convoluted polyvinylchloride tubing (38.1 mm inner diameter and ∼1.52 m long). Wheelrevolutions were counted using a magnetic detection system withmonitoring by a hardware/software computer interface (MedAssociates, Georgia, VT). Hamsters were acclimated/trained to thisapparatus for one week prior to and after cannulation (see below).

We used an acclimation/training regimen that minimizeschanges in body mass and food intake that can occur when ini-tially housed in the foraging and hoarding apparatus. Specifically,hamsters were given free access to food pellets and were able toearn a food pellet for every 10 wheel revolutions. After the firsttwo days the free access to food was removed and all food hadto be earned (1 pellet/10 wheel revolutions) for 5 d, during whichbody mass, wheel revolutions, pellets earned (food foraging), foodintake, and food hoarding were measured daily. After the 7 d accli-mation/training period, animals were placed temporarily back intothe shoebox cages before cannula implantation (see below).

2.3. Foraging groups and measurement of foraging, foodhoarding, and food intake

Three foraging groups were used as in our first of many reportsof these groups [17]. When foraging effort is required beyondtraversing the tubing, then completion of a programmed number ofwheel revolutions triggers food pellet delivery, usually 10, as ≥10inhibits hoarding due to decreased payoff–this is the 10 revolutionper pellet group (10REVS). Two non-foraging conditions critical tointerpreting the 10REVS foraging results were included. In the FreeWheel (FW) condition, food (300 pellets) was presented in the cagenon-contingently and independent of wheel running, but wheelrunning was allowed (controlling for non-specific locomotor stim-ulation/inhibition thereby providing insight into earned food by the10REVS group). In the Blocked Wheel (BW) condition, food (300pellets) also was presented non-contingently, but the wheel wasblocked (controlling for locomotor activity-induced changes – i.e.,sedentary controls).

Foraging (pellets earned) was defined as the number of pel-lets earned (10REV) and food hoarding was defined as the numberof pellets found in the bottom cage plus those removed from thecheek pouches. For the BW and FW groups where food was givennon-contingently, food intake was defined as the number of pelletssupplied (300 pellets/day) minus the total pellets hoarded or leftin the top cage (surplus pellets). In the 10REV group, food intakewas defined as the number of pellets earned minus the total pel-lets hoarded or left in the top cage (surplus pellets). The electronicscale used to weigh the food pellets was set to “parts” measure-ment, resulting in one 75 mg food pellet = 1 with fractions of pelletscomputed by the scale.

2.4. Cannula implantation, injections, and verification

Cannulae were stereotaxically implanted aimed unilaterally atthe ventromedial aspect of the Arc (posterior to bregma: −1.4 mm,lateral to midline: 0.3 mm, and ventral to skull: −8.0 mm), because

this region shows the densest NPY-Y2R expression in rats [37] andmice [23] under isoflurane (Aerrane, Baxter Healthcare Corpora-tion, Deerfield, IL) inhalation anesthesia as previously described[19]. In brief, each animal had hair removed from the top of their

22 B.J.W. Teubner, T.J. Bartness / Peptides 47 (2013) 20–28

Fig. 1. Cannula location plotted onto schematic brain section diagrams (taken from Paxinos and Watson [38]) with filled circles (�) representing the tip of a correctlyplaced cannula and filled diamonds (�) representing misplaced cannula. (A) Plate 43, (B) Plate 44, (C) Plate 45, (D) Plate 46, (E) Plate 47 (F) Plate 49 and (G) Plate 50.S cleus;A halamd othala

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ome circles represent more than one cannula. 3V, third ventricle; Arc, Arcuate nurcuate nucleus; ArcMP, medioposterior Arcuate nucleus; DM, dorsomedial hypotorsomedial ventromedial hypothalamus; VMHVL, ventrolateral ventromedial hyp

ead, skull exposed, and were placed into a stereotaxic surgicalpparatus (David Kopf Instruments, Tujunga, CA). A guide cannula26 gauge stainless steel; Plastics One, Roanoke, VA) was lowerednto place and secured to the skull using cyanoacrylate ester gel,/16 mm jeweler’s screws, and dental acrylic. The opening in theuide cannula was sealed using a removable obturator throughouthe experiment except during parenchymal injections. Hamsterseceived buprenorphine (0.2 mg/kg body mass, s.c.) to minimizeiscomfort and apple slices to facilitate food and water intake

mmediately following surgery and for the following 2 d. Animalsere housed in shoebox cages for 2 weeks following surgery before

eing returned to the foraging and hoarding apparatus.

Each animal was “mock-injected” daily in the week before a

est day, where the obturator was removed and the animal wasightly restrained for 1 min to acclimate the animal to the injectionrocedure. On test days, an inner cannula (33 gauge stainless steel,

ArcD, dorsal Arcuate nucleus; ArcL, lateral Arcuate nucleus; ArcLP, lateroposteriorus; ME, median eminence; VMHC, central ventromedial hypothalamus; VMHDM,mus.

Plastics One, Roanoke, VA) was connected to a Hamilton syringevia PE-20 tubing and inserted into the guide cannula, extending0.5 mm below the guide cannula tip. All injections were given atlight offset (1330 EST). Each injection (200 nl) of neurochemical orvehicle was delivered over 30 s and the injection needle remainedin place for ∼30 s before removal, as done previously [e.g., [15,19]].

Following the final test day, animals were injected with 300 nlbromophenol blue dye to mark the location of the cannula tipand animals were then given an overdose of pentobarbital sodium(100 mg/kg), transcardially perfused with 100 ml of heparinizedsaline followed by 125 ml of 4% paraformaldehyde in phosphatebuffered saline, pH = 7.4. The brains were then removed and post

fixed in a 4% paraformaldehyde solution for 2 d, followed by a 30%sucrose solution until sectioning, replacing the sucrose solutionafter 24 h. Brains were sectioned at 80 �m for cannula location ver-ification using light microscopy. Cannulae were considered an Arc

ess / Peptides 47 (2013) 20–28 23

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B.J.W. Teubner, T.J. Bartn

it if the blue dye was visible in the ventromedial aspect of the Arcnd only these animals were included in the analyses (n = 75, seeig. 1 for cannula locations).

.5. Baseline data and foraging groups

At the conclusion of the acclimation/training period animalsere separated into one of the three foraging groups (10REV, FW,W) described above. Animals were separated into the groupsatched for body mass, food intake, and food hoarding and were

llowed 2 weeks to acclimate to their foraging treatment group.

.6. Experiment 1: NPY-Y2R antagonism by BIIE0246 in fednimals

Arc injections consisted of one of three doses of BIIE0246 (0.1,.0, 5.0 nmol in 200 nl) or vehicle (5% DMSO), with vehicle choicend doses based on effective Arc delivered drug in laboratory rats1]. Each animal received all injections in a counterbalanced-withinubjects design. A washout period of 1 wk separated individualnjections to ensure all measures had returned to baseline val-es similar to our previous work [29]. On injection days, animalsere provided with a clean burrow cage and access to food wasrevented by blocking access to the top cage 2 h before injec-ions. Animals were injected at light offset and access to food waseturned. Wheel revolutions, food foraging, food hoarding, and foodntake were measured at 1, 2, 4, 24 h and each day post-injectionntil the next test day (final group sizes BW: n = 21, FW: n = 22, and0REV: n = 26).

.7. Experiment 2: NPY-Y2R agonism by PYY(3-36) inood-deprived animals

After the week-long washout period following the final BIIE0246est day, animals were maintained in their foraging treatmentroup for an additional week and then assigned to an injectionreatment group (see below) balanced for body weight, food intake,nd food hoarding as above. The animals were then food deprivedor 56 h (IACUC approved), a time length previously shown to max-mize food hoarding [4,18]. Before access to food was returned atight offset, half of the animals received an injection of PYY(3-36)0.1 nmol in 200 nl), the active form of the peptide for satiation52], into the Arc and the other half received the saline vehicle.

heel revolutions, food foraging, food intake, and food hoardingere measured at 1, 2, 4, 24 h and each day post-injection until all

nimals returned to pre-injection levels. After the animals returnedo behavioral baseline, brain tissue was collected to verify cannulaocation (Fig. 1; 69 hits and 11 misses or removed their cannula;nal group sizes: PYY(3-36): BW: n = 12, FW: n = 11, and 10REV:

= 13 and vehicle: BW: n = 9, FW: n = 11, and 10REV: n = 13).

.8. Statistics

Raw data from Experiment 1 were transformed for each indi-idual into percent change from vehicle before statistical analysessing the formula: [((X-Vehicle)/Vehicle) × 100], where “X” equalshe value measured in response to the dose of BIIE0246 andVehicle” equals the value measured for that individual after vehi-le injection. No statistical comparisons were made among theime intervals because the intervals were of unequal duration.o statistical comparisons are reported across test days in thisounterbalanced-within subject design, as repeated measures two-

ay ANOVA (foraging treatment × Arc-injection) showed no effect

f injection order. The data were analyzed using a two-way ANOVAforaging treatment × Arc-injection; 3 × 4). For Experiment 2, dataere not transformed into percent change from vehicle, because

requirement and a functional running wheel [Free Wheel (FW)] and (B) forag-ing [pellets earned; in hamsters with a 10 wheel revolutions per pellet foragingrequirement (10REV)].

animals only were food deprived once and therefore could notserve as their own control, and the absolute values were analyzedusing a two-way ANOVA (foraging treatment × Arc-injection; 3 × 2)within each individual time point for the same reason as above.All statistical analyses were performed using NCSS (version 2007,Kaysville, UT). Exact probabilities and test values were omitted forsimplicity and clarity of presentation. Differences were consideredstatistically significant if P < 0.05. Tukey-Kramer Multiple Compari-son Tests were used for post hoc tests when appropriate. Misplacedcannulae were not included in the final statistical comparisons.

3. Results

3.1. Experiment 1: NPY-Y2R antagonism with BIIE0246 in fedanimals

Wheel running. At each time interval, Arc injection of BIIE0246did not significantly stimulate wheel running activity compared

to vehicle injection at any of the three doses tested (0.1, 1.0and 5.0 nmol; Fig. 2A). The lack of wheel running increase in theFW group, where food delivery was not contingent upon wheel

2 ess / Peptides 47 (2013) 20–28

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4 B.J.W. Teubner, T.J. Bartn

unning, suggests that there was not non-specific stimulation ofocomotor activity.

Food foraging. Arc injection of BIIE0246 did not significantlyncrease food foraging (wheel running contingent food delivery) atny time point examined (Fig. 2B) above vehicle-treated animals.

Food intake. Food intake was not stimulated above vehicle at anyime interval examined (0–1, 1–2, 2–4, 4–24 h) by Arc injection ofIIE0246 for all three foraging treatments (10REV, FW, and BW;ig. 3A–C).

Food hoarding. BIIE0246 injection into the Arc did not stim-late food hoarding compared to vehicle injection at any timeoint examined for each foraging treatment (10REV, FW, and BW;ig. 4A–C). During the 2–4 h interval after the 5.0 nmol BIIE0246njection, hoarding in the 10REV group approached significanceP = 0.059) when compared with vehicle injection.

.2. Experiment 2: NPY-Y2R agonism using PYY(3-36) inood-deprived animals

Wheel running. PYY(3-36) treatment inhibited the foodeprivation-induced increases in wheel running during the–1 h interval (Fig. 5A). The inhibition of wheel running is not

ndicative of malaise caused by PYY(3-36), because the inhibitionlso was not seen in the 10REV group (see below).

Food foraging. Arc injection of PYY(3-36) did not result in a sig-ificant inhibition of food foraging compared to saline injection atny time point measured (0–1, 1–2, 2–4, 4–24 h as well as duringhe next 6 d; Fig. 5B).

Food intake. Arc injection of PYY(3-36) attenuated food intakefter food deprivation compared with Arc saline injection at 0–1nd 1–2 h for the 10REV foraging treatment (Fig. 6C), but not in theW or FW group (Fig. 6A and B); no significant differences wereresent after the first two hours of refeeding for any group.

Food hoarding. In the 10REV group, agonism of the NPY-Y2Rn the Arc using PYY(3-36) inhibited food hoarding upon refeedingompared with saline injection at 1–2 h and approached signifi-ance at 0–1 h (P = 0.07; Fig. 7C). Significant differences were noteen at any other time point or foraging treatment (Fig. 7A and B).

. Discussion

Controlling ingestive behavior is a vital aspect of prevent-ng/treating obesity and accordingly a concerted effort has been

ade to describe the mechanisms involved in food intake. NPYs the most potent central orexigenic neurochemical in labora-ory rats [13,33,43] with marked increases in food hoarding andntake occurring with stimulation of Y1-R and Y5-R, respectivelyn Siberian hamsters [20]. The NPY-Y2R agonism/antagonism hadot been tested for its role in the appetitive ingestive behav-

ors of food hoarding or foraging [14] in any species before theresent study. Here we found for the first time that agonism ofhe Y2-R by PYY(3-36) inhibited food intake and hoarding earlyfirst few hours) after refeeding following food deprivation andhat antagonism of the Y2-R by BIIE0246 did not affect appeti-ive or consummatory ingestive behaviors in fed hamsters. Theseesults suggest a possible inhibitory role of PYY(3-36) in food hoard-ng.

Antagonism of the Y2-R in the Arc using BIIE0246 causes shorterm increases in food intake by laboratory rats [1], effects similaro those of NPY Y1-R and Y5-R agonism [e.g., [24,45]]. NPY-Rs have

dual role in the control of food intake, where Y2-R and Y4-R

gonism is anorexigenic and Y1-R and Y5-R agonism is orexigenicn other rodents [3,21,32]. This dualism only partially extendedo Siberian hamsters here as PYY(3-36) microinjections into therc inhibited food intake and especially food hoarding, but the

tionary running wheel [Blocked Wheel (BW)], (B) no foraging requirement and afunctional running wheel [Free Wheel (FW)], and (C) a 10 wheel revolutions perpellet foraging requirement [10 revolutions/pellet (10REV)].

B.J.W. Teubner, T.J. Bartness / Peptides 47 (2013) 20–28 25

Food Hoard edA. Blocked Wheel/ Fre e Foo d

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Fig. 4. Mean ± SEM percent change from vehicle (5% DMSO) in response to BIIE0246(0.1, 1.0, and 5.0 nmol) of food hoarded with (A) no foraging requirement and astationary running wheel [Blocked Wheel (BW)], (B) no foraging requirement anda functional running wheel [Free Wheel (FW)], and (C) a 10 wheel revolutions perpellet foraging requirement [10 revolutions/pellet (10REV)].

B. Food F oraging

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Fig. 5. Mean ± SEM of Arc injection of PYY(3-36) (0.1 nmol) or Saline post-fooddeprivation of (A) wheel revolutions in hamsters with no foraging requirements

and a functional running wheel [Free Wheel (FW)] and (B) foraging (pellets earned)in hamsters with a 10 wheel revolutions per pellet foraging requirement [10 revo-lutions/pellet (10REV)]. *P < 0.05 vs saline.

NPY-Y2R antagonist BIE0246 did not stimulate food foraging,intake, or hoarding. This lack of effect of BIIE0246 on baseline foodintake also has been reported for laboratory rats [40]. It is possiblethat our BIIE0246 dose was insufficient to block endogenous Y2signaling, although this seems somewhat unlikely because we useda dose 5-times greater than a dose effective in rats [1]. In two pilotstudies, we injected the highest dose of BIIE0246 (5.0 nmol) usedhere into the Arc followed 2–3 min later by PYY(3-36) peripherally(7.5 nmol/kg) or into the Arc (0.1 nmol). In both studies, BIIE0246co-administered with PYY(3-36) resulted in no significant changein ingestive behaviors when compared to saline-treated Siberianhamsters. These data suggest that our dose BIIE0246 is able to pre-vent the inhibition of ingestive behaviors caused by Y2 agonism.The present data suggests that there is not a chronic stimulation ofY2 signaling in non-energetically challenged (i.e., ad libitum-fed)Siberian hamsters similar to laboratory rats [40], which is unlikethe apparent underlying inhibition of ingestive behaviors by leptin

[30] and cholecystokinin [46] in Siberian hamsters.

It is worth noting the large standard error values found inmost of the variables measured after Arc administration of the Y2antagonist. The animals exhibited a dichotomous split into high

26 B.J.W. Teubner, T.J. Bartness / Peptides 47 (2013) 20–28

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Fig. 6. Mean ± SEM of Arc injection of PYY(3-36) (0.1 nmol) or saline post-fooddeprivation of food intake with (A) no foraging requirement and a stationary run-ning wheel [Blocked Wheel (BW)], (B) no foraging requirement and a functionalrunning wheel [Free Wheel (FW)], and (C) a 10 wheel revolutions per pellet foragingrequirement [10 revolutions/pellet (10REV)]. *P < 0.05 vs saline.

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Fig. 7. Mean ± SEM of Arc injection of PYY(3-36) (0.1 nmol) or saline post-fooddeprivation of food hoarded with (A) no foraging requirement and a stationary run-ning wheel [Blocked Wheel (BW)], (B) no foraging requirement and a functionalrunning wheel [Free Wheel (FW)], and (C) a 10 wheel revolutions per pellet foragingrequirement [10 revolutions/pellet (10REV)]. *P < 0.05 vs saline.

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B.J.W. Teubner, T.J. Bartn

nd low levels of food foraging, intake, and hoarding, but hamstershowing high or low levels of one behavior did not necessarilyredict high or low levels of the other behaviors as seems apparentor food hoarding by Syrian hamsters [12]. In addition, the exactocation of the cannula within the Arc also was not associated

ith a particular ingestive behavioral response or the magnitudef the response. Large variations in food hoarding both within andetween animals from day-to-day are common, quite unlike thatf food intake studies in this species in our experience. The cause ofhe variations in spontaneous food hoarding by Siberian hamstersemains a mystery presently and is not due to differences in bodyat (for example: fat hamsters hoarding less than lean animalsecause they possess greater internal energy stores).

The second experiment was designed to test the inhibitory rolef the Y2-R signaling using the naturally occurring NPY Y2-R ago-ist PYY(3-36). PYY(3-36) has potent anorexigenic effects whetherdministered peripherally or centrally in laboratory rats and micefor review: [35]], with few exceptions [47]. The anorexic effectsf PYY(3-36) are diminished when animals are stressed [26], whichay account for the studies where PYY(3-36) was unable to inhibit

ood intake [47]. Because of the possible effects of stress inter-cting with the PYY(3-36) treatment, our animals were habituatedo the injection protocol. As noted above, when food deprivediberian hamsters are refed, large increases in food hoarding andoraging occur persisting for ∼7 d, whereas food intake does notncrease beyond the first few h [18]. Arc injected PYY(3-36) inhibitedood intake and food hoarding in the true foraging group (10REV)uring the first few hours of refeeding, a timeframe of effective-ess similar to that of 24 h food-deprived-refed laboratory ratsfter PYY(3-36) treatment for food intake [9]. The finding that theffect of PYY(3-36) only was seen in the hamsters ‘earning’ theirood via foraging (wheel running, i.e., 10REV) is consistent withur findings with other anorexigenic peptides that exhibit theirreatest inhibition in the 10REV group including the NPY Y1-Rntagonist 1229U91 [29], leptin [30], melanocortin 4-R agonismmelanotan II [28]] as well as triggering the greatest increasesn food hoarding for orexigenic peptides administered centrallyNPY [15,20], AgRP [19]]. The reason that ‘earned’ food elicits botharger decreases and increases in food hoarding is not clear. Its not that these animals are in any greater increase in nega-ive energy balance due to the wheel running because the FWroup runs approximately the same number of wheel revolu-ions as the 10REV group, although food is not contingent onhe wheel running. Thus, some factor(s) associated with foraged‘earned’) food rather than freely available food seems in play inhe present and our previous studies that certainly warrants furthertudy.

Collectively, the present data indicate that NPY Y2-R agonismnhibits food intake and hoarding, albeit in the short term (0–2 h)

ith refeeding after food deprivation. In addition, there does notppear to be underlying NPY Y2-R signaling inhibiting ingestiveehaviors in this species because the antagonism of NPY Y2-Rignaling does not increase appetitive or consummatory ingestiveehaviors in ad libitum-fed hamsters. The short term nature ofYY(3-36) is not unique to this study, and as such seems to be limitedn its ability to decrease foraging/hoarding in Siberian hamsters.onger lasting NPY Y2-R agonists are being developed [36], how-ver, some of which may have the potential for therapeutic use tourtail food intake and hoarding in humans.

cknowledgments

The authors thank the Department of Animal Resources ateorgia State University for expert animal care and Dr. Cheryl. Vaughan, Danni Liu, Alex Thomas, Daniel Vizcaino, Dominiq

[

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eptides 47 (2013) 20–28 27

Okoduwa, Melissa Chaney, and Shasmine Kelly for assistance indata collection.

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