The effect of intraperitoneal administration of leptin on short-term food intake in rats

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gy 580 (2008) 143–152www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

The effect of intraperitoneal administration of leptin on short-term foodintake in rats

Jayesh D. Patel a, Ivor S. Ebenezer a,b,⁎

a Neuropharmacology Research Group, School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT, England, United Kingdomb Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DZ, England, United Kingdom

Received 2 May 2007; received in revised form 10 October 2007; accepted 16 October 2007Available online 25 October 2007

Abstract

The effects of intraperitoneal (i.p.) injection of leptin (1, 5, and 10 μg/kg) were investigated on the food consumption during a 60 min test mealin 21 h fasted rats. All doses of leptin produced significant reductions in cumulative food intake during the first 15 min and 30 min (at least,Pb0.05) after administration. Similarly, i.p., but not subcutaneous (s.c.), administration of leptin (25 μg/kg) reduced food intake in 21 h fastedrats. Leptin (10 and 25 μg/kg, i.p.) did not reduce water intake in 16 h water-deprived rats, nor did leptin (25 μg/kg) produce aversion in a two-bottle conditioned taste aversion test indicating that the hypophagic effect of leptin is (i) behaviourally specific for food and not water intake, and(ii) not due to drug-induced malaise. Moreover, leptin (10 and 25 μg/kg, i.p.) did not significantly alter food intake in non-deprived rats whenmeasured at 30 min intervals over a period of 24 h. Chemical vagotomy with capsaicin abolished the inhibitory effects of leptin (25 μg/kg, i.p) onfood intake in fasted rats and suggest that the hypophagic effect is dependent on intact vagal afferent nerves. Furthermore, the hypophagia inducedby leptin (10 μg/kg, i.p.) in fasted rats was not attenuated by systemic administration of the peripherally acting cholecystokinin1 receptorantagonist, 2-naphthalenesulphanyl-L-aspartyl-2-(phenethyl) amide (2-NAP; 2 mg/kg, i.p.), indicating that the suppressant effects of leptin on foodconsumption are not secondary to the release of endogenous peripheral cholecystokinin.© 2007 Elsevier B.V. All rights reserved.

Keywords: Leptin; Satiety; Food intake; Taste aversion; Water intake; Vagotomy

1. Introduction

Leptin is a 16-kDa protein product of the ob gene (Zanget al., 1994) and is expressed mainly in white adipose tissuefrom where it is secreted into the blood and acts as a feedbacksignal to control energy balance and body weight (Campfieldet al., 1995; Halaas et al., 1995). It has been demonstrated inrodents that leptin is carried into the brain by a specific transportsystem and decreases long-term food intake (and hence bodyweight) by modulating the secretion of orexigenic (e.g.neuropeptide-Y, melanin-concentrating hormone and orexins)and anorexigenic (e.g. glucagon-like peptide-1, melanocortin

⁎ Corresponding author. Neuropharmacology Research Group, School ofPharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth,PO1 2DT, England, United Kingdom. Tel.: +44 2392842661.

E-mail address: [email protected] (I.S. Ebenezer).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.10.046

and neurotensin) transmitter substances within the hypothalmus(Friedman and Haalas, 1998; Kalra et al., 1999; Elmquist et al.,1999). Defects in the leptin signalling system can result inobesity. Thus, obesity in the ob/obmouse is due to a mutation inthe ob gene that results in an absence in the secretion of leptinfrom white adipose tissue, while obesity in the db/db mouse isas a result of a mutation in the leptin receptors (see Friedmanand Haalas, 1998).

Most of the recent work on leptin and feeding has focused onits central role and its role in the long-term control of feeding andbodyweight. In general, workers have given large (sub-milligramto milligram) doses of leptin systemically, usually by thesubcutaneous (s.c.) route, to rodents and have found that thereis normally a decrease in food intake starting about 4 to 6 h afteradministration, and continuing for at least 24 h (Campfield et al.,1995; Pellymounter et al., 1995; Barrachina et al., 1997a,b;Luheshi et al., 1999; Wang et al., 2000). There is convincing

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144 J.D. Patel, I.S. Ebenezer / European Journal of Pharmacology 580 (2008) 143–152

evidence that the systemically administered leptin acts within thehypothalamus to suppress feeding behaviour (see Kalra et al.,1999; Elmquist et al., 1999). Others have injected low(microgram) doses of leptin centrally, either intracerebroventri-cularly (i.c.v.) or directly into the acuate nucleus of the hypo-thalamus, and reported similar long-term actions of leptin on foodintake (Buchanan et al., 1998; Friedman and Haalas, 1998).

By contrast, however, relatively little attention has been paidto a peripheral action of the protein on ingestive behaviour, norits control of individual meals (short-term control of feeding).Recently, it has been demonstrated that leptin is produced inepithelial cells of the rat and human stomach (Bado et al., 1998;Sobhani et al., 2000) and in the stomach of non-mammalianvertebrates (Muruzabal et al., 2002). It has been found that thisleptin is released into the circulation in response to food in thestomach (Bado et al., 1998) or administration of the gastro-intestinal peptides cholecystokinin (Bado et al., 1998), secretinor pentagastrin (Sobhani et al., 2000). It has also been shownthat there are leptin receptors in afferent neurones of the vagusnerve (Buyse et al., 2001; Burdyga et al., 2002) and that leptincan activate gastric vagal afferents (Wang et al., 1997; Yuanet al., 1999). Although the physiological role of leptin in the gutis not known, as it is released from the stomach in response tofood and hormones related to feeding, it raises the intriguingquestion as to whether leptin itself acts peripherally to elicitshort-term satiety, by signalling the brain via vagal afferents, ina manner that is similar to that that has been suggested forcholecystokinin (see Baldwin et al., 1998). In a preliminarystudy, which was presented to the joint meeting of theAustralasian, British, Canadian and Western PharmacologySocieties in Vancouver in 2001 and published in abstract form(Patel and Ebenezer, 2001), we showed that leptin, in the doserange 1–10 μg/kg, administered i.p. to fasted rats, produced ashort-lasting reduction in food intake. The experiments reportedhere were therefore undertaken to investigate the effects ofintraperitoneal administration of leptin on short-term foodintake in rats and gain insight into its possible mechanism ofaction. Brief rationales for each of the 8 experiments carried outin this study are given below.

Experiment 1 was undertaken to investigate the effects ofintraperitoneal (i.p.) injection of leptin (1, 5, and 10 μg/kg) onfood consumption during a 60 min test meal in 21 h fasted rats.The results show that leptin produced a dose-dependentreduction in cumulative food intake during the first 15 minand 30 min after administration. As systemic administration ofhigh sub-milligram to milligram doses of leptin has been shownto decrease food intake in non-deprived animals with slow onsetand long duration of action (see above), Experiment 2 wascarried out to investigate the effects of low microgram doses ofleptin on acute and long-term food intake in non-deprived rats.A series of experiments (Experiments 3, 4 and 5) weresubsequently carried out to confirm the behavioural specificityof the hypophagic effects of the protein. In Experiment 3, leptinwas denatured to establish whether the short-term reduction infood intake is due to the protein itself or to possible non-peptidecontaminants, such as lipopolysaccharide (LPS), which canreduce food intake in animals (see Parrott and Vellucci, 1998).

Further tests for behavioural specificity were carried out todetermine whether i.p. administration of leptin (i) reduces waterintake in water-deprived rats (Experiment 4), and (ii) decreasesfood intake by producing malaise or sickness in the animals(Experiment 5). Experiments were also undertaken to gaininformation on the possible site and mechanism by which lowmicrogram doses of leptin decrease food intake in hungry rats.As mentioned above, leptin is released from the stomach duringa meal (Bado et al., 1998; Sobhani et al., 2000) and raises thequestion as to whether leptin may act in the abdominal area tostimulate vagal afferents to signal the brain to produce satiety.Thus, in Experiment 6, we examined the effects of s.c. and i.p.administration of leptin on food intake in hungry rats to see if itshypophagic actions on short-term food consumption are due tocirculating levels of the protein (as would be produced by s.c.injection) or are the results of a localised effect in the abdominalarea (as would be produced by i.p. injection) (see Ebenezer,1999), while in Experiment 7 we investigate the possibility thatleptin may act by activating vagal afferents in the abdominalarea to signal hypothalamic areas in the brain to suppress foodintake. The rationale for the final experiment was based on thefinding that systemic administration of leptin can produce smallincreases in the plasma concentration of the putative peripheralsatiety factor cholecystokinin in rats (Guilmeau et al., 2002). Ifthe suppressant effects of leptin on food consumption aresecondary to the release of endogenous peripheral cholecysto-kinin, we reasoned that it would be abolished by pre-treatmentwith the peripherally acting cholecystokinin1 receptor antago-nist, 2-naphthalenesulphanyl-L-aspartyl-2-(phenethyl) amide(2-NAP; see Ebenezer and Baldwin, 1995). This possibilitywas tested in Experiment 8.

Some of this work has been presented at meetings of learnedsocieties and published in abstract form as conferenceproceedings (Patel and Ebenezer, 2001, 2003).

2. Methods

The protocols used in this study were approved by theEthical Review Committee at the University of Portsmouth.

2.1. The subjects

Male Wistar rats (body weights: 240–320 g for Experiments1–5 and Experiment 7, 330–360 g for Experiment 6 and 380–450 g for Experiment 8) were used in these experiments. Theanimals were housed in cages in groups of 4 and kept on a 12 hlight/dark cycle (lights on at 9.00 h, light off at 21.00 h). Theexperiments, with the exception of Experiment 6, began at14.00 h. The rats had free access to water at all times, unlessotherwise stated (see Experiments 5 and 6). Different groups ofrats were used in each of the experiments.

2.2. Experiment 1. Effects of intraperitoneal administration ofleptin on food intake

Male Wistar rats (n=8) were deprived of food for 21 h eachday and received four training sessions during which time they

145J.D. Patel, I.S. Ebenezer / European Journal of Pharmacology 580 (2008) 143–152

were placed singly in experimental cages measuring 32×25×19 cm and allowed free access to their normal pelleted food(food composition: protein 20%, oil 4.5%, carbohydrate 60%,fibre 5%, ash 7%+traces of vitamins and metals) and water. Thefood was presented to the rats in shallow cylindrical cups.During the experimental sessions that followed, each rat wasinjected i.p. with either vehicle solution or leptin (1, 5, and10 μg/kg) immediately prior to being placed singly in theexperimental cages, and cumulative food consumption mea-sured in intervals during the 60 min test meal, as describedpreviously (Ebenezer, 1990). A repeated measures design wasused with each animal receiving all treatments; 3 days separatedsuccessive trials.

2.3. Experiment 2. The effects of microgram doses of leptinadministered intraperitoneally on 24 h food intake in non-deprived rats

Non-deprived male Wistar rats (n=8) were given 2habituation sessions when they were placed singly in exper-imental cages with free access to food and water for 24 h.During the subsequent experimental trials, the animals wereinjected i.p. with either vehicle or leptin (10 or 25 μg/kg) at thebeginning of the light cycle (9.00 h) and placed singly inseparate experimental cages with free access to food and water.Cumulative food intake was measured in 30 min time bins overthe 24 h test period. A repeated measures design was used witheach animal receiving all treatments; 3 days separated suc-cessive trials.

2.4. Experiment 3. Effect of denatured leptin administeredintraperitoneally on food intake

A solution of leptin (25 μg/kg) was heated to 100 °C for10 min to denature the protein. Male Wistar rats (n=8) weredeprived of food for 21 h each day and received similar trainingsessions as described for Experiment 1. During the subsequentexperimental sessions, each rat was injected i.p. with eithervehicle or the denatured leptin (25 μg/kg), immediately prior tobeing placed singly in the experimental cages. The amount offood consumed by each animal was measured at 15, 30, 60 and120 min. A cross-over design was used with each animalreceiving both treatments; 3 days separated successive trials.

2.5. Experiment 4. Effect of intraperitoneal administration ofleptin on water intake

Male Wistar rats (n=8) were deprived of water for 16 h a dayand received 5 training sessions to drink water from a graduatedwater bottle in experimental cages, as described previously(Ebenezer et al., 1992). During experimental sessions thatfollowed, each rat was injected i.p. with either vehicle solutionor leptin (10 or 25 μg/kg), immediately prior to being placedsingly in the experimental cages. Water intake for each animalwas measured in 5 min bins for 60 min. A repeated measuresdesign was used with each animal receiving all treatments;3 days separated successive trials.

2.6. Experiment 5. Effect of intraperitoneal administration ofleptin in a conditioned taste aversion experiment

A method described previously (Ebenezer et al., 1992) wasused. Rats (n=24) were deprived of water for 16 h a dayduring the course of this experiment. They were randomlydivided into a vehicle treatment group (n=8), a lithium chloride(LiCl) treatment group (n=8), and a leptin treatment group(n=8). Each animal was given 3 training sessions in test cageswhere they were presented with tap water and sucrose solution(12% w/v) in two separate graduated 20 ml drinking bottles.During the third training session the total intake of tap water andsucrose solution was measured during the first 15 min afterpresentation. The next day, the rats were given 15 min ac-cess to sucrose solution only in the test cages, and werethen injected i.p. with either vehicle, LiCl (100 mg/kg) or leptin(25 μg/kg) according to their designated treatment groups.Following the injection they were replaced in the test cages forfurther 5 min before being returned to their home cages. Twentyfour hours later they were presented with tap water and sucrosesolution in the test cages, and their water and sucrose intakemeasured at 30 min after presentation.

2.7. Experiment 6. The effects of subcutaneous andintraperitoneal administration of leptin on food intake

Male Wistar rats (n=8) were deprived of food for 21 h eachday and received similar training sessions as described forExperiment 1. During the experimental sessions that followed,each rat was injected s.c. with either vehicle or leptin (25 μg/kg),immediately prior to being placed singly in the experimentalcages. The amount of food consumed by each animal wasmeasured at 15, 30, 60 and 120min.A cross-over designwas usedwith each animal receiving both treatments; 3 days separatedsuccessive trials. After a 7 day dry-out period, the experimentalprocedure was repeated on the same animals, except that thevehicle solution (control) and leptin (25 μg/kg) were administeredi.p. instead of s.c.

2.8. Experiment 7. The effect of denervation of vagal sensoryafferents on leptin-induced suppression of food intake

A slightly modified procedure described previously byBrzozowski et al. (1996) was used to chemically lesion thesensory afferent nerves. Rats (n=6) were pre-treated withatropine sulphate (1 mg/kg, s.c.) and were anaesthetised withEquithesin (Lumb, 1963) daily over 3 consecutive days.Following induction of anaesthesia, the animals were slowlyinfused i.p. with the following doses of capsaicin: 25 mg/kg(day 1), 50 mg/kg (day 2) and 50 mg/kg (day 3). All injectionsof capsaicin were performed under anaesthesia to counteract thepossible respiratory impairment associated with injection of thisagent. The vagal sensory denervation procedure was carried out2 weeks prior to the start of the feeding experiments. Controlrats (n=6) underwent the same experimental proceduredescribed above, but were injected with vehicle, instead ofcapsaicin.

Fig. 1. The effects of i.p. administration of leptin (1, 5 and 10 μg/kg) on foodintake in rats (n=8) that were fasted for 21 h. Vertical lines represent +S.E.M.⁎⁎Pb0.01, ⁎Pb0.05.


The rats were deprived of food for 21 h each day andreceived similar training sessions as described for Experiment 1.During experimental sessions that followed, the rats in eachgroup were injected i.p. with either vehicle or leptin (25 μg/kg),immediately prior to being placed singly in the experimentalcages. The amount of food consumed by each animal wasmeasured at 15, 30 and 60 min. A cross-over design was usedwith each animal receiving both treatments; 3 days separatedsuccessive trials.

The major aim was to chemically lesion the gastric vagalafferents. In order to achieve this, the capsacin was slowly infusedi.p., rather than s.c. as described by Brzozowski et al. (1996), toavoid collateral damage. To check the effectiveness of the sensorynerve denervation produced by capsaicin, the rats were injectedwith 0.01% acetic acid i.p. They did not display abdominalconstrictions to the injection indicating that the vagal afferentswere successfully lesioned. In addition, at the end of theexperiments with leptin (see above) the effects of cholecystokininwere examined on food intake in both the capsaicin-treated andsham-treated rats to provide further confirmation that the vagalafferent denervation procedure was successful (see Baldwin et al.,1998). The experimental procedure was similar to that describedabove except that the animals received i.p. injections of saline andcholecystokinin (2 μg/kg).

2.9. Experiment 8. The effect of pre-treatment with thecholecystokinin1-receptor antagonist 2-NAP on leptin-inducedsuppression of feeding

Male Wistar rats (n=6) were deprived of food for 21 h eachday and received similar training sessions as described forExperiment 1. During the experimental sessions that followed,each rat was injected i.p. with either vehicle followed by saline,saline followed by leptin (10 μg/kg), 2-NAP (2 mg/kg) followedby vehicle, or 2-NAP (2 mg/kg) followed by leptin (10 μg/kg).The two injections were separated by 30 min. Immediately afterthe second injection, the animals were placed singly in theexperimental cages and food intake measured for 15 min. Theanimals received all treatments in a repeated measures designand at least 3 days separated successive trials.

2.10. Drugs used

Recombinant mouse leptin was purchased from SigmaBiochemicals, Dorset, England. The leptin (1 mg) wasreconstituted in buffer consisting of 0.5 ml 15 mM HCl,followed by 0.3 ml 7.5 mM NaOH and made up to a volume of1 ml with physiological saline. The stock solution was aliquotedinto Eppendorf vials and the vials were stored at −20 °C.During experiments, the solution was thawed and made up inphysiological saline to give an injection volume of 0.1 ml per100 g body weight. The vehicle consisted of the buffer solutionmade up in physiological saline. Capsaicin was purchased fromSigma Biochemicals, Dorset, England and made up as asuspension in 10% ethanol, physiological saline and tween, togive an injection volume of 0.1 ml per 100 g body weight. Thesulphated octapeptide of cholecystokinin was purchased from

Sigma Biochemicals, Dorset, England and made up inphysiological saline solution (NaCl, 0.9% w/v) to give aninjection volume of 0.1 ml per 100 g body weight. Phys-iological saline solution was used in control experiments.

2.11. Statistical analysis

The data from Experiments 1, 3, 4, 6, 7 and 8 were analysedby analysis of variance and the Newman–Keuls post-hoc test(Winer, 1971). The cumulative food intake data at each timepoint for Experiment 2 were analysed by using the paired t-test(two-tailed). Comparisons between consumption of water andsucrose solution for Experiment 5 were analysed by using thepaired test (two-tailed).

3. Results

3.1. Experiment 1. Effects of intraperitoneal administration ofleptin on food intake

I.P. injection of leptin (1, 5 and 10 μg/kg) reduced food intakein 21 h fasted rats (see Fig. 1). The onset of the inhibitory effectwas apparent during the first 15 min after administration (F(3,21)=16.9840, Pb0.01). Post-hoc tests revealed that all dosessignificantly suppressed food intake during this period(Pb0.01, in each case). The hypophagic effects of leptin werestill apparent at 30 min (F(3,21)=6.2909, Pb0.01). However, theinhibition of feeding elicited by these doses was fairly short-lasting and cumulative food consumption returned to near controlvalues by 60 min (F(3,21)=1.7910, n.s.). None of the dosesproduced any overt abnormal behavioural effects in the animals.

3.2. Experiment 2. The effects of microgram doses of leptinadministered intraperitoneally on 24 h food intake in non-deprived rats

The results are illustrated in Fig. 2. Statistical analysis of thefeeding data show that leptin (10 and 25 μg/kg) had nosignificant effects on cumulative food intake at any of theintervals over the 24 h test period.

Fig. 2. The effects of i.p. administration of leptin (10 and 25 μg/kg) on foodintake in non-deprived rats measured in 30 min intervals over 24 h. See text forfurther details. Vertical lines represent +S.E.M.


3.3. Experiment 3. Effect of denatured leptin administeredintraperitoneally on food intake

I.P. injection of a denatured solution of leptin (10 μg/kg) didnot reduce food consumption in 21 h fasted rats compared withvehicle treatment at any of the measurement intervals. Thus, thecumulative food intake (mean±S.E.M.) of rats injected withvehicle solution at 15 min, 30 min, 60 min and 120 min was 5.3±0.3 g, 8.2±0.2 g, 12.9±0.5 g, and 17.3±1.3 g respectively, whilethe cumulative food intake (mean±S.E.M.) of rats injected withthe denatured leptin solution at the same time intervals was5.3±0.3 g, 7.9±0.4 g, 12.3±0.8 g, and 17.5±1.2 g respectively.

3.4. Experiment 4. Effect of intraperitoneal administration ofleptin on water intake

The results are illustrated in Fig. 3. Under control conditions,the 16 h water-deprived rats consumed most of their waterduring the first 10 to 20 min after presentation. Leptin (10 and

Fig. 3. The effects of i.p. administration of leptin (10 and 25 μg/kg) on waterconsumption in rats (n=8) that were deprived of water for 16 h. Vertical linesrepresent +S.E.M.

25 μg/kg), administered i.p. did not have any significant effectson water intake in these animals.

3.5. Experiment 5. Effect of intraperitoneal administration ofleptin in a conditioned taste aversion experiment

Fig. 4A shows that on day 3 of the training sessions, the ratsin all 3 groups displayed a preference for the sucrose solutioncompared with tap water. However when consumption ofsucrose solution was paired with injection of LiCl, a knownaversive agent (Ebenezer et al., 1992), the rats avoided drinkingthe sucrose solution when presented with sucrose and water onexperimental day 5 (Fig. 4B). By contrast, when consumptionof sucrose solution was paired with either vehicle or leptin, therats in both groups did not display an aversion to the sucrosesolution on day 5 and drank significantly more sucrose solutionthan tap water (Fig. 4B). These data show that i.p. administra-tion of leptin (25 μg/kg) is not aversive to the rats.

Fig. 4. (A) Tap water and sucrose solution intake in 16 h water-deprived ratsassigned to a saline treatment group (n=6), a leptin treatment group (n=6), and aLiCl treatment group (n=6) during the third training session. Water and sucrosesolution intake was measured during the first 30 min after presentation. (B) Tapwater and sucrose solution intake in 16 h water-deprived rats 24 h after pairingthe ingestion of the sucrose solution with i.p. injection of either saline, leptin(25 μg/kg) or LiCl (100 mg/kg). Water and sucrose solution consumption wasmeasured during the first 30 min after presentation. See text for further details.Vertical lines represent +S.E.M. ⁎⁎Pb0.01 (tap water intake compared withsucrose solution intake).


3.6. Experiment 6. The effects of subcutaneous and intraperitonealadministration of leptin on food intake

S.C. administration of leptin, at a dose of 25 μg/kg, did notsignificantly reduce cumulative food intake at 15 min, 30 min,60 min 120 min and 180 min post-injection in 21 h food-deprived rats (Fig. 5A). By contrast, i.p. administration of leptin(25 μg/kg) significantly reduced cumulative food intake at15 min and 30 min in 21 h fasted animals (Pb0.05, in each case;Fig. 5B). However, the decrease in food consumption was notapparent at 60, 120 or 180 min.

3.7. Experiment 7. The effect of denervation of sensoryafferents on leptin-induced suppression of food intake

Control rats that had received capsaicin vehicle displayed areduction in cumulative food intake during the first 15 and30 min after i.p. administration of leptin (25 μg/kg) comparedwith vehicle treatment (Fig. 6A). By contrast, rats that had beentreated with capsaicin did not display a reduction in food intakeafter i.p. administration of leptin (25 μg/kg) compared withvehicle treatment (Fig. 6B). These animals displayed no overtadverse behaviours during the 2 week interval between

Fig. 5. (A) The effects of s.c. administration of leptin (25 μg/kg) on food intakein rats (n=8) that were fasted for 21 h. (B) The effects of i.p. administration ofleptin (25 μg/kg) on food intake in rats (n=8) that were fasted for 21 h. Verticallines represent +S.E.M. ⁎Pb0.05.

Fig. 6. A. The effects of i.p. administration of leptin (25 μg/kg) on food intake insham-treated rats (n=6) that were fasted for 21 h. B. The effects of i.p.administration of leptin (25 μg/kg) on food intake in chemically vagotomisedrats (n=6) that were fasted for 21 h. See text for further details. Vertical linesrepresent +S.E.M. ⁎⁎Pb0.01, ⁎Pb0.05.

capsaicin treatments and the start of the experiments. They ateand gained weight normally.

In order to validate successful denervation of the vagalafferents, the rats in both the sham-treatment and capsaicin-treatment groups were injected i.p. with saline and cholecys-tokinin (2 μg/kg). The results are shown in Fig. 7. cholecys-tokinin produced a significant hypophagic response in thesham-treated rats (Fig. 7A). By contrast, cholecystokinin didnot depress feeding in the capsaicin-treated animals (Fig. 7B).

3.8. Experiment 8. The effect of pre-treatment with thecholecystokinin1-receptor antagonist 2-NAP on leptin-inducedsuppression of feeding

The results are shown in Fig. 8. Leptin (10 μg/kg)significantly suppressed food intake during the 15 minmeasurement period (Pb0.01). Pre-treatment with 2-NAP(2 mg/kg) did not reverse the inhibitory effect of leptin onfood intake.

4. Discussion

The major finding of this study is that i.p. administration oflow microgram doses of leptin, in the range 1–25 μg/kg,suppresses food intake in fasted rats. Leptin, administered i.p.,has a fairly rapid onset and a relatively short duration of action.Thus, the hypophagic effect of the protein is apparent during thefirst 15 to 30 min after administration and is no longer evident

Fig. 8. The effect of pre-treatment with 2-NAP (2 mg/kg, i.p.) on leptin-inducedsuppression of food intake in rats (n=6) that were fasted for 21 h. Vertical linesrepresent +S.E.M. ⁎⁎Pb0.01 compared with Saline–Vehicle.

Fig. 7. A. The effects of i.p. administration of leptin (2 μg/kg) on food intake insham-treated rats (n=6) that were fasted for 21 h. B. The effects of i.p.administration of cholecystokinin (2 μg/kg) on food intake in chemicallyvagotomised rats (n=6) that were fasted for 21 h. See text for further details.Vertical lines represent +S.E.M. ⁎⁎Pb0.01, ⁎Pb0.05.


60 min later. Comparison of the pattern of feeding of the ratsafter i.p. administration of leptin with that of the vehicleindicates that, while there was decreased feeding during the 0–15 min and 15–30 min measurement intervals, there wasrebound feeding during the 30–60 min measurement interval.This pattern of feeding is similar to that described for theanorectic peptide cholecystokinin, where it has been observedthat low doses administered i.p. decrease feeding in hungry ratsduring the first 15 to 30 min and this is followed by reboundfeeding as the suppressant effects of the peptide on food intakedecline (see Baldwin et al., 1998). It is noteworthy that in thepresent experiments, maximal reductions in food intake incumulative food intake occurred during the first 15 min afterdrug administration and the maximum suppression of feedingdid not exceed 40% of baseline feeding with the higher doses(i.e. 10 and 25 μg/kg). It is also worth mentioning that we havefound that i.p. administration of leptin in the dose range 0.1–0.5 μg/kg did not affect short-term feeding in fasted rats (Pateland Ebenezer, unpublished results). In general agreement withour findings, Peters et al. (2005) have recently demonstratedthat intra-celiac arterial administration of low microgram dosesof leptin (in the range 1–10 μg) decreased the consumption of a15% w/v solution of sucrose during the 15 min test period.These data are in marked contrast to the suppression of foodintake observed after systemic administration of high (sub-milligram to milligram) doses of leptin, which has a slow onset(between 4 and 6 h) and long duration (up to 24 h) of action(Campfield et al., 1995; Pellymounter et al., 1995; Barrachina

et al., 1997a,b; Luheshi et al., 1999; Wang et al., 2000).Moreover, in the experiments quoted above, non-deprivedanimals were used and first measurement of food intake was atleast 1 h after administration of leptin. By contrast, hungry ratswere used in this study and the main effects reported wereobserved in the first 15 and 30 min. Furthermore, the resultsfrom Experiment 6 show that the low microgram doses of leptinused in this study had no effects on cumulative food intake innon-deprived rats when measured in 30 min time bins over 24 h(see Fig. 6), suggesting that these systemic doses are too low tohave any measurable long-term effects on food intake.

It is possible that i.p. administration of low doses of leptindoes not specifically affect food intake but causes a generaldepression in all behaviours which results in the observedreduction in feeding. If this is indeed true, then i.p.administration of leptin should also reduce other consummatorybehaviours such as water intake (see Ebenezer, 1996; Smith andGibbs, 1998; Baldwin et al., 1998). The results obtained inExperiment 4 show that leptin (10 and 25 μg/kg; i.p.) has noeffect on water intake in thirsty rats, indicating that thesuppressant effects of i.p. administered leptin on food intakeare behaviourally specific.

Although the results from Experiment 4 indicate that theinhibitory effect of leptin is specific to the ingestion of food,Smith and Gibbs (1998) have pointed out that it is still possiblethat such a substance may interact “with some otherconsequence of food to produce a non-observable, slight toxiceffect similar to human nausea”. In order to eliminate thepossibility that the hypophagic effects observed may be due totoxic effects, two experiments were carried out. (a) Therecombinant leptin used in this study was harvested fromE. coli and, according to the suppliers, may contain traceamounts (b0.03%) of lipopolysaccharide (LPS). LPS producessickness in animals and although it is highly improbable thatsuch low contamination with LPS will affect feeding (seeParrott and Vellucci, 1998) we tested this possibility inExperiment 3. A solution containing leptin (10 μg ml) wasdenatured by heating the solution to 100 °C; such treatmentdoes not affect LPS activity (Luheshi et al., 1999). I.P. injectionof the denatured leptin did not reduce food intake in rats, thusconfirming that the leptin-induced hypophagia is not caused byLPS contamination. (b) A second experiment was carried out totest the possibility that i.p. administered leptin could act as anunconditioned stimulus for the acquisition of a conditioned taste


aversion (see Baldwin et al., 1998). To test this possibility, theeffect of leptin was investigated in the two-bottle conditionedtaste aversion test (Experiment 5). This test is commonly usedin rats to determine whether an injected substance is aversive(Ebenezer et al., 1992; Houston et al., 2002). The rationale forthe experiment is that the candidate chemical substance servesas an unconditional stimulus for the acquisition of a conditionedtaste aversion to a “preferred” sucrose solution, based on theassumption that the acquisition of a conditioned taste aversiononly occurs when the unconditioned stimulus produces malaiseor illness in the animal. The data obtained show that i.p.administration of the maximum dose of leptin used in this studydid not produce conditioned taste aversion to the sucrosesolution, indicating that leptin does not produce aversive effectsin these animals. By contrast, LiCl, a known aversive agent (seeEbenezer, 1993; Ebenezer et al., 1992), which served as apositive control in the experiment, caused learned taste aversionto the sucrose solution (Fig. 4). Thus, the results obtained inExperiments 3 and 5 suggest that the hypophagic effect of i.p.administered leptin is not caused by drug-induced malaise orother non-specific toxic effects.

It is unlikely that i.p. administration of leptin elicits itsinhibitory action on feeding by a central mode of action because itis has been estimated that less than 1000th of the circulatingconcentration enters the brain (Shiraishi et al., 1999). Thus, for the1 μg/kg dose, which was the lowest i.p. dose of leptin found tosignificantly suppress feeding, we estimate that less than 20 pg ofleptin would get into the brain [This calculation is based on acirculating blood plasma leptin concentration of approximately0.65 ng ml in rats following i.p. injection of 1 μg/kg leptin(Brzozowski et al., 1999) and a blood volume of 25 ml and notconsidering other pharmacokinetic factors]. Furthermore, 10 and25 μg/kg doses of leptin, administered i.p., had no effects oncumulative food intake measured over a 24 h period in non-deprived rats (Experiment 6), suggesting that these systemic dosesare too low to affect centralmechanisms.We have found that directmicroinjection of a 1 μg dose of leptin into the lateral ventricle of22 h fasted rats did not reduce food intake at 15, 30 and 60 minafter administration (Patel and Ebenezer, unpublished results), andEmond et al. (1999) have reported that intracerebroventricular (i.c.v.) administration of a 10 μg dose of leptin into the 3 rd ventricledid not affect short-term feeding in hungry rats. However, Seeleyet al. (1996) have shown that i.c.v. administration of 3.5 μg ofleptin into the 3rd ventricle decreased 4 h food intake in both fastedand non-fasted rats. Thus, the rapid and short duration of the leptin-induced hypophagia after i.p. injection is more consistent with aperipheral, rather than a central, site of action.

The results obtained in Experiment 2 show that when arelatively high dose of leptin, i.e. 25 μg/kg, was injected s.c., itdid not inhibit food intake in fasted rats, but when this dose wasgiven i.p., it suppressed food intake. These findings suggestthat merely increasing circulating levels of leptin by s.c.injection has no effect on short-term food intake. The datafurther indicate that a high local concentration of leptin isrequired in the abdominal area to effectively suppress feeding,as would be achieved by i.p., rather than s.c., administration ofthe protein (see Ebenezer, 1999 for further information). This

may also explain why other workers who administered highdoses of leptin s.c. to rodents did not observe decreases in foodintake when measured 1 h after administration (Barrachinaet al., 1997a,b; Luheshi et al., 1999; Wang et al., 2000).Recently, Peters et al. (2005, 2006) have reported that lowmicrogram doses of leptin administered into the celiac arteryreduced intake of a sucrose solution, but had no effect onsucrose solution consumption when administered into thesystemic venous circulation. These results concur with ourfindings that increasing circulating levels of leptin have noeffect on short-term food consumption.

It is well established that vagal afferent fibres are the mainlink between the gastrointestinal tract and the central nervoussystem networks regulating food intake (Schwartz et al., 1991).The recent demonstration that leptin receptors are present onascending vagal afferents in rat and man (Buyse et al., 2001;Burdyga et al., 2002), suggests that the protein may signalhypothalamic areas in the brain to suppress food intake after i.p. administration by activating vagal afferents. Electrophysi-ological studies are consistent with vagal afferents beinginvolved in the activity of leptin. In an in vivo stomach-gastric–vagus-gastric–artery attached preparation, intra-arte-rial administration of leptin increased gastric vagal afferentactivity (Wang et al., 1997), and Yuan et al. (1999)demonstrated in a neonatal stomach–vagus–brainstem prepa-ration that leptin applied to the stomach increased the activityof nucleus tractus solatarius (NTS) neurones in a concentra-tion-dependent manner. It has been previously established thatstimulation of vagal afferents can increase the activity of relayneurones in the NTS which in turn can activate afferentpathways to the hypothalamus to inhibit feeding (see Morley,1987; Baldwin et al., 1998 for details). In the present study weendeavoured to chemically lesion vagal afferent fibres withcapsaicin. The success of the c-fibre lesion of the afferent vaguswas confirmed by the absence of abdominal constrictions to i.p.administration of 0.01% acetic acid. Furthermore, it has beenpreviously established that the hypophagic effect of i.p.administered cholescystokinin is abolished by either surgicalor chemical vagotomy (see Baldwin et al., 1998). In the presentstudy we found that capsaicin treatment abolished thehypophagic effect elicited by i.p. administration of cholecys-tokinin (see Fig. 7), providing further evidence that the afferentvagus in the abdominal area was successfully lesioned.Although it is possible that capsaicin treatment also affectedother sensory nerves besides vagal afferents, we tried tominimise such collateral damage by administering thecapsaicin by the i.p., rather than the s.c. route, so that themain effects would be on the gastric vagal afferents. The resultsobtained in Experiment 6 show that chemical ablation of the c-afferent vagal fibres by systemic administration of capsaicinabolished the leptin-induced hypophagia. These data thusindicate that i.p. administration of leptin suppresses food intakein hungry rats by signalling the brain networks involved in thecontrol of feeding by activating vagal afferents. Recently,Peters et al. (2005, 2006) have shown that the hypophagiainduced by intra-celiac arterial leptin was abolished by eithersub-diaphragmatic vagotomy or capsaicin treatment, giving


further support for the view that peripheral leptin signals thebrain via vagal afferents to decrease feeding.

However, it is also possible that i.p. administration of leptinmay act indirectly to stimulate vagal afferents by releasing otherperipheral satiety factors, such as cholecystokinin. It has beenrecently demonstrated that i.p. administration of a relatively highdose of leptin (i.e. 100 μg/kg) produces a small increase in theplasma concentration of the putative peripheral satiety factorcholecystokinin in rats (Guilmeau et al., 2002). However, weconsider it unlikely that i.p. administration of leptin exerts itssuppressant effects on food intake by this mode of action because(i) the minimum effective dose of leptin in this study was 100 foldlower than that shown by Guilmeau et al. (2002) to releasecholecystokinin, and (ii) the results obtained in Experiment 7show that the cholecystokinin1 receptor antagonist 2-naphthale-nesulphanyl-L-aspartyl-2-(phenethyl) amide (2-NAP) (Hull et al.,1993; Ebenezer and Baldwin, 1995) does not block thehypophagic effects of leptin (10 μg/kg, i.p.). We have previouslydemonstrated that pre-treatment with this dose of 2-NAP willreverse the suppressant effects of cholecystokinin (Ebenezer andBaldwin, 1995; Romans and Ebenezer, 1997; Patel and Ebenezer,2000).

In conclusion, data obtained in this study indicate that lowmicrogram doses of leptin decrease short-term food intake inhungry rats by a behaviourally specific mechanism of action.The data further indicate that a high local concentration ofleptin is required in the abdominal area to effectively suppressfeeding, as would be achieved by i.p., rather than s.c.,administration of the protein, and that the hypophagic effectof leptin is dependent on intact vagal afferents. As leptin isreleased from rat stomach in response to food (Bado et al.,1998) and leptin receptors are present on vagal afferent fibrethat innervate the stomach (Buyse et al., 2001), the currentfindings tentatively suggest the possibility that endogenousleptin, released from the stomach in response to food, may actin a paracrine fashion to stimulate ascending vagal afferents inthe abdominal area to signal the brain to produce satiety (seeGibbs et al., 1973; Smith and Gibbs, 1992, 1998; and Baldwinet al., 1998 for details on criteria for a peripheral satiety factor).Thus, it is conceivable that, in addition to its central role in thecontrol of energy balance and body weight (Friedman andHaalas, 1998; Kalra et al., 1999; Elmquist et al., 1999) leptinalso plays a role as a short-term peripherally acting satietyfactor regulating the amount of food eaten during a meal.However, further studies need to be undertaken to determine ifendogenous peripheral leptin does have a role as a short-termsatiety factor.

References

Bado, A., Levasseur, S., Attoub, S., Kermorgant, S., Laigneau, J.P.,Bortoluzzi, M.N., Moizo, L., Lehy, T., Guerre-Millo, M., Marchand-Brustel, Y.L., Lewin, M.J.M., 1998. The stomach is a source of leptin.Nature 394, 790–793.

Baldwin, B.A., Parrott, R.F., Ebenezer, I.S, 1998. Food for thought: a critique onthe hypothesis that cholecystokinin is a physiological satiety factor.Prog. Neurobiol. 55, 447–507.

Barrachina, M.D., Martinez, V., Wang, L., Wei, J.Y., Tache, Y., 1997a.Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice. Proc. Natl. Acad. Sci. U. S. A. 94,10455–10460.

Barrachina, M.D., Martinez, V., Wei, J.Y., Tache, Y., 1997b. Leptin-induceddecrease in food intake is not associated with changes in gastric emptying inlean mice. Am. J. Physiol. 272, R1007–R1011.

Buchanan, C., Mahesh, V., Zamorano, P., Brann, D., 1998. Central nervoussystem effects of leptin. TEM Anal. 9, 146–150.

Burdyga, G., Spiller, D., Morris, R., Lal, S., Thompson, D.G., Saeed, S.,Dimaline, R., Varro, A., Dockray, G.J., 2002. Expression of the leptinreceptor in rat and human nodose ganglion neurones. Neuroscience 109,339–347.

Buyse, M., Ovesjo, M.-L., Goiot, H., Guilmeau, S., Peranzi, G., Moizo, L.,Walker, F., Lewin, M.J.M., Meister, B., Bado, A., 2001. Expression andregulation of leptin receptor proteins in afferent and efferent neurons of thevagus nerve. Eur. J. Neurosci. 14, 64–72.

Brzozowski, T., Konturek, S.J., Sliwowski, Z., Pytko-Poloncyzk, J., Szlachcic, A.,Drozdowics, D., 1996. Role of capsaicin-sensitive sensory nerves ingastroprotection against acid independent and acid-dependent ulcerogens.Digestion 57, 424–432.

Brzozowski, T., Konturek, P.Ch., Konturek, S.J., Pajdo, R., Duda, A.,Pierzchalski, P., Bielanski, W., Hahn, E.G., 1999. Leptin in gastroprotec-tion induced by cholecystokinin or by a meal. Role of vagal and sensorynerves and nitric oxide. Eur. J. Pharmacol. 374, 263–276.

Campfield, L.A., Smith, F.J., Guisez, Y., Devos, R., Burn, P., 1995. Recombinantmouse OB protein: evidence for a peripheral signal linking adiposity andcentral neural networks. Science 269, 546–549.

Ebenezer, I.S., 1990. The effect of intracerebroventricular administration ofbaclofen on food intake in rats. NeuroReport 1, 73–76.

Ebenezer, I.S., 1993. Effects of arginine-8 vasopressin and lithium chloride on anappetitively motivated task in rats. Med. Sci. Res. 21, 97–98.

Ebenezer, I.S., 1996. Intraperitoneal administration of cholecystokinin inhibitsoperant water intake in rats: implications for the CCK-satiety hypothesis. Proc.Royal Soc. B. 263, 491–496.

Ebenezer, I.S., 1999. Intraperitoneal but not subcutaneous administration ofcholecystokinin (CCK) inhibits operant and non-operant food intake in rats:implications for the CCK-satiety hypothesis. Methods Find. Expt. Clin.Pharmacol. 21, 161–171.

Ebenezer, I.S., Baldwin, B.A., 1995. 2-Naphthalenesulphanyl-L-aspartatyl-2-(phenethyl) amide (2-NAP) and food intake in rats: evidence that endogenousperipheral CCK does not play a major role as a satiety factor. Br. J. Pharmacol.116, 2371–2374.

Ebenezer, I.S., Houston, A.J., Crook, T.J., 1992. The effects of systemicadministration of baclofen on water intake in rats. Gen. Pharmacol. 23,375–379.

Emond, M., Schwartz, G.J., Ladenheim, E.E., Moran, T.H., 1999. Central leptinmodulates behavioral and neural responsivity to CCK. Am. J. Physiol. 276,R1545–R1549.

Elmquist, J.K., Elias, C.F., Saper, C.B., 1999. From lesions to leptin: hypothalamiccontrol of food intake and body weight. Neuron 22, 221–232.

Friedman, J.M., Haalas, J.L., 1998. Leptin and the regulation of body weight inmammals. Nature 395, 763–770.

Gibbs, J., Young, R.C., Smith,G.P., 1973. Cholecystokinin decreases food intake inrats. J. Comp. Physiol. Psychol. 84, 448–495.

Guilmeau, S., Nagain-Domaine, C., Buyse, M., Tsocas, A., Roze, C., Bado, A.,2002. Modulation of exocrine pancreatic secretion by leptin through CCK1-receptors and afferent vagal fibres in the rats. Eur. J. Pharmacol. 447, 99–107.

Halaas, J.L., Gajiwala, K.S., Maffei, M., Cohen, S.L., Chait, B.T., Rabinowitz, D.,Lallone, R.L., Burley, S.K., Friedman, J.M., 1995. Weight-reducing effects ofthe plasma protein encoded by the obese gene. Science 269, 543–546.

Houston, A.J., Wong, J.C., Ebenezer, I.S., 2002. Effects of subcutaneousadministration of the gamma-aminobutyric acid-A receptor agonist muscimolon water intake in water-deprived rats. Physiol. Behav. 177, 445–450.

Hull, A.D., Shankley, N.P., Harper, E.A., Gerskowitch, V.P., Black, J.W., 1993. 2-Naphthalenesulphanyl-L-aspartatyl-2-(phenethyl) amide (2-NAP)—a selec-tive cholecystokinin CCK-A receptor antagonist. Br. J. Pharmacol. 108,734–740.


Kalra, S.P., Dube, M.G., Pu, S., Xu, B., Horvath, T.L., Kalra, P.S., 1999.Interacting appetite-regulating pathways in the hypothalamic regulation ofbody weight. Endoc. Rev. 20, 68–100.

Luheshi, G.N., Gardner, J.D., Rushforth, D.A., Loudon, A.S., Rothwell, N.J.,1999. Leptin actions on food intake and body temperature are mediated byIL-1. Proc. Natl. Acad. Sci. U. S. A 96, 7047–7052.

Lumb, W.V., 1963. Small Animal Anaesthesia. Lea and Fibiger, Philadelphia.Morley, J.E., 1987. Neuropeptide regulation of appetite and weight. Endoc. Rev.

8, 256–287.Muruzabal, F., Fruhbeck, G., Gomez-Ambrosi, J., Archanco, M., Burrell, M.A.,

2002. Gen. Comp. Endocrinol. 128, 149–152.Parrott, R.F., Vellucci, S.V., 1998. Comparison of the antipyretic actions of

indomethacin and L-745,337, a selective cyclooxygenase-2 inhibitor, inendotoxin-treated prepubertal pigs. Gen. Pharmacol. 30, 65–69.

Patel, J.D., Ebenezer, I.S., 2000. Effect of 2-NAP on food intake in rats:implications for the cholecystokinin-satiety hypothesis. Br. J. Pharmacol.129 149P.

Patel, J.D., Ebenezer, I.S., 2001. Does leptin act as a peripheral satiety factor?Proc. Western Soc. Pharmacol. 44, 225.

Patel, J.D., Ebenezer, I.S., 2003. The effects of chemical vagotomy on thesuppression of food intake produced by intraperitoneal administration ofleptin. Br. J. Pharmacol. 140 61P.

Pellymounter, M.A., Cullen, M.J., Baker, M.B., Hecht, R., Winters, D., Boone, T.,Collins, F., 1995. Effects of the obese gene product on body weight regulationin ob/ob mice. Science 269, 540–543.

Peters, J.H., McKay, B.M., Simasko, S.M., Ritter, R.C., 2005. Leptin-inducedsatiation mediated by abdominal vagal afferents. Am. J. Physiol. 288,R879–R884.

Peters, J.H., Simasko, S.M., Ritter, R.C., 2006. Modulation of vagal afferentexcitation and reduction of food intake by leptin and cholecystokinin.Physiol. Behav. 89, 477–485.

Romans, K., Ebenezer, I.S., 1997. The effects of the cholecystokinin-A receptoranatgonist 2-naphthalenesulphanyl-L-aspartyl-2-(phenethyl) amide (2-NAP)on food intake in rats. J. Endocrinol. 155 (Suppl. 2) 40pp.

Schwartz, G.J., McHughes, P.R., Moran, T.H., 1991. Integration of vagalafferent responses to gastric loads and cholecystokinin in rats. Am. J.Physiol. 261, R64–R69.

Seeley, R.J., van Dijk, G., Campfield, L.A., Smith, F.J., Burn, P., Nelligan, J.A.,Bell, S.M., Baskin, D.G., Wood, S.C., Schwartz, M.W., 1996. Intraventric-ular leptin reduces food intake and body weight of lean rats but not obeseZucker rats. Horm. Metab. Res. 28, 664–668.

Shiraishi, T., Sasaki, K., Niijima, A., Oomura, Y., 1999. Leptin effects onfeeding-related hypothalamic and peripheral neuronal activities in normaland obese rats. Nutrition 15, 576–579.

Smith, G.P., Gibbs, J., 1992. The development and proof of the CCK-8Shypothesis of satiety. In: Dourish, C.T., Cooper, S.J., Iversen, S.D.,Iversen, L.L. (Eds.), Multiple Cholecystokinin Receptors in the CNS.Oxford University Press, Oxford, pp. 162–182.

Smith, G.P., Gibbs, J., 1998. The satiating effects of cholecystokinin andbombesin-like peptides. In: Smith, G.P. (Ed.), Satiation from gut to brain.OUP, Oxford.

Sobhani, I., Bado, A., Vissuzaine, C., Buyse, M., Kermorgant, S., Laigneau, J.P.,Attoub, S., Lehy, T., Henin, D., Mignon, M., Lewin, M.J., 2000. Leptinsecretion and leptin receptor in the human stomach. Gut 47, 178–183.

Wang, Y.H., Tache, Y., Sheibel, A.B., Go, V.L.W., Wei, J.Y., 1997. Two types ofleptin-responsive gastric afferents terminals: an in vitro single unit study inrats. Am. J. Physiol. 273, R833–R837.

Wang, L., Barachina, M.D., Martinez, V., Wei, J.Y., Tache, Y., 2000. Synergisticinteraction between CCK and leptin to regulate food intake. Regul. Pept. 25,79–85.

Winer, B.J., 1971. Statistical principles in experimental design. McGraw-Hill,New Yok.

Yuan, C.S., Attele, A.S., Wu, J.A., Zhang, L., Shi, Z.Q., 1999. Peripheral gastricleptin modulates brain stem neuronal activity in neonates. Am. J. Physiol.277, G626–G630.

Zang, Y., Proenea, R., Maffei, M., Barone, M., Leopold, L., Friedman, J.M.,1994. Nature 372, 425–432.

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The effect of intraperitoneal administration of leptin on short-term food intake in rats