dose-dependent effects of central leptin gene therapy on genes that regulate body weight and...

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MOLECULARTHERAPYVol. 4, No. 2, August 2001

opyright The American Society of Gene Therapy

SN 1525-0016/01 $35.00

139

doi:10.1006/mthe.2001.0427, available online at http://www.idealibrary.com on IDEAL

Dose-Dependent Effects of Central Leptin GeneTherapy on Genes That Regulate Body Weight

and Appetite in the Hypothalamus

Harveen Dhillon,1,* Satya P. Kalra,2, and Pushpa S. Kalra1

Departments of1Physiology and2Neuroscience, University of Florida McKnight Brain Institute, College of Medicine,

Box 100244, Gainesville, Florida 32610, USA

*Current address: Beth Israel Deaconess Medical Center and Harvard Medical School, Division of Endocrinology, RN-325,

99 Brookline Avenue, Boston, Massachusetts 02215, USA

To whom correspondence and reprint requests should be addressed. Fax: (352) 294-0191.E-mail: [email protected].

We have examined the dose-dependent effects and central action of intraventricular adminis-

tration of a recombinant adeno-associated virus encoding rat leptin (rAAVleptin) in suppress-ing body weight (BW) gain in adult female rats. A low dose of rAAVleptin (5 1010 particles)suppressed weight gain (15%) without changing daily food intake (FI), but a twofold higher dosedecreased BW by 30% along with a reduction in daily FI. Reduced BW was due to a loss in bodyadiposity because serum leptin was reduced. Serum insulin levels were decreased (96%) by onlythe high dose along with a slight reduction in glucose. Uncoupling protein-1 (UCP-1) mRNAexpression in brown adipose tissue (BAT), reflecting energy expenditure through thermogene-sis, was upregulated to the same magnitude by the two rAAVleptin doses. We analyzed by insitu hybridization the expression in the hypothalamus of genes encoding the appetite-regulat-ing neuropeptides. Only the high dose decreased expression of neuropeptide Y (NPY), the orex-igenic peptide, and increased proopiomelanocortin (POMC), precursor of the anorexigenic pep-tide, -MSH. Our studies show for the first time that increased availability of leptin within the

hypothalamus through central leptin gene therapy dose-dependently decreases weight gain,adiposity, and serum insulin by increasing energy expenditure and decreasing FI. The decreasein FI occurs only when NPY is reduced and -MSH is increased in the hypothalamus by the highdose of rAAVleptin. Delivery of the leptin gene centrally through rAAV vectors is a viable ther-apeutic modality for long-term control of weight and metabolic hormones.

Key Words: leptin, neuropeptide Y, melanocortin, UCP1, brown adipose tissue, insulin,glucose, rAAV, gene therapy, hypothalamus

NTRODUCTIONeptin, a 16-kD adipocyte hormone, is a key peripheralormonal signal from adipocytes to the hypothalamus forody weight (BW) homeostasis [1,2]. Peripheral or centraldministration of leptin in leptin-deficient obese Lepob/ob

mice decreased BW by suppressing the daily food intakeFI) and increasing energy expenditure, but no effect waseen in obeseLeprdb/db mice or fatty Zucker fa/fa rats, whichack functional leptin receptors [35]. Leptin is believed toe transported across the bloodbrain barrier to hypo-halamic target sites where it acts on the appetite regulat-ng network (ARN) to inhibit FI, and on the sympathetic

ervous system (SNS) to augment energy expenditurehrough brown adipose tissue (BAT) thermogenesis1,2,610]. Whether this negative relationship between

adipocytes and hypothalamic systems exists in wild-typelaboratory rodents for the daily management of BW home-ostasis is not clear. Leptin administration, either as a bolusor slow infusion, to normal wild-type rodents was onlytransiently effective at supraphysiological dosages inreducing BW and there was little or no reduction in FI[1114]. The increase in endogenous leptin levels was alsofound to be ineffective for preventing the age-related adi-posity and weight gain in rodents [15,16]. The endoge-nous leptin titers are elevated in these rats but are unableto inhibit FI or to augment energy expenditure. Similarly,hyperleptinemia resulting from consumption of a high-

energy diet fails to activate the compensatory hypothala-mic mechanisms involved in weight homeostasis[2,1720].

ARTICLE


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Copyright The American Society of Gene Therapy40

ARTICLEdoi:10.1006/mthe.2001.0427, available online at http://www.idealibrary.com on IDEAL

Among the several potential mechanisms advanced toccount for this apparent ineffectiveness or resistance to lep-

n [1,2,7,21], we have examined the possibility that leptinnsufficiency at target sites in the hypothalamus mayccount for the loss of control on weight gain. We recentlyeported that when leptin availability was enhanced locallyn hypothalamic neurons following an intracerebroventric-lar injection (icv) of recombinant adeno-associated virusector encoding rat leptin (rAAVleptin), the age-related

weight gain in normal outbred male and female Sprague-awley rats was blocked for the entire six-month durationf the experiment [22]. Similarly, central administration ofep (encoding leptin) by means of rAAV was recentlyeported to be effective in preventing weight gain in obese

epob/ob mice [23,27]. In the rats there was a depletion of whiteat deposits without decreases in daily food consumption.his diminution in fat deposition apparently resulted selec-vely from increased energy expenditure through BAT ther-

mogenesis [22], an important factor in the development ofbesity [1,24,25]. The blockade of age-related BW gain andat deposition without any effect on FI was unanticipatednd raised the following questions: Would a higher dose ofAAVleptin suppress both BW gain and FI? Is there a dose-ependent effect of central rAAVleptin therapy? What is the

mechanism of long-term control of weight and energyxpenditure following central rAAVleptin gene therapy?

A large body of evidence shows that leptin target neu-ons within the hypothalamic ARN, the orexigenic neu-opeptide Y (NPY) and agouti-related peptide (AGRP)

producing neurons, and the anorexigenic -melanocytestimulating hormone (-MSH) producing proopiome-

lanocortin (POMC) neurons, are located in the arcuatenucleus (ARC) and are functional antagonists [1,2,26].We previously reported that an intravenous injection ofrAAVleptin to Lepob/ob mice that raised plasma leptinlevels and decreased FI and BW attenuated NPY and

AGRP mRNA expression and increased POMC mRNAexpression [27].

Here we investigated the dose-dependent effects of cen-tral rAAVleptin on BW, FI, and the metabolic hormonesleptin and insulin in wild-type Sprague-Dawley (SD) rats.To determine the mechanism of central leptin action, weevaluated UCP-1 mRNA in BAT and mRNA expression of

NPY, AGRP, and POMC in the hypothalamus.

RESULTSEffects of rAAVLeptin on BW and FIRats injected with rAAVUF5 showed a 15.7% weight gainfrom the pre-injection weights during the 6-week durationof the experiment (Fig. 1). In contrast, one rAAVleptin injec-tion (5 1010 particles) resulted in maintenance of the pre-injection weight. At week 6 postinjection, BW of therAAVleptin treated rats was 15% lower than that of controlrats, but 24-hour FI was similar to that of controls during the

entire experimental period (Fig. 1B).A higher viral infection attained by two injections of

rAAVleptin (total dose = 10 1010 particles) evoked

FIG. 1. Effects of the low dose (5 1010

particles) icv rAAVleptin injection on body

weight (A), food intake (B), serum leptin

(C), insulin (D), and glucose (E) at week 6

postinjection. The arrow denotes the time

of injection. *P 0.05 versus rAAVUF5.

A B

C D E


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opyright The American Society of Gene Therapy141


uantitatively different effects on BW and 24-hour FI (Fig.). BW gain was not affected by injections of the controlector (13.6% gain from pre-injection level; Fig. 2A) asndicated by a pattern similar to that of the untreated con-

rol rats. Two rAAVleptin injections significantlyecreased BW from the pre-injection level within 1 weekP 0.05) with an overall weight loss of 16.4% (initial24.1 5.4 versus final 188.4 11.1 g) at week 6 postin-ection. Compared with controls there was a 30% weightoss in rAAVleptin treated rats. Twenty-four hour FI wasuppressed at one week postinjection (P 0.05) andemained low for the remainder of the experiment (aver-ge 13.6 0.1 g versus 16.6 0.4 g controls;P 0.05; Fig.B).

The group of rats pair-fed with the rAAVleptin treatedats displayed a similar weight loss for the first four weeks;

hereafter they slowly regained weightut only up to their pre-injection levelsP 0.05 versus rAAVUF5 andntreated controls at week 6).

ffects of rAAVLeptin on Serumeptin, Insulin, and Glucose

ConcentrationsA dose-related reduction in serum leptinwas also evident in rAAVleptin treatedats. Serum leptin levels were reduced by6% in rats receiving the low dose of rAAV

P 0.05; Fig. 1C) and by 96% in responseo the higher dose (P 0.01; Fig. 2C).

Maintenance of weight and FI for six

weeks produced no change in serum insulin and glucose lev-els in the low dose rAAVleptin treated rats (Figs. 1D and 1E).In contrast, in response to the higher rAAVleptin titer (Fig.2D), serum insulin levels were reduced by 96% (P 0.05 ver-

sus untreated and rAAVUF5 treated controls). Glucose con-centrations were slightly reduced (21.2%, P 0.05 versusrAAVUF5 injected rats; Fig. 2E) but were normoglycemic(127 3.3 mg/dl). There was no change in serum leptin,insulin, or glucose levels in pair-fed rats (Figs. 2C2E).

Effect of rAAVLeptin on UCP1 mRNA in BATWe found that rAAVleptin injection upregulated UCP1mRNA in BAT compared with the respective rAAVUF5treated controls (P 0.05; Fig. 3). However, this increasewas not dose-dependent because the magnitude of increasewas approximately twofold following either dose of

IG. 2. Effects of high dose (10

010 particles) icv rAAVleptin

dministration on body weight

A), food intake (B), serum leptin

C), insulin (D), and glucose (E) at

week 6 postinjection. The arrows

enote the time of injection. **P

0.01 and *P 0.05 versus allroups.

FIG. 3. Effects of low dose (5 1010 par-

ticles) and high dose (10 1010 particles)

rAAVleptin on BAT UCP-1 mRNA expres-

sion at week 6 postinjection.*P 0.05 versus respective rAAVUF5

control. aP 0.05 versus other groups.

A B

C D E


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AAVleptin. On the other hand, as reported earlier [12],air-feeding induced a significant reduction in UCP-1

mRNA (P 0.05).

ffects of rAAVLeptin on HypothalamicNeuropeptide mRNA Expression

he results of the in situ hybridization analyses ofNPY,AGRP,ndPOMC mRNA in the hypothalamic ARC are depicted inigs. 4 and 5. Low dose rAAVleptin injection, which sup-ressed BW gain and reduced serum leptin without affect-

ng FI, failed to modify the mRNA levels of these peptidesFig. 4). In contrast, in association with the reduction in BW,I, and serum leptin produced by the high dose rAAVlep-n, both NPYandPOMC mRNA levels were significantly

ltered (Figs. 4 and 5). Whereas NPYmRNA was downregu-ated (P 0.05),POMC mRNA was elevated over the controlAAVUF5 treated rats (P 0.05). A similar reduction in FI

in pair-fed rats failed to alter gene expression ofNPY and POMC in the ARC. AGRP mRNAexpression was not altered by any treatment.

DISCUSSIONIn agreement with our previous reports[22,28,29], the results of this study show thatlow dose rAAVleptin injected icv suppressedweight gain without affecting energy intake.This weight maintenance, accompanied bydepletion of adipose tissue and reduced serumleptin levels, may be due primarily to aug-mented energy expenditure through increasedthermogenesis as indicated by upregulation ofUCP-1 mRNA in BAT [22]. The current findingsextend these observations to show that a

twofold increase in the rAAVleptin doseenhanced its effectiveness with a steady reduc-tion in body weight along with a significantdecrease in FI. The low and high doses ofrAAVleptin produced an equivalent increase,possibly a maximal response, in BAT UCP-1mRNA and hence equivalent energy expendi-ture through thermogenesis. Therefore, it islikely that BW suppression in response to tworAAVleptin injections, unlike BW maintenanceafter one injection, was solely a result of theattenuated energy intake. These findings,

together with previous results fromLepob/ob

miceand SD rats [22,2729], support the notion thattwo distinct neural mechanisms, with differentthresholds for responsiveness to leptin, exist inthe hypothalamus. Seemingly, the neural net-works that augment thermogenesis throughactivation of BAT UCP-1 mRNA [8,10,24,25]and, possibly, general activity to enhance energyexpenditure [5] have a relatively lower thresh-old to leptin than those that regulate appetite

or energy intake. The sustained effects of different doses ofrAAVleptin injected centrally are apparently due to the

paracrine/autocrine effects of augmented leptin generatedlocally [21,22]. Using immunocytochemical analysis of greenfluorescent protein (GFP) encoded in the rAAVUF5 controlvector, we have demonstrated GFP expression in neurons inthe ARC and other hypothalamic sites after an icv injection[22,28]. RTPCR analysis of leptin mRNA revealed signifi-cant upregulation in the hypothalamus of rAAVleptintreated rats [22]. Furthermore, reduced plasma leptin levelsin these rats argues against the possibility of increased periph-eral contributions to hypothalamic targets.

In previous studies, hyperleptinemia produced by sys-temic injections of either rAAVleptin [30] or adenovirus

encoding leptin [14,31] attenuated hyperinsulinemia andhyperglycemia inLepob/ob mice and decreased blood insulinin adult rats [3234]. In our earlier study, serum leptin

G. 4. Dose-dependent effects of icv rAAVleptin on NPY(A and D),AGRP(B and E), andPOMC

C and F) mRNA expression in the arcuate nucleus at week 6 postinjection. Low dose =

1010 particles; high dose = 10 1010 particles. *P 0.05 versus rAAVUF5 controls and

FC (pair-fed control).

A

B

C

D

E

F


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evels were suppressed rapidly but theeduction in serum insulin was seen onlyt six months after low dose rAAVlep-n administration [22]. As reported here,owever, suppression of serum insulin

was apparent at six weeks after the highose of rAAVleptin in association withxtremely low circulating leptin levels.

We have observed that micro-injectionf rAAVleptin into the hypothalamicaraventricular nucleus, the feeding rel-vant site, led to an immediate decreasen serum leptin and insulin (unpub-shed data). This suggests that periph-ral leptin by itself may not be respon-ible for diminished pancreatic insulinecretion. A feedback relationship

etween leptin and insulin secretion haseen suggested by in vivo and in vitroxperiments. Leptin can either stimulater inhibit insulin release in rodents20,3539] and insulin directly affectseptin secretion from adipocytes [37,40].Our results advocate a central compo-

ent of leptin action to inhibit serumnsulin levels. Although it is possiblehat the reduction in FI and serum lep-n in rats receiving the high dose

AAVleptin accounts for the suppression of serum insulin

evels, the reduction in serum insulin levels seen in rats withnaltered FI after low dose icv rAAVleptin injection suggestshat insulin suppression may not be related to reduced FI.urthermore, there was no reduction in serum insulin inair-fed rats that consumed an equivalently reduced amount.

In this context, it is noteworthy that the reduction inerum insulin was associated with normal levels of serumlucose in rAAVleptin treated rats in this and a previoustudy [22]. Therefore, it is possible that rAAVleptinnjected rats develop increased sensitivity to insulin for glu-ose homeostasis. However, additional investigations are

warranted to support a role of central leptin action in mod-

lating insulin sensitivity.To identify the neural factors responsible for the dose-

ependent effects of rAAVleptin on energy consumptionnd expenditure, we examined the gene expression of aew key hypothalamic neuropeptides implicated in weightegulation [1,2]. In leptin-deficient Lepob/ob mice [27], weeported significant modulatory effects of rAAVleptin onhe hypothalamic mRNA expression of NPY, AGRP, andOMC. NPY is a powerful stimulator of feeding [2,41,42].

A subpopulation of NPY-producing neurons in the ARCxpresses the biologically active long form of the leptineceptor [43] and leptin has been shown to downregulate

NPYmRNA expression in the ARC [2]. Our results show-ng decreased NPYmRNA expression in the ARC only afterhe high dose of rAAVleptin, which suppressed FI, imply

that downregulation of ARC NPYexpression attenuated

appetite.The functional antagonist of NPY involved in the medi-

ation of leptins effects on appetite regulation in the ARN isthe melanocortin system [1,2,26]. POMC is produced by neu-rons in the ARC and is the precursor of-MSH, the anorec-tic peptide. These ARC POMC neurons express the leptinreceptor and leptin enhances POMC mRNA expression inwild-type rodents,Lepob/ob mice [1,2], andLepob/ob mice treatedintravenously with rAAVleptin [27]. Upregulation of ARCPOMC mRNA after the high dose icv rAAVleptin injectionsuggests that suppression of FI may be the combined effectof decreased NPY and increased melanocortin signalings.

These simultaneous shifts in opposite directions in NPYandPOMC mRNA expression are likely a direct effect of leptingenerated locally in the hypothalamus and not secondary tothe inhibition in FI, because a similar 1314% reduction inconsumption in pair-fed rats affected neither NPYnorPOMCexpression. NPY neurons apparently exert a tonic restrainton melanocortin signaling because they are synapticallylinked with POMC neurons in the ARC [2,44], POMC neu-rons express NPY receptors [45] and NPY injected icvdecreasedPOMC mRNA expression and diminished -MSHrelease from the hypothalamus in vitro [2,46]. These lines ofexperimental evidence are in accord with the postulated two-

prong action of leptin in inhibiting appetite [2]. Leptininhibits NPY and augmentsPOMC mRNA expression directlythrough leptin receptors located on each of these neuronal

FIG. 5. NPY, AGRP, and POMC mRNA expression in the arcuate nucleus at week 6 postinjection in

rAAVUF5 (control), pair-fed, and rAAVleptin (10 1010 particles)-injected rats.


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opulations. The resultant diminished NPYergic signaling,n turn, further curtails the restraint on POMC neurons,hereby allowing increased melanocortin signaling [27].

AGRP is coexpressed in most NPY neurons in the ARCnd, like NPY, AGRPmRNA expression is increased after fast-ng and decreased by leptin injection [1,47]. AGRP stimulateseeding by antagonizing the action of-MSH at MC4 recep-ors [1,2]. Systemic rAAVleptin injection in Lepob/ob miceecreasedAGRPmRNA [27]. Our failure to discern any effectnAGRPmRNA expression in the current study was unex-ected. It is possible that, under the current experimentalonditions, the amount of leptin produced by the rAAVlep-n was ineffective in suppressingAGRPmRNA expressionut effective in downregulating NPYmRNA expression.

Our results show that long-term expression ofLepob in theypothalamus can, depending upon the dose of rAAVlep-n administered, either arrest the age-related weight gain or

uppress BW in SD rats maintained on ad libitum rat chow.his dose-dependent effect on BW is apparently achievedther by increased energy expenditure through themogen-sis alone or coupled with reduced food consumption.dditional consequences of augmented central leptin actionre a dose-dependent diminution in circulating leptin andnsulin levels in the presence of normoglycemia, a responsendicative of increased insulin sensitivity. Diminution ofppetite in response to a high level of centrally available lep-n may be due to the simultaneous decrease in orexigenic

NPYergic signaling and increase in anorexigenicmelanocortin signaling. Because rAAV vectors are non-path-

genic and non-immunogenic [48], delivery of leptinhrough rAAV is potentially an efficient therapeutic modal-y for long-term weight control.

MATERIALS AND METHODSnimals. Adult female SD rats (200225 g) were purchased from Harlan

prague Dawley, Inc. (Indianapolis, IN), and housed individually in a specific

athogen-free (SPF) environment in plastic cages fitted with specially designed

od cups to collect spillage. Food and water were available ad libitum and

ghts were on from 0500 to 1900 h. The animal protocols were approved by

he Institutional Animal Care and Use Committee (IACUC).

onstruction and packaging of rAAV vectors. The rAAV vectors were con-

ructed at the University of Florida Gene Therapy Center as described [27].iefly, the vector pTRCBAObEcoRI fragment of pCRrOb (a gift from Roger

. Unger, University of Texas, Dallas, TX) containing rat leptin cDNA was sub-

oned into rAAV vector plasmid pAAVGEnh after deletion of theEcoRI frag-

ent carrying -glucuronidase cDNA sequence. Vectors were packaged, puri-

ed, concentrated, and titered essentially as described [49]. The titer of

AAVCBAOb vector, hereafter referred to as rAAVleptin, was 1 1013 phys-

al particles/ml and the ratio of physical-to-infectious particles was 100.

AAV vectors, purified using iodixanol gradient/heparin-affinity chromatog-

phy, were 99% pure. The control vector, rAAVUF5, was similarly con-

ructed and encodes GFP [49]. The titer of the rAAVUF5 was 1 1013 phys-

al particles/ml.

xperimental design. Rats were anesthetized (ketamine 100 mg/kg BW +

ylazine 15 mg/kg BW) and permanent cannulae were stereotaxically

mplanted in the third cerebroventricle as described [22]. Cerebrospinaluid efflux served as an indicator of the accuracy of cannula placement.

ats were allowed 710 d to recover from surgery before use in the following

wo experiments.

For experiment 1, rats were divided into two weight-matched groups

(69 rats/group) and injected icv with either rAAVUF5 (control, 5 ml) or

rAAVleptin (5 1010 particles in 5 l). Body weights were recorded before

the icv injection and weekly thereafter. Once per week, preweighed

amounts of food were placed in the food cups at 1000 h. Twenty-four

hours later, the remaining food and spillage in the cup was weighed and

24 h food consumption was calculated.

For experiment 2, rats were divided into two weight-matched groups(68 rats/group) and injected twice (48 h apart) with either rAAVUF5 (con-

trol, 5 l) or rAAVleptin (5 1010 particles/5 l/injection). Body weight

and 24 h food intake were recorded weekly for 6 weeks as described above.

Two additional control groups of unoperated rats were included in this

experiment: an untreated group (n = 6) and a group of rats pair-fed to the

amount consumed by the rAAVleptin treated rats (n = 6).

To avoid excessive weight loss per IACUC guidelines, all rats were sac-

rificed at six weeks post-injection when the rAAVleptin injected rats in

experiment 2 displayed a 30% loss in BW. Rats were sacrificed between 0900

and 1200 h. Brain and BAT were rapidly dissected out and frozen at 80C.

BAT UCP1 mRNA expression was assessed by dot-blot analysis. Three

brains per group were processed for NPY, AGRP, andPOMC mRNA by in

situ hybridization. Serum from trunk blood was collected for analyses of

leptin, insulin, and glucose.

Leptin. Serum leptin levels were assayed in duplicate with a rat leptin RIA

kit (Linco Research, Inc., St Charles, MO) with a sensitivity of 0.5 ng/ml

and a range of 0.550 ng/ml. In experiment 2, serum leptin was reduced

and was measured with a rat/mouse leptin RIA kit (ALPCO, Windham,

NH) with a sensitivity of 6 pg/ml and 12.5800 pg/ml range of detection.

Insulin. Insulin was measured with a rat insulin RIA kit (Linco Research,

Inc., St. Charles, MO). The sensitivity of the assay was 0.1 ng/ml and 0.110

ng/ml range. Samples from experiment 2 were assayed using a more sen-

sitive rat insulin RIA kit (Linco Research, Inc., St Charles, MO) with a sen-

sitivity of 0.02 ng/ml and 0.021.0 ng/ml range of detection.

Glucose. Glucose was measured colorimetrically with Trinder (Sigma).

Dot-blot analysis for BAT UCP-1. The full-length cDNA clone for uncoupling

protein-1 (UCP-1) was provided by Leslie Kozak (Jackson Laboratory, Bar

Harbor, ME) and verified by northern analysis. Total RNA was isolated fromBAT with STAT-60 (Teltest Inc., Friendswood, TX) and dot-blot hybridiza-

tion analysis of UCP-1 mRNA was conducted as described [9]. To minimize

variability all samples from one experiment were run on the same blot.

In situ hybridization. We performed in situ hybridization for NPY, AGRP,

and POMC mRNA in the brain as described [27,50]. A 396-bp complete

mouse AGRP cDNA (GenBank acc. no. U89484), inserted into pBSK +/ vec-

tor, was provided by Roger Cone (Oregon Health Science University,

Portland, OR). ThePOMC probe was constructed by cloning a 478-bp cDNA

fragment (5 psn 220, 3 psn 697, GenBank acc. no. J00759) into pGEM(T

vector [50]. The NPYprobe was constructed using a plasmid containing a

511-bp rat NPYfragment provided by Steven L. Sabol (NIH, Bethesda, MD).

Antisense riboprobes were transcribed in the presence of 35S-UTP

(Amersham Life Sciences, Inc., Arlington Heights, IL) using T7 (AGRP,

POMC) or T3 (NPY) RNA polymerase.Fresh frozen coronal sections (16 mm) encompassing the hypothalamus

were cut in a cryostat at 20C and thaw-mounted on poly-L-lysine (Sigma)

coated microscope slides in four series and stored at 80C. Sections were

post-fixed in 3% paraformaldehyde and acetylated and hybridized with 35S-

labeled antisense probes (1 106 cpm) at 50C for 1620 h in a humidified

chamber. Slides were exposed to Kodak Biomax MR autoradiography film for

5 d, followed by dipping in Kodak NTB2 emulsion for 15 d at 4C and coun-

terstained with 0.1% cresyl violet. Slides from control and rAAVleptin treated

rats were processed together to eliminate procedural variations.

We estimated the relative optical density (ROD), calculated as total

target area multiplied by the integrated optical density for AGRP, NPY, and

POMC, from autoradiograms with the MCID image analysis system

(Imaging Research, St. Catherine, Ontario, Canada). We analyzed 12

matched sections containing the ARC from each brain. The backgroundoptical density in an area adjacent to the ARC was subtracted from the tar-

get optical density. The ROD of 12 sections in the same brain were aver-

aged and expressed relative to the average ROD from the control group.


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tatistical analyses. We used two-way ANOVA with time and treatment

s variables for comparison of the weekly BW and FI data. In experiment

differences between the four treatment groups at each time point were

ompared with one way ANOVA, followed by Neuman-Keuls post-hoc test.

erum leptin, insulin, and glucose levels and BAT UCP-1 mRNA, brain

PY, POMC, and AGRP mRNA expression were compared by Students t test

xperiment 1) or ANOVA and Neuman-Keuls test (experiment 2).P 0.05

as considered significant for all analyses.

ACKNOWLEDGMENTShis research was supported by grants from the National Institutes of Health (HD

K 37273 and NS 32727). We thank Nicholas Muzyczka (Director of Gene Therapy

enter) and Sergei Zolotukhin (Director of the Vector Core Laboratory, University of

orida) for preparation of rAAVleptin vectors. Presented in part at the 30th Annual

Meeting of the Society for Neuroscience, New Orleans, Louisiana, Nov. 49, 2000.

ECEIVED FOR PUBLICATION APRIL 12; ACCEPTED JUNE 25, 2001.

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