Ž .Brain Research 816 1999 38–46

Research report

Disruption in neuropeptide Y and leptin signaling in obese ventromedialhypothalamic-lesioned rats 1

Michael G. Dube a,) , Bin Xu b, Pushpa S. Kalra a, Charles A. Sninsky c, Satya P. Kalra b

a Department of Physiology, POB 100274, UniÕersity of Florida College of Medicine, GainesÕille, FL 32610, USAb Department of Neuroscience, POB 100244, UniÕersity of Florida College of Medicine, GainesÕille, FL 32610, USA

c Department of Medicine, POB 100277, UniÕersity of Florida College of Medicine, GainesÕille, FL 32610, USA

Accepted 15 September 1998

Abstract

Ž .Electrolytic lesions placed in the ventromedial hypothalamus VMH of rats induce instant hyperphagia and excessive weight gain.Ž .Since neuropeptide Y NPY is a potent hypothalamic orexigenic signal, and leptin secreted by adipocytes regulates NPY output, we

tested the hypothesis that altered NPYergic-leptin signaling may underlie hyperphagia in VMH-lesioned rats. VMH-lesioned ratsexhibiting hyperphagia and excessive weight gain in a time-related fashion were sacrificed on days 2, 7, and 21 post-surgery. Quite

Ž .unexpectedly, NPY concentrations in the hypothalamic paraventricular nucleus PVN , a major site of NPY release for stimulation offeeding, and in other sites, such as the dorsomedial nucleus, lateral hypothalamic area and median eminence-arcuate nucleus decreased,with the earliest diminution occurring on day 2 in the PVN only. In vitro basal and Kq-evoked NPY release from the PVN ofVMH-lesioned rats was significantly lower than that of controls. Analysis of hypothalamic NPY gene expression showed that althoughthe daily decrease in NPY mRNA from 0800 to 2200 h occurred as in control rats, NPY mRNA concentrations were markedly reduced atthese times in the hypothalami of VMH-lesioned rats. Leptin synthesis in adipocytes as indicated by leptin mRNA levels was alsoprofoundly altered in VMH-lesioned rats. The daily pattern of increase in adipocyte leptin mRNA at 2200 h from 0800 h seen in controlswas abolished, higher levels of leptin gene expression at 2200 h were maintained at 0800 h. The pattern of increase in serum leptin andinsulin levels diverged in VMH-lesioned rats. Serum insulin concentration increased to maximal on day 2 and remained at that level onday 21-post-lesion; serum leptin levels on the other hand, increased slowly in a time-related fashion during this period. These resultsdemonstrate that hyperphagia and excessive weight gain in VMH-lesioned rats are associated with an overall decrease in hypothalamicNPY and augmented leptin signaling to the hypothalamus. The divergent time course of increases in serum leptin and insulin levelssuggest independent mechanisms responsible for their augmented secretion, and neither these hormones nor VMH lesions altered the dailyrhythm in NPY gene expression. These observations underscore the existence of an independent mechanism controlling the daily rhythmin hypothalamic NPY gene expression and suggest that leptin feedback action requires an intact VMH. q 1999 Elsevier Science B.V. Allrights reserved.

Keywords: Body weight; Hyperphagia; Paraventricular nucleus; Adipocyte; Insulin

1. Introduction

Damage to the basomedial hypothalamus results inw xgross obesity in rodents and humans 5,42 . In rats, abla-

Ž .tion of the ventromedial hypothalamus VMH leads tow xhyperphagia and a profound weight gain 5,20,42,52 in

association with altered autonomic, endocrine, andw xmetabolic functions 5,42 . While several behavioral as-

) Corresponding author. Fax: q1-352-846-0270; E-mail:mdube@phys.med.ufl.edu

1 This study was presented in part at the 22nd Annual Meeting of theSociety for Neuroscience, Anaheim, CA, in October 1992.

pects of hyperphagia and obesity due to ablation of theVMH have been studied extensively in rats, the underlyingmodifications in various neurotransmittersrneuro-modulators known to affect feeding behavior are poorlyunderstood.

Ž .Compelling evidence shows that neuropeptide Y NPYis an important physiological signal in the hypothalamicregulation of the daily pattern of ingestive behavior in ratsw x Ž .28,29 . NPY produced in the arcuate nucleus ARC and

Ž .released in the paraventricular nucleus PVN and sur-rounding regions of the hypothalamus is believed to be

w xinvolved in stimulation of feeding 9,16,27–29 . Wheninjected centrally, NPY elicited a robust feeding response

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 98 00985-8

( )M.G. Dube et al.rBrain Research 816 1999 38–46 39

w x10 and chronic infusion resulted in hyperphagia andw xobesity in rats 1,8,26,61 . In fasted rats, NPY levels

increased selectively in the PVN and refeeding returnedw xPVN NPY levels to baseline values 45 . NPY release,

measured in vivo or in vitro, was increased in fasted ratsw x13,27 as well as in streptozotocin-induced diabetic ratsw x46,47 . In addition, passive immunoneutralization of NPYin normal and VMH-lesioned rats significantly reduced

w xfood intake 12,15 thereby suggesting a physiological rolefor NPY in the induction of normal feeding behavior andin hyperphagic VMH-lesioned rats. Further, a daily rhythmin hypothalamic NPY gene expression was seen in associa-tion with the daily feeding pattern characterized by feeding

w xduring the dark-phase of the light–dark cycle 58,59 .Recently, it was shown that leptin, a 16-kD protein

secreted by adipocytes, is involved in the hypothalamicw xcontrol of energy homeostasis 7,29,62 . Peripheral and

central administration of leptin inhibited food intake andsuppressed body weight in normal and genetically obeserodents. Leptin administration reduced NPY gene expres-sion in the ARC of mice and rats and decreased in vitroNPY release from the mouse hypothalamus primed with

w xcorticosterone 6,7,19,29,41,53 . Also, leptin receptor andw xNPY mRNA were colocalized in neurons in the ARC 36 .

These findings demonstrated that diminution in NPY sig-naling may account for the inhibitory effects of leptin onfood intake. Further, when neural signaling in the hypo-thalamus was disrupted either by application of the neuro-toxin colchicine or ablation of the VMH, adipocyte leptingene expression and secretion were markedly elevatedw x18,21,33,50 .

Therefore, we undertook the following experiments tovalidate the hypothesis that an altered pattern of NPYer-gic-leptin signaling may underlie hyperphagia and exces-sive body weight gain after VMH ablation. To evaluateNPY signaling in VMH-lesioned rats, we analyzed hy-pothalamic NPY gene expression, NPY concentration invarious hypothalamic sites and the in vitro NPY releasefrom the PVN. In addition, to evaluate the effects onleptin, adipocyte leptin mRNA and serum leptin levelswere examined. Because of the existence of daily rhythmsin hypothalamic NPY and adipocyte leptin gene expressionw x48,58,59 , the levels of gene expression during both thelight and dark phases were examined in VMH-lesionedrats. In view of the emerging evidence of a causal relation-

w xship between leptin and insulin secretion 17,30,32,39,63 ,we also investigated the changes in serum insulin levels inthese rats.

2. Materials and methods:

2.1. Animal handling and surgical procedures

ŽAdult male Sprague–Dawley rats Charles River Labo-ratories, Wilmington, MA, for experiments 1 and 2; Harlan

.Sprague–Dawley, Indianapolis, IN, for experiment 3weighing 250–325 g were used. Animals were housed

Žindividually and maintained under controlled light lights-. Ž .on 0500–1900 h and temperature 21–248C conditions

with continuous access to lab chow pellets and water.Animal protocols used in this project were approved by theUniversity of Florida Institutional Animal Care and UseCommittee in accordance with the ‘Guide for the Care andUse of Laboratory Animals’.

Following approximately 1 week of adaption to thelaboratory, surgery was performed under sodium pento-

Ž .barbital anesthesia 50 mgrkg . Bilateral electrolytic le-sions of the VMH were produced by passing anodal direct

Ž .current 2.5 mA for 15 s through the exposed tip of anw xinsulated stainless steel pin 12 . With the nosebar set 3.3

mm below the interaural line, the electrode was positioned2.6 mm behind bregma, 0.5–0.6 mm lateral of the midlineand lowered to the base of the brain and then raised 0.5mm. In sham-lesioned rats the electrode was lowered butno current was passed. Animals in the intact groups wereweighed only and returned to their cages.

2.2. Experiment 1: determination of NPY content in spe-cific hypothalamic nuclei

NPY concentration in various hypothalamic nuclei wasdetermined on days 2, 7 and 21 following brain surgery.At each time interval three groups were examined, aVMH-lesioned group and two control groups, including asham-operated group and an unoperated intact group. Bodyweight was monitored from the time of surgery untilsacrifice and only those rats which demonstrated excessweight gain were used for the VMH-lesioned groups. Inthe case of the 2 day post-lesion group, food intake wasalso measured for the period 24–48 h following surgery.

Animals were weighed and decapitated during the mid-dle of the lights-on phase. Serum from blood was storedfrozen until leptin and insulin determinations by radioim-

Ž .munoassay RIA . The brains were rapidly removed andfrozen on powdered dry ice. Serial coronal sections of 300

w xmm were cut in a cryostat microtome at y108C 45 andthe following hypothalamic nuclei were micropunched fol-

w xlowing the atlas of Paxinos and Watson 40 : medialŽ . Ž .preoptic area MPOA , lateral preoptic area LPOA , PVN,

Ž .lateral hypothalamic area LHA , dorsomedial nucleusŽ . Ž .DMN and perifornical area PFA . In the case of the7-day post-lesion group, the median eminence-arcuate nu-

Ž .cleus ME-ARC was quickly removed at the time ofsacrifice using microdissecting scissors under a binocularmicroscope before the whole brain was frozen. This wasdone to ensure that in this group the entire ME-ARC wasanalyzed since we found in the 2 and 21 day groups thatthe ME and ARC were often lost during sectioning due tothe fragility of the connection of this area to the brain inVMH-lesioned rats. Thus, the ME-ARC data is presented

( )M.G. Dube et al.rBrain Research 816 1999 38–4640

for the 7-day group only. The micropunches were placedin 0.1 N HCl, frozen at y808C, then thawed and cen-trifuged in a microfuge for 2 min. The supernatant waswithdrawn and lyophilized for NPY determination by RIA.The pellet was dissolved in 50 ml of 0.2 N NaOH for

w xprotein determination using the method of Bradford 3 andthe Bio-Rad protein dye reagent. The dissected ME-ARCfrom the 7-day post-lesion group were homogenized in100 ml 0.1 N HCl, centrifuged and the supernatant re-moved and lyophilized. The pellet was dissolved in 100 ml0.2 N NaOH for protein determination as above. Followingpunching, the sections were dried and fixed in formolsaline. They were later stained with carbol fuchsin and theextent of the lesions was determined by direct projection

w xof the sections onto the Paxinos and Watson atlas 40 .

2.3. Experiment 2: in Õitro NPY release from the PVN

The in vitro NPY release from the PVN was examinedin three groups of experimental and control rats on day 7post-lesion. As in the above experiment, body weight wasmonitored. During the middle of the lights-on phase therats were weighed and then decapitated. As described

w xpreviously 13,23 , the brains were quickly removed,blocked on ice and mounted on a specimen block. The

Žspecimen block was aligned in a vibratome TPI, St. Louis,.MO , immersed in 48C Kreb’s Ringer bicarbonate solution

Ž .KRB, pH 7.4 containing 0.01% BSA, 0.003% bacitracinand 0.1% glucose, and cut into 300 mm thick sections. ThePVN was bilaterally punched with a 1.0 mm diametermicropunch and placed in the wells of a tissue cultureplate containing 250 ml KRB at 48C. The sections werethen mounted on microscope slides, dried and fixed withformol saline for histological determination of the lesionsites as described above. The tissue culture plate wasplaced in a Dubnoff metabolic incubator and incubated at378C in an atmosphere of 95% O –5% CO with constant2 2

Ž .shaking 55 cyclesrmin . After 30 min of preincubation,the micropunches were incubated in fresh KRB for twoconsecutive 30-min periods and the medium was collected

Žto assess the basal rate of NPY release basal I and basal.II . Fresh medium containing 45 mM KCl, substituted

iso-osmotically for NaCl, was then added and the microp-unches incubated for an additional 30 min to assess Kq-stimulated NPY release. Media samples were acidifiedwith 10 ml 1.0 N HCl, lyophilized, and stored at y808Cuntil estimation of NPY by RIA. The micropunches weredissolved in 100 ml 0.2 N NaOH for protein determinationas above. Results were expressed as pg NPYrmg protein.

2.4. Experiment 3: relationship of NPY mRNA and leptinmRNA

Approximately 3–4 weeks post-surgery, VMH-lesionedand sham-lesioned rats were weighed and sacrificed by

Ž .decapitation at 0800 h light phase the time of nadir andŽ .at 2200 h dark phase the time of peak in adipocyte leptin

w xmRNA and hypothalamic NPY mRNA 48,58,59 . Thebrains were quickly removed and the medial basal hypo-

Ž .thalamus MBH was dissected out and frozen in dry icefor later analysis of preproNPY mRNA by RNase protec-

Ž .tion assay RPA . A sample of epididymal fat was re-moved and frozen for analysis of leptin mRNA by RPA.

2.5. Radioimmunoassays

w xAs described previously 14 , the NPY RIA employedŽporcine NPY as standard Peninsula Laboratories, Bel-

.mont, CA and NPY antiserum against human NPY pre-pared in rabbits that was generously supplied by Dr.

ŽHarold Spies Oregon Regional Primate Research Center,.Beaverton, OR . Radioactive iodinated NPY was pur-

Ž .chased from Amersham Life Science Chicago, IL . In-sulin was assayed using an RIA kit obtained from IncstarŽ .Stillwater, MN . Rat leptin RIA was performed with a kit

Ž .obtained from Linco Research St. Charles, MO .

2.6. RNase protection assay for NPY and leptin

Complementary DNA to preproNPY was obtained fromŽ .Dr. S.L. Sabol NIH, Bethesda, MD . The NPY and leptin

w xriboprobes were prepared as described previously 57,60 .As internal standards, cyclophilin and b-actin antisenseriboprobes were synthesized using cDNA templates pur-

Ž .chased from Ambion Austin, TX . Total RNA extractedŽfrom the MBH with RNA-STAT 60 Tel-Test,

. 32Friendswood, TX was hybridized with P-labeled NPYand cyclophilin riboprobes. Similarly, total RNA extractedfrom fat tissue was hybridized with 32 P-labeled leptin andb-actin antisense riboprobes. After overnight hybridizationat 458C, RNase ArT1 digestion was performed for 1 h at378C. Protected hybrids were isolated by ethanol precipita-tion and quantitated using a Molecular Dynamics Phospho-

Ž .rImager Sunnyvale, CA . The NPY mRNA values werenormalized to cyclophilin mRNA and leptin mRNA levelswere expressed relative to the b-actin value.

2.7. Statistical analysis

Data are presented as mean"S.E.M. The tissue con-centrations of NPY were analyzed using a one-way

Ž .ANOVA lesion=sham= intact for each day studied.One-way ANOVA was used for comparison of bodyweight, food intake, serum insulin, serum leptin and NPYin vitro release data. ANOVAs were followed by Fisher’sPLSD test for individual paired comparisons. NPY andleptin mRNA data were analyzed by two-way ANOVAwith time of day and treatment as variables followed byNewman–Keuls multiple comparison test. Only differ-ences with p-0.05 were considered significant.

( )M.G. Dube et al.rBrain Research 816 1999 38–46 41

3. Results

3.1. Effects of VMH lesions on body weight, food intake,serum insulin and serum leptin leÕels

w xConsistent with our previous studies 12 , histologicalexamination of the brains from experiments 1 and 2 re-vealed that the lesioned animals had significant damagethat was limited to the area of the ventromedial hypothala-mus while sham rats did not display such damage. Asdepicted in Fig. 1A, in experiment 1 VMH-lesioned ratsgained significantly greater weight at the three post surgery

Žintervals than the controls. Rats in experiments 2 7 days. Ž .post surgery and 3 2–3 weeks post surgery exhibited

similar changes in body weight with mean increases per

Ž .Fig. 1. A Cumulative body weight change of rats sacrificed at threedifferent intervals following surgery. ): Lesioned rats had significantly

Ž .greater body weight increases than shams and intacts p-0.05 . †: Shamrats had significantly lower body weight increases than lesion and intact

Ž . Ž .rats on these days p-0.05 . Number of ratss5–8 ratsrgroup. BFood intake 24–48 h post-lesion for the three groups sacrificed on day 2

Žpost-lesion. Different superscripts indicate significant differences p-.0.05 . Numbers in bars indicate the number of rats.

Ž . Ž .Fig. 2. Serum insulin levels A and serum leptin levels B for ratssacrificed at three different time intervals following surgery. )): p-

0.01, ))): p-0.001 lesion rats significantly higher than control groups.†: p-0.01 for sham group relative to intact and lesion groups. ns5–8ratsrgroup.

day of 7.3"0.78 g in VMH-lesioned and 3.5"0.5 g inŽ .sham-lesioned rats p-0.05 lesion vs. shams . The food

intake for the 2 day post-lesion group, 24–48 h followingŽ .surgery Fig. 1B , was higher in lesioned vs. control rats.

Ž .Serum insulin levels Fig. 2A exhibited a three-fold in-crease in the VMH-lesioned groups over control values onday 2 and remained at this level on days 7 and 21post-lesion. In contrast, serum leptin levels exhibited aprogressive increase over the 21 day post-lesion periodŽ .Fig. 2B , with a small but significant increase starting onday 2 after VMH lesions. The day 2 sham group, whichexperienced weight loss and significantly reduced foodintake relative to intact rats, had significantly lower serumleptin levels as would be expected immediately followingbody weight loss.

3.2. Effects of VMH lesions on NPY leÕels in hypothalamicnuclei

NPY concentrations in various hypothalamic nuclei wereŽ .differentially affected by VMH lesions Fig. 3 . The PVN

( )M.G. Dube et al.rBrain Research 816 1999 38–4642

Fig. 3. NPY concentration in various hypothalamic nuclei 2, 7 and 21days following surgery. ): p-0.05, )): p-0.01 for lesion grouprelative to control groups. PVNsparaventricular nucleus, DMNsdorsomedial nucleus, LHA s lateral hypothalamic area. n s 5–8ratsrgroup.

NPY concentration in the VMH-lesioned rats was signifi-cantly decreased on day 2, with the maximum diminution

Ž .occurring on day 7 26–28% of control , a response main-tained on day 21. The DMN and LHA NPY concentrationswere also significantly lower than controls on day 7 and 21post-lesion. However, unlike the PVN, diminution in NPYwas delayed until day 7. NPY concentration was also

Ž .lower in the ME-ARC 7 days post-surgery Fig. 4 . TheŽ .other nuclei examined MPOA, LPOA, PFA did not

Žexhibit significant changes in NPY concentration data not.shown .

Fig. 4. NPY concentration in the median eminence-arcuate nuclei of ratssacrificed 7 days post-lesion. )): p-0.01 relative to control groups.Numbers in bars indicate the number of rats.

3.3. Effects of VMH lesions on in Õitro NPY release fromthe PVN

The in vitro release of NPY from the PVN was alsoŽ .suppressed by VMH lesions Fig. 5 . Both basal and

Kq-stimulated NPY release were significantly lower fromthe PVN of lesioned rats relative to the sham and intact

Ž .groups p-0.05 .

3.4. Effects of VMH lesions on daily rhythms in hypothala-mic NPY mRNA and adipocyte leptin mRNA

w xAs expected 58,59 , in the control rats NPY mRNAŽ .levels decreased at 2200 h vs. 0800 h Fig. 6 . Ablation of

the VMH markedly decreased NPY mRNA levels at bothŽ .0800 and 2200 h p-0.05 relative to controls. However,

the daily rhythm in NPY mRNA expression was main-

Fig. 5. In vitro basal and potassium-stimulated NPY release from theparaventricular nucleus of rats sacrificed 7 days following surgery. ):p-0.05, ))): p-0.0001 relative to control groups. Numbers inparentheses indicate the number of rats.

( )M.G. Dube et al.rBrain Research 816 1999 38–46 43

Ž .Fig. 6. A NPY mRNA levels in the medial basal hypothalamus of lesionand sham-operated rats. Different superscripts indicate significant differ-

Ž . Ž .ences p-0.05 . Numbers in bars indicate number of rats. B LeptinmRNA levels in epididymal fat taken from lesion and sham-operated rats.

Ž .Different superscripts indicate significant differences p-0.05 . Num-bers in bars indicate the number of rats.

tained in the VMH-lesioned animals as the values at 2200Ž .h were significantly lower than at 0800 h p-0.05

paralleling the pattern of control rats. As reported previ-w x Ž .ously 29,48 , leptin mRNA in the control groups Fig. 6B

displayed a daily change; levels increased at 2200 h fromŽ .those seen in the morning 0800 h . However, leptin gene

expression in VMH-lesioned rats was elevated signifi-cantly from that of controls at 0800 h and remainedelevated at 2200 h. Thus, the daily light phase to darkphase increase in adipocyte leptin mRNA was abolished inVMH-lesioned rats.

4. Discussion

NPY producing neurons located in the ARC project intothe PVN and various other regions of the hypothalamusw x9,16 . Experimental evidence has implicated the ARC-

PVN NPY axis in the stimulation of feeding behaviorw x9,16,28,29 . The current observations of a reduction inhypothalamic NPY gene expression along with diminutionin PVN NPY stores and release in vitro suggest thathyperphagia and rapid rate in body weight gain after VMHablation were associated with reduced NPY signaling inthe ARC-PVN axis. Seemingly, both the synthesis andNPY release from the PVN nerve terminals are drasticallycurtailed after VMH lesion. A similar diminution in NPYsynthesis and release in the ARC-PVN axis occurred whenneural signaling in the VMH was disrupted by colchicinemicroinjections, a procedure that inflicted little tissue dam-

w xage 22 . In mice, hyperphagia and increase in body weightproduced by VMN lesions with gold thioglucose were alsoaccompanied by suppressed NPY mRNA in the ARC,

w xmeasured during the lights-on period 2,35 . Consequently,one can reason that disruption in hypothalamic signalingproduced either by VMH ablation or neurotoxin injectionwith little neural tissue damage results in diminished NPYsynthesis and release in the ARC-PVN axis. This apparentinverse relationship between hyperphagia and NPYergicsignaling runs counter to numerous studies implying that itis the increased NPY neurosecretion that augments feedingbehavior. For example, increases in PVN NPY release andNPY gene expression in the ARC were evoked by fasting-

w xinduced appetite 4,13,27,34,38,55 and immunoneutraliza-w xtion of NPY attenuated feeding in fasted rats 15 . Simi-

larly, ARC NPY gene expression and PVN NPY levelsw xand release were increased in hyperphagic Zucker 11,49

w xand diabetic rats 46,47,56 . These observations togetherwith the reports that continuous intraventricular NPY infu-sion induced hyperphagia and increased body weightw x8,26,61 clearly support the existence of a positive rela-tionship of NPY action with appetite and body weightgain. However, it is important to note that despite down-regulation of NPY, the hyperphagia in VMH-lesioned ratswas dependent upon endogenous NPY because passiveimmunoneutralization of NPY rapidly suppressed feeding

w xin these rats 12 . Thus, it appears that both upregulationand downregulation of NPYergic signaling in the ARC-PVN NPY axis may underlie hyperphagia and increase inthe rate of body weight gain.

Several possibilities can be advanced to account forhyperphagia occurring along with a diminution in orexi-genic NPY signaling in the ARC-PVN axis in these rats. Itis possible that ablation of the VMH may increase partici-

w xpation of other hypothalamic orexigenic signals 28,29 .Indeed, we have observed that neurotoxin-induced disrup-tion in VMH neurotransmission increased hypothalamicgene expression and peptide levels of the orexigenic pep-

w xtide, galanin 24,43 . It is also possible that hypothalamicanorexigenic signals, such as corticotropin releasing hor-

Ž .mone CRH , are curtailed to allow increased feeding inVMH-lesioned rats. However, at least in the case of CRH,this is unlikely because the hypothalamic CRH–pituitary–

w xadrenal axis is hyperactive in VMH-lesioned rats 5 .

( )M.G. Dube et al.rBrain Research 816 1999 38–4644

The other possibility, supported by several reportsw x22,44 , is that NPY deficiency may lead to post-synapticsupersensitivity so that the endogenously released NPY,albeit at a low rate, results in exaggerated feeding. Inter-ruption of NPY supply to the PVN from the brainstemrendered rats hyperresponsive to NPY and these rats dis-

w xplayed hyperphagia and increased body weight gain 44 .Disruption of neural transmission by bilateral injections ofcolchicine into the VMH produced rapid hyperphagia inconjunction with NPY deficiency. These rats exhibitedsupersensitivity to NPY to the extent that extremely lowdoses of NPY, which were virtually ineffective in normal

w xrats, evoked near maximal food intake 22 . The increasedsensitivity to NPY was attributable to increased NPYreceptors and the NPY Y1 receptor antagonist, 1229U91,suppressed feeding, thereby affirming the view that hyper-phagia in these rats was dependent on endogenous NPYw x22 . Thus, the similarities in the hyperphagic syndromeproduced by either VMN colchicine injection or VMHlesions, support the possibility that enhanced NPY receptorsensitivity may contribute towards unregulated, excessivefood intake.

Hyperphagia in VMH-lesioned rats may also be causedby a lack of the inhibitory effects of leptin on food intakew x w x7,29 . Indeed, our findings and those of others 18,21,50show that in conjunction with unabated feeding, synthesisand secretion of leptin were markedly elevated in VMH-le-sioned rats. Central administration of leptin in doses thatare normally effective failed to reduce feeding in these twomodels of VMH-based obesity suggesting that these rats

w xdeveloped resistance to leptin 21,50 . Further, recently wereported that ectopic hyperleptinemia induced by adenovi-ral transfer of leptin cDNA decreased feeding and body

w xweight in normal, but not VMH-lesioned rats 31 . Appar-ently, the VMH is an integral component of the hypothala-mic network that mediates leptin-induced inhibition offood intake, and either destruction or signaling impairmentin the VMH abrogates the leptin-induced restraint. Basedon these observations, it is possible to suggest that at leasttwo modalities in hypothalamic signaling, increased sensi-tivity to NPY, possibly due to high abundance of Y1

receptors, and development of leptin resistance, contributeto the hyperphagic syndrome in VMH-lesioned rats.

Also noteworthy is the observation that in VMH-le-sioned rats circulating insulin levels rose rapidly to amaximum paralleling the time course in the rate of bodyweight gain despite the slow rates of leptin secretion.These differences in the rates of insulin and leptin hyper-secretion are important in view of the several reportssuggestive of a feedback loop whereby insulin stimulates

w xleptin secretion 48,63 , and leptin inhibits insulin releasew x17,30,32 . If this is the case, then our findings indicatethat VMH ablation disrupts this tight feedback control andthat the integrity of neural elements in the VMH is anecessary component in the manifestation of central con-trol. However, the evidence that leptin can inhibit insulin

release directly from pancreatic islets is not in agreementw xwith this inference 32,39 . Additional studies are war-

ranted to explain these discrepancies and to preciselydetermine the relationship between insulin and leptin se-cretion in normal and VMH-lesioned rats.

Previous work has also implied an inverse relationshipbetween circulating insulin levels and hypothalamic NPY-

Ž .ergic signaling. In rats injected with streptozotocin STZto induce insulinopenia, PVN NPY concentrations andrelease and ARC NPY mRNA levels increased and these

w xresponses were normalized by insulin replacement 46,47 .It is known that intraventricular insulin infusion inhibitsfood intake and the fasting-induced increase in ARC NPY

w xgene expression 51 . However, as evident in this andw xprevious studies 5,29,42,52 , hyperinsulinemia in VMH-

lesioned rats is unable to curb food intake and bodyweight.

Another new finding of this study is that despite thediminution in NPY mRNA in the ARC, the daily rhythmic-ity in NPY gene expression was retained in VMH-lesionedrats. Seemingly, manifestation of this daily rhythmicity isnot related to feeding behavior and is probably driven by

w xthe endogenous clock entrained to the photoperiod 37,54 ,w xpossibly to regulate other neuroendocrine functions 25 . In

contrast, the daily pattern in adipocyte leptin gene expres-sion was abolished in VMH-lesioned rats and leptin geneexpression was maintained at an elevated range during the24 h period. Presumably, high levels of leptin gene expres-sion in the VMH-lesioned rats correlates, as we and othersw x18,50 have observed, directly with body weight andadiposity, and absence of the normal daily pattern may beattributed to unabated feeding. However, the remote possi-bility that VMH-lesions may have shifted the daily rhythmin adipocyte leptin gene expression, that was not detectedby our limited sampling times, cannot be ruled out.

Finally, retention of the daily pattern in hypothalamicNPY gene expression concomitant with constantly ele-vated adipocyte leptin gene expression in VMH-lesionedrats argues against the prevailing assumption that hypotha-lamic NPY signaling is tightly regulated by leptin feed-

w xback 7,29 . Our results suggest an intrinsic daily pattern ofgene expression in NPY neurons in the ARC that ismanifested independently of adipocyte signals. It has beensuggested that distinct subpopulations of NPY-producingneurons in the ARC may be involved in the regulation of

w xvarious neuroendocrine and behavioral effects 25,29 . Ourobservations raise the intriguing possibility, for whichthere is currently little supporting evidence, that the sub-population of NPY-producing neurons that display dailyrhythms are distinct from those that are leptin targets andproject to the PVN and other hypothalamic sites involvedin the control of food intake.

In summary, these results show that the hyperphagiaand abnormal increase in body weight in VMH-lesionedrats occurs along with a diminution in NPYergic signaling,but the normal daily pattern of fluctuations in NPY gene

( )M.G. Dube et al.rBrain Research 816 1999 38–46 45

expression is unaffected. In contrast, leptin secretion andgene expression is markedly elevated and the normal dailypattern of fluctuations is obliterated in VMH-lesioned rats.These results are in accord with the emerging view thathyperphagia and increased rate of body weight gain mayresult from disruption in hypothalamic signaling leading toeither a high or low abundance of NPY in the hypothala-mic sites implicated in the control of food intake and thedevelopment of leptin resistance.

Acknowledgements

We thank the National Institute of Health for financialŽ .support DK37273 and NS32727 and Mrs. Dawn Stewart

for secretarial assistance. The excellent technical assistanceof T. Edwards is greatly appreciated.

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