AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats

Peptides 26 (2005) 2567–2578

AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats Erin Keen-Rhinehart a,b , Satya P. Kalra b , Pushpa S. Kalra a,∗ a

Department of Physiology and Functional Genomics, Box 100274, University of Florida, Gainesville, FL 32610-0274, USA b Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA Received 14 April 2005; received in revised form 24 May 2005; accepted 25 May 2005 Available online 15 July 2005

Abstract Leptin is a hormone secreted primarily by white adipocytes that regulates energy homeostasis and reproduction via CNS receptors. Koletsky (f/f) rats with a leptin receptor (OB-Rb) gene mutation are obese, diabetic and infertile. We employed recombinant adeno-associated viral (rAAV) vectors to transfer the human OB-Rb gene into the brains of female Koletsky rats to identify sites of leptin action in the brain. rAAV-OB-Rb was microinjected into the medial preoptic area (MPOA), the paraventricular nucleus (PVN), the ventromedial hypothalamus, the arcuate nucleus (ARC), or the dorsal vagal complex in the brainstem. Food intake and body weight were monitored bi-weekly for 55 days. Vaginal cytology was examined daily to assess estrous cyclicity. After sacrifice, uncoupling protein-1 (UCP-1) mRNA in brown adipose tissue and serum concentrations of leptin, insulin, glucose, estradiol and progesterone were measured. Expression of OB-Rb was documented by RT-PCR and site specificity of microinjection was verified by immunohistochemical detection of green fluorescent protein following a control microinjection of rAAV-GFP. OB-Rb installation in the ARC reduced food intake, however, energy expenditure, assessed by UCP-1 mRNA expression, was increased by OB-Rb installation in all sites except the PVN. When injected into the MPOA and ARC, rAAV-OB-Rb stimulated the reproductive axis as evidenced by normalization of estrous cycle length and increased luteinizing hormone releasing hormone concentrations in the hypothalamus. These studies show that long-term installation of a functional leptin receptor in the CNS is achievable using rAAV vectors and further show that leptin acts on specific sites in the brain to produce differential effects on food intake, energy expenditure and reproduction. © 2005 Elsevier Inc. All rights reserved. Keywords: Recombinant adeno-associated virus; Hypothalamic-pituitary-gonadal axis; Hypothalamic appetite regulating network; Energy expenditure; Neuropeptide Y; Proopiomelanocortin

1. Introduction Leptin, encoded by the ob gene, is secreted primarily from white adipose tissue and binds to receptors in the CNS to regulate a variety of physiological functions [11,12,48]. Leptin stimulates energy expenditure, inhibits appetite [11] and increases insulin sensitivity [33]. Leptin stimulates the reproductive axis by increasing hypothalamic luteinizing hormone releasing hormone (LHRH), that in turn stimulates pituitary gonadotropin release and reproductive organ function [47]. Genetic defects in leptin signaling result in obesity, diabetes ∗

Corresponding author. Tel.: +1 352 392 4169; fax: +1 352 294 0191. E-mail address: [email protected] (P.S. Kalra).

0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.05.027

and infertility, as seen in ob/ob mice with a leptin gene mutation and in db/db mice, fa/fa Zucker rats and f/f Koletsky rats with leptin receptor gene mutations [7,9,15,18,25,26,49]. We and others have shown that much of leptin action occurs through CNS receptors (for review see [20,22]). Leptin inhibits appetite by decreasing the hypothalamic orexigenic neuropeptide, neuropeptide Y (NPY) [11,20,43], and stimulating the anorexigenic neuropeptides, ␣-melanocyte stimulating hormone (␣-MSH) and cocaine and amphetamine related transcript (CART [14,20,28,38]). Central leptin action increases energy expenditure by stimulating sympathetic nervous system (SNS) outflow to brown adipose tissue (BAT), as evidenced by increased uncoupling protein-1 (UCP-1) mRNA expression [12,42]. The orexi-

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genic neuropeptide NPY normally inhibits SNS outflow to BAT in addition to stimulating appetite [4]. Thus, leptin induced decreases in hypothalamic NPY may contribute to the increase in energy expenditure via sympathetic activation of BAT. Leptin also may act through changes in NPY to modulate the reproductive axis. A chronic increase in NPY signaling inhibits LHRH [19,23]; therefore, a leptin induced decrease in hypothalamic NPY may mediate the stimulatory effects of leptin on LHRH secretion. In obese (f/f) Koletsky rats, the leptin receptor is nonfunctional due to a mutation causing a premature stop codon in the extracellular domain [44]. Similar to other genetic models of leptin signaling deficiency, Koletsky rats are obese, diabetic and infertile [15,25,26]. We have recently reported alterations in levels of hypothalamic neuropeptides that regulate energy balance and reproduction in obese male and female f/f Koletsky rats [25]. Hypothalamic NPY levels were elevated while anorexigenic ␣-MSH and CART were decreased. We also observed decreased UCP-1 mRNA in BAT, indicating decreased energy expenditure. In female obese Koletsky rats, the reproductive axis was adversely affected with abnormally long estrous cycles and reduced levels of hypothalamic LHRH and serum estradiol and progesterone [25]. In wild type animals, the leptin receptor has been localized to several hypothalamic nuclei including the ventromedial hypothalamus (VMH), medial preoptic area (MPOA), paraventricular nucleus (PVN), and the arcuate nucleus (ARC) along with the dorsal vagal complex (DVC) in the brainstem [13,30,31,41,46]. Leptin receptor mRNA is expressed in NPY/AGRP neurons as well as in POMC/CART neurons in the ARC [3,16]. We hypothesized that populations of leptin receptors in differing sites in the CNS may be key to the pleiotropic actions of leptin. The feasibility of leptin receptor installation in the central nervous system sites with the aid of viral vector gene therapy has been demonstrated. Morton et al. [32], used adeno-virus to for short-lived expression of leptin receptor in the ARC or lateral hypothalamus of Koletsky rats and determined that leptin signaling in the ARC was sufficient to manifest leptin effects on food intake. We used recombinant adeno-associated viral (rAAV) vectors for longterm expression of the leptin receptor gene in fatty (fa/fa) Zucker rats and demonstrated that leptin signaling in the ARC was critical for restoring estrous cyclicity [24]. In order to identify the CNS sites specifically involved in leptin action on food intake, energy expenditure and reproduction we have now used rAAV to install the leptin receptor in several CNS sites in the leptin receptor deficient female f/f Koletsky rat.

2. Materials and methods Female obese (f/f) Koletsky rats, 3–4 weeks of age purchased from Vassar College (Poughkeepsie, NY) were housed in individual cages in air-conditioned rooms (22 ◦ C) with lights on from 05:00–19:00 h. Standard rat chow and tap water were available ad libitum. Animal procedures were

approved by the Institutional Animal Care and Use Committee. 2.1. Viral vectors As detailed earlier [24], one rAAV transfer vector (rAAVOB-Rb) carried the human leptin receptor transgene under the control of a tet-responsive promoter (tetO). The human longform leptin receptor cDNA cloned from pCDNA3.1-OB-Rb (a gift from Dr. Hsiung, Lilly Research Laboratories, Indianapolis, IN) was subcloned into pTR-UF13 plasmid vector downstream of tetO. The second promoter vector, designated rAAV-rtTA/rTS, was constructed to encode two chimeric genes, a reverse-tet-regulatable trans-activator (rtTA) which activates gene transcription in the presence of dox, and a tetcontrolled trans-suppressor which silences gene transcription in the absence of dox. In the presence of dox, the rtTA protein product binds dox, undergoes a conformational change and homodimerizes to initiate gene transcription of OB-Rb. The third vector, designated rAAV-UF11-GFP contains the green fluorescent protein (GFP) gene driven by the same chicken ␤-actin promoter. All vectors were packaged and titered at the University of Florida Powell Gene Therapy Center as previously described [11,24,36]. 2.2. Experimental design Under pentobarbital anesthesia (60 mg/kg body weight) rats were fitted into a stereotaxic apparatus. With the aid of a Kopf microinjector, rats were microinjected bilaterally as described earlier [24] with 0.25 ␮l rAAV-OBRb (2.4 × 1010 infectious particles/ml) plus 0.25 ␮l rAAVrtTA/rTS (4.5 × 103 infectious particles/ml) into one of the following nuclei: ARC (2.7 mm posterior to bregma, 0.2 mm lateral of the sinus, and 9.8 mm ventral to the dura), VMH (2.7 mm posterior to bregma, 0.5 mm lateral of the sinus, and 8.8 mm ventral to the dura), PVN (1.8 mm posterior to bregma, 0.4 mm lateral of the sinus, and 7.5 mm ventral to the dura), MPOA (0.3 mm posterior to bregma, 0.6 mm lateral of the sinus, and 7.8 mm ventral to the dura), or the DVC (13.5 mm posterior to bregma, 0.5 mm lateral of the sinus, and 1.5 mm ventral to the dura) stereotaxic coordinates were determined from the rat brain atlas [35]. There were 5–6 rats/group/site. During stereotaxic surgery a 60-day slow release doxycycline pellet (dox, Innovative Research Inc., Sarasota, FL) was implanted subcutaneously to release dox continuously over a period of 60 days. Dox was required to conformationally change the tet-responsive trans-activator, allowing it to bind to the tet-responsive promoter in rAAVOB-Rb and cause OB-Rb gene transcription. In our earlier study [24] we had detected small expression of the leptin receptor even in the absence of dox, therefore, in the present study groups of 8–9 control f/f rats were microinjected in each site with 0.5 ␮l rAAV-GFP and implanted with a dox pellet sc. Body weight and food intake were measured bi-weekly, and estrous cycles were monitored daily by examination of vagi-

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nal cytology. In addition, a group of six lean, age-matched female Koletsky control rats, without a leptin receptor mutation were purchased at 150–180 g and monitored in the same manner during the 55-day experiment. Approximately 55 days post-injection, rats in diestrus were killed by decapitation between 10:00 am and 2:00 pm, brains were rapidly removed and the hypothalami were excised. The hypothalamic fragment extended from the optic chiasm rostrally to the mammillary bodies caudally and to the lateral sulci. The hypothalamic fragment was divided longitudinally into halves. One half was homogenized in 1 ml of 0.1 N HCl for neuropeptide analyses and stored at −20 ◦ C. The other half was snap-frozen on dry ice for mRNA quantitation. Serum from trunk blood was stored at −20 ◦ C until hormone analyses. BAT was dissected, snap-frozen in dry ice and stored at −80 ◦ C until mRNA extraction for UCP-1 mRNA analysis by dot blot hybridization. In addition, ovaries and uteri were removed, weighed, and fixed in Bouin’s solution. Forty micrometers thick sections of ovaries and uteri were stained with hematoxylin and eosin for histological examination. To verify the injection sites, three rats from each rAAV-GFP microinjected group were deeply anesthetized with pentobarbital and perfused intracardially with 4% paraformaldehyde. Brains were removed and post-fixed in 4% paraformaldehyde and subsequently immersed in 20% sucrose overnight. Forty micrometers thick sections were stored in cryoprotectant at −20 ◦ C until they were processed for immunohistochemical localization of GFP as previously described, using a primary antibody from Clontech Laboratories Inc. (Palo Alto, CA) [36]. 2.3. Serum measurements Blood hormone levels were analyzed by RIA in duplicate in single assays. Serum leptin and insulin levels were assayed in 50 ␮l of serum diluted 1:10 in glass tubes using radioimmunoassay (RIA) kits purchased from Linco Research, Inc. (St. Charles, MO), according to manufacturer’s instructions. The sensitivity was 0.5 ng/ml for the leptin assay and 0.1 ng/ml for the insulin assay. Serum glucose levels were measured using a glucometer with a sensitivity of 20 mg/dl (Glucometer Elite XL, Bayer Corp., Pittsburgh, PA). Serum steroids were measured with RIA kits from Diagnostic Systems Laboratories (Webster, TX), according to the manufacturer’s instructions. Estradiol was measured in 200 ␮l of undiluted serum extracted with diethyl ether using the third generation estradiol RIA kit, and the sensitivity of the assay was 0.6 pg/ml. Serum progesterone was measured in 10 ␮l of unextracted serum and the sensitivity of the assay was 0.10 ng/ml.

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tent was determined using a Bradford protein assay (Bio-Rad, Richmond, CA), and results from RIAs of the hypothalamic extracts are expressed as pg per ␮g protein. NPY concentration in 50 ␮l of neutralized hypothalamic extract was determined by RIA as previously described [39], using porcine NPY as the reference standard (Peninsula Laboratories, Belmont, CA) and iodinated NPY (Amersham Biosciences, Piscataway, NJ). Antiserum to NPY (RA31) was generously supplied by Dr. William Crowley (University of Utah, Salt Lake City, UT). The range of the assay was 1.95–1000 pg per tube. Alpha-melanocyte stimulating hormone (␣-MSH) was measured in 50 ␮l of neutralized hypothalamic extract using an RIA kit from Peninsula Laboratories (Belmont, CA), according to the manufacturer’s instructions. The range the assay was 1–128 pg per tube. LHRH levels in 50 ␮l aliquots of neutralized hypothalamic extract were measured by RIA, as previously described [5], using synthetic LHRH (Peninsula Laboratories, Belmont, CA) as reference standard and as iodinated hormone (Amersham Biosciences, Piscataway, NJ). LHRH antiserum (EL-14) was kindly supplied by Drs. W.E. Ellinwood and M. Kelly (Oregon Regional Primate Center, Portland, OR). The range of the assay was 0.1–80 pg per tube. 2.5. UCP-1 mRNA expression in BAT Total cellular RNA was extracted using an RNeasy Kit (Qiagen, Chatsworth, CA) and analyzed for UCP-1 mRNA expression by dot blot hybridization as described earlier [40]. The full-length cDNA clone was provided by Dr. Leslie Kozak, Jackson Laboratory, Bar Harbor, ME and verified by Northern analysis. 2.6. RT-PCR for human OB-Rb mRNA Human leptin receptor gene expression was performed as previously described [24]. Briefly, total hypothalamic RNA was extracted and DNase treated using an RNeasy Kit (Qiagen, Chatsworth, CA). One microgram of RNA was reversetranscribed using random hexamer primer Taqman reagents (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Primers for the human leptin receptor gene were designed using the sequence of the plasmid pCDNA3.1 + hOBR (Lilly Research Laboratories, Indianapolis, IN). The gene was amplified using Perkin-Elmer Biosystems reagents (Foster City, CA) from a single RT reaction using the following parameters: denaturation at 90 ◦ C, 1 min, annealing at 60 ◦ C, 1 min, extension at 72 ◦ C, 1 min for 38 cycles [24]. 2.7. Quantitative real time PCR

2.4. Hypothalamic neuropeptides Acid extracts of the hypothalami were neutralized with 0.1 N NaOH and 50 ␮l aliquots were lyophilized. Protein con-

Hypothalamic expression of NPY, POMC, CART and AGRP was quantified by real time PCR, as previously described [24,25]. Briefly, total hypothalamic RNA was

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extracted and DNase treated using an RNeasy Kit (Qiagen, Chatsworth, CA). One microgram of RNA was reversetranscribed using random hexamer primers and Taqman reagents (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Synthesized cDNA, corresponding to 50 ng total RNA, was used for real time quantitative PCR. Gene specific primers and probes were designed for POMC (Genebank Acc#NM 139326), CART (Genebank Acc#NM 017110), AGRP (Genebank Acc#AF20601), and NPY (Genebank Acc#NM 012614) using Primer Express Software (PE Applied Biosystems) according to the software guidelines. All primers and Taqman probes were purchased from PE Applied Biosystems. A Taqman PCR assay for each was performed in duplicate on cDNA samples in 96-well plates on an ABI Prism 7000 Sequence Detection System (PE Applied Biosystems). 18S assays, designed and purchased through PE Applied Biosystems, were run in parallel for each sample. Each 25 ␮l reaction contained 12.5 ␮l Taqman 2× Master Mix (PE Applied Biosystems), 1.0 ␮l cDNA, 2.5 ␮l sense primer (8 ␮M), 2.5 ␮l antisense primer (8 ␮M), 0.3 ␮l probe (100 nM), and 6.2 ␮l PCR grade water. The PCR parameters were 95 ◦ C for 10 min, 1 cycle, then 60 ◦ C for 1 min, 95 ◦ C for 15 s for 40 cycles. 2.8. Statistical analyses All values are expressed as mean ± S.E.M. For each variable, statistical comparisons between control and treated groups for each site were performed by Student’s t-test. For the analysis of body weight and food intake over time, statistical comparisons between the control and rAAV-OB-Rb injected groups for each site were performed by two-way ANOVA (time × treatment) to test for overall significance with treatment and time as variables, and Tukey’s post hoc test for individual significant differences. Statistical analyses utilized Graph Pad Prism software (Graph Pad software, San Diego, CA). The level of significance was set at p < 0.05 for all analyses. For the real time quantitative PCR data, all statistics were done on delta Ct values normalized to 18S before the data were transformed to percent differences and expressed as percent of lean controls.

3. Results 3.1. OB-Rb mRNA expression Human OB-Rb mRNA expression in hypothalami of rats microinjected with viral vectors encoding OB-Rb and the promoter vectors was assessed by RT-PCR (Fig. 1). The RT-PCR assay was specifically designed to assess the presence or absence of human leptin receptor mRNA with no cross reactivity to rat or mouse leptin receptor mRNA. This design allowed for detection of OB-Rb mRNA specifically driven by the rAAV-OB-Rb vector system induced by doxycycline. As shown in Fig. 1, there was no detectable human

Fig. 1. Leptin receptor mRNA expression following microinjection of the viral vector encoding the human leptin receptor into specific central sites of obese Koletsky rats (data shown from two representative rats in each group). CYC: cyclophilin; OB-R: leptin receptor; GFP: green fluorescent protein; ARC: arcuate nucleus; VMH: ventromedial hypothalamus; PVN: paraventricular nucleus; MPOA: medial preoptic area; DVC: dorsal vagal complex.

OB-Rb mRNA in control rats injected with rAAV-GFP. Significant expression of human leptin receptor mRNA was detected all groups of rats microinjected with rAAV-OB-Rb and implanted subcutaneously with dox pellets. 3.2. Green fluorescent protein expression Gene expression following rAAV microinjections was verified by immunohistochemical localization of GFP. GFP was localized in neurons and fibers at each of the injected sites (for example, ARC and NTS: Fig. 2A and B). GFP positive cells were seen bilaterally and uniformly around the microinjection sites in cells mostly displaying neuronal morphology and in axon and dendrites. Results of these control injections were similar to other experiments performed in our laboratory showing GFP expression confined to the site of microinjection with minimal spread to adjacent areas [1,2]. These control microinjections have been shown previously to illustrate the areas of CNS transfected by serotype-2 rAAV vectors without alterations in transfection area caused by independent properties of the transgene [1,2]. 3.3. Effect of OB-Rb installation on energy homeostasis Fig. 3A–E shows the body weight of rats in each experimental group during the 55-day experiment. In rats microinjected with rAAV-OB-Rb in the ARC, VMH, MPOA, and

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Fig. 2. Photomicrograph (4× and 20×) of examples of representative coronal sections showing immunohistochemical localization of green fluorescent protein for (A) arcuate nucleus (ARC) and median eminence (ME) and (B) the nucleus tractus solitarius (NTS) and area postrema (AP); 3v: third ventricle, lPBN: lateral parabrachial nucleus, XII: hypoglossal nucleus.

DVC, but not the PVN, small but significant reductions in body weight gain were first apparent on day 10 post-injection and lasted for the duration of the experiment. This resulted in significantly lower body weights at day 55 post-injection as compared to that of obese rAAV-GFP injected rats (p < 0.05, Fig. 3F). However, in no group was there a reduction in body weight from initial weight or a reversal of obesity. Fig. 4A–E shows the 24 h food intake measured at 3day intervals for each group throughout the experimental period. A significant decrease in food intake was detected only in rats microinjected with rAAV-OB-Rb in the ARC. This reduction was seen throughout the observation period resulting in a significant decrease in average 24 h food intake (Fig. 4F, p < 0.05). Although body weight gain of rats injected with rAAV-OB-Rb into the VMH, MPOA, and DVC was decreased, there was no decrease in food intake.

In obese Koletsky control rats injected with rAAV-GFP, UCP-1 mRNA in BAT, a measure of thermogenic capacity, was reduced as compared to lean controls (p < 0.05, Fig. 5). rAAV-OB-Rb microinjection into all nuclei, except the PVN significantly elevated UCP-1 mRNA expression compared to their respective obese rAAV-GFP injected controls (Fig. 5, p < 0.05). Following rAAV-OB-Rb microinjection into the ARC, VMH, MPOA, or DVC, UCP-1 mRNA levels were in the range of those of lean, control rats. 3.4. Metabolic hormone levels (Table 1) As expected, obese rats microinjected with rAAV-GFP were severely hyperleptinemic with serum leptin levels several fold higher than in lean control rats. rAAV-OB-Rb microinjection in the ARC or VMH significantly reduced

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Fig. 3. Effect of rAAV-OB-Rb microinjection at various sites on body weight in female Koletsky rats. (A–E) Open circles = rAAV-GFP treated group and closed squares = rAAV-OB-Rb treated group. * p < 0.05 vs. control GFP injected group in this and subsequent figures.

serum leptin levels compared to respective obese rAAV-GFP microinjected controls (p < 0.05). rAAV-OB-Rb microinjection into the PVN, MPOA or DVC did not affect serum leptin concentrations. All groups of obese Koletsky rats had significantly higher serum leptin levels compared to the untreated lean group, regardless of treatment. The obese Koletsky rats were also hyperinsulinemic and hyperglycemic compared to lean control rats (Table 1). Insulin levels ranged from 9.4 to 28.1 ng/ml in different groups of obese rAAV-GFP microinjected control rats compared to 3.3 ± 1.5 ng/ml in the lean control rats. Microinjection of rAAV-OB-Rb into the ARC and DVC reduced serum insulin concentrations (p < 0.05). Inexplicably, rAAVOB-Rb microinjection into the MPOA elevated serum insulin concentrations. There was no effect of rAAV-OB-Rb in the VMH or PVN on serum insulin concentrations. Despite these wide variations in insulin response, rAAV-OB-Rb installation into all nuclei except the PVN significantly reduced serum glucose levels; in the ARC and VMH groups serum

Table 1 Effects of rAAV-OB-Rb microinjection on serum leptin, insulin and glucose levels Site group Lean ARC-GFP ARC-OB-Rb VMH-GFP VMH-OB-Rb PVN-GFP PVN-OB-Rb MPOA-GFP MPOA-OB-Rb DVC-GFP DVC-OB-Rb

Leptin (ng/ml) 8.56 83.7 50.1 70.1 47.3 94.0 75.0 60.0 72.4 77.5 69.3

± ± ± ± ± ± ± ± ± ± ±

1.2a 7.0 8.6* 3.4 0.9* 4.5 3.8 4.7 13 4.1 4.0

Insulin (ng/ml) 3.3 20.7 9.3 9.4 10.1 13.6 17.8 9.8 17.1 28.1 19.1

± ± ± ± ± ± ± ± ± ± ±

1.5a 3.0 2.7* 1.0 2.0 0.9 1.4 2.0 1.9* 3.4 1.6*

Glucose (mg/dl) 137 ± 5b 150 ± 4 125 ± 4* 161 ± 2 141 ± 8* 152 ± 7 168 ± 3 180 ± 4 161 ± 4* 191 ± 8 162 ± 6*

All values are mean ± S.E.M. a p < 0.05 vs. other groups. b p < 0.05 vs. GFP injected obese control groups. * p < 0.05 compared to the GFP control group for that nucleus.

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Fig. 4. Effect of rAAV-OB-Rb microinjection at various sites on food intake in female Koletsky rats. (A–E) Open circles = rAAV-GFP treated group and closed squares = rAAV-OB-Rb treated group.

glucose levels were reduced to the range of normoglycemia seen in lean control rats. These two groups exhibiting normoglycemia showed concurrent decreases in serum leptin concentrations.

Fig. 5. Effect of leptin receptor installation on uncoupling protein-1 (UCP1) mRNA expression in brown fat in female Koletsky rats assessed by dot blot hybridization and expressed as percent of lean controls. a p < 0.05 vs. GFP injected obese rats in this and subsequent figures.

3.5. Effect of OB-Rb installation on reproduction (Table 2) In agreement with our previous study, estrous cycle length (Table 2) was significantly longer in the obese Koletsky rats microinjected with rAAV-GFP compared to lean control rats [25]. Estrous cycle duration was unchanged in rats microinjected with rAAV-OB-Rb in the VMH, PVN or DVC. However, rAAV-OB-Rb microinjection into either the ARC or MPOA significantly shortened estrous cycle length to the normal 4–5 day duration. In the latter group rats there was a marked increase in ovarian weight compared to the obese rAAV-GFP microinjected controls as well as the lean rats (p < 0.05). There was no effect of rAAV-OB-Rb on ovarian weights in the other groups. Obese Koletsky rats had slightly elevated uterine weights compared to lean control rats and leptin receptor installation did not affect uterine weight except for a small decrease in ARC microinjected rats. Lep-

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Table 2 Effects of rAAV-OB-Rb microinjection on reproductive cycles, organs and hormones Treatment group

Days/estrous cycle

Lean ARC-GFP ARC-OBR VMH-GFP VMH-OBR PVN-GFP PVN-OBR MPOA-GFP MPOA-OBR DVC-GFP DVC-OBR

4.4 6.0 4.8 5.5 5.1 5.4 5.1 5.6 4.6 5.6 5.2

± ± ± ± ± ± ± ± ± ± ±

0.2 0.4 0.2* 0.2 0.1 0.1 0.1 0.3 0.2* 0.3 0.1

Ovarian weight (g) 51.0 52.6 56.4 42.0 43.3 40.0 42.8 63.9 104.0 42.9 43.3

± 3.1 ± 3.1 ± 3.9 ± 1.5 ± 1.7 ± 1.3 ± 1.4 ± 9.4 ± 4.3* ± + 1.2 ± 1.4

Uterine weight (g)

Estradiol (pg/ml)

317 ± 10 468 ± 34 366 ± 21* 416 ± 11 433 ± 21 440 ± 24 425 ± 14 504 ± 40 595 ± 46 402 ± 30 457 ± 23

33.5 3.9 8.5 3.6 3.6 3.6 3.8 2.0 5.3 7.5 17.7

± ± ± ± ± ± ± ± ± ± ±

2.0 0.8 3.8 1.1 1.7 1.0 1.3 0.3 2.5 2.3 5.3

Progesterone (ng/ml) 7.3 14.6 7.0 15.3 6.8 22.5 23.1 13.5 6.4 18.0 18.8

± ± ± ± ± ± ± ± ± ± ±

1.9 2.5 1.4* 3.1 1.8* 5.7 3.2 0.7 1.0* 4.9 5.0

All values are mean + S.E.M. * p < 0.05, compared to the GFP control group for that nucleus.

tin receptor installation in any site did not affect ovarian or uterine histology (data not shown). As expected [25], serum estradiol was significantly lower in obese rats compared to lean control rats (Table 2), and despite improvements in estrous cyclicity with microinjection in the ARC or MPOA, estradiol levels were not altered, regardless of the microinjection site. Serum progesterone levels were higher in obese rats compared with lean controls. Viral vector induced leptin receptor expression in the ARC, VMH, or MPOA significantly reduced serum progesterone (p < 0.05). 3.6. Effect of OB-Rb on hypothalamic neuropeptides Hypothalamic LHRH concentrations in obese rats were lower than in the lean control rats (Fig. 6). rAAV-OB-Rb microinjection in the ARC or MPOA that normalized estrous cycle length significantly elevated hypothalamic LHRH concentrations to the range seen in the lean controls. There was no effect of rAAV-OB-Rb microinjection in the VMH, PVN, or DVC on LHRH concentration in the hypothalamus. Both NPY mRNA expression and NPY peptide concentration in the hypothalami of obese Koletsky rats were higher than in the lean control rats, similar to our previously reported results (Fig. 7 [25]). NPY mRNA expression, assessed by quantitative real time PCR, is represented as percent change from the lean control group. rAAV-OB-Rb microinjection in

Fig. 6. Effect of leptin receptor installation on hypothalamic luteinizing hormone releasing hormone (LHRH) peptide levels, as determined by radioimmunoassay and expressed as pg/␮g protein.

the ARC decreased NPY mRNA (p < 0.05) to 40% of that seen in the obese rAAV-GFP microinjected control rats. The decrease in NPY mRNA was accompanied by a corresponding decrease in hypothalamic NPY peptide concentration (4.5 ± 0.3 versus 9.7 ± 2.2 pg/␮g protein, p < 0.05), bringing the NPY concentration in the hypothalami of obese rAAVOB-Rb ARC microinjected rats to the range seen in the lean control rats. While there was no effect of rAAV-OBRb microinjection into the MPOA or DVC on NPY mRNA expression, there was a significant reduction in hypothalamic NPY peptide concentration in these two groups. rAAV-OBRb microinjection in the VMH or PVN altered neither NPY mRNA expression nor NPY peptide concentration in the hypothalamus. Obese rats had lower hypothalamic POMC mRNA expression and ␣-MSH peptide compared to the lean control rats (Fig. 8 A and B). Generally, there was no effect of leptin

Fig. 7. Effect of leptin receptor installation on hypothalamic neuropeptide Y (NPY) (A) mRNA expression determined by quantitative real time PCR and (B) peptide levels determined by radioimmunoassay.

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Fig. 8. Effect of leptin receptor installation on hypothalamic proopiomelanocortin (POMC) mRNA expression determined by quantitative real time PCR and ␣-melanocyte stimulating hormone (␣-MSH) peptide levels determined by radioimmunoassay.

receptor installation on POMC gene expression except for the 150% increase seen in rats microinjected with rAAVOB-Rb in the VMH, resulting in similar POMC mRNA expression to that seen in the lean control rats (Fig. 8A). This increase did not translate into increased ␣-MSH, a protein product of POMC mRNA. Although POMC mRNA expression was unchanged, rAAV-OB-Rb microinjection in the ARC increased ␣-MSH concentration (2.2 ± 0.1 versus 1.5 ± 0.2 pg/␮g protein in GFP controls) in the hypothalamus to levels similar to those seen in the lean control rats (Fig. 8B). Leptin receptor installation in the VMH, PVN, MPOA, or DVC did not alter hypothalamic ␣-MSH peptide concentration. In contrast to the selective effects of OB-Rb installation on NPY and POMC, neither orexigenic AGRP nor anorexigenic CART expression were affected by rAAV-OB-Rb microinjection in any of the four hypothalamic sites or the DVC in the hindbrain (data not shown).

4. Discussion This study in leptin receptor deficient Koletsky rats confirms our earlier study in Zucker rats showing that dox inducible rAAV vectors can be used to successfully express the leptin receptor gene in the CNS [24]. We used rAAV vectors for long-term gene expression to delineate sites in the CNS that signal the various physiological actions of leptin on food intake, energy expenditure and reproduction. The human leptin receptor was successfully expressed for the 55 days duration of the experiment in each of the five sites

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tested as evidenced by RT-PCR detection of human OB-Rb gene transcripts and the observed physiological effects of receptor function. Immunohistochemical analysis in rAAVGFP injected rats confirmed the proper placement of the viral vectors and expression was confined to the sites of injection with little spread to adjacent areas, suggesting that receptor expression in rAAV-OB-Rb microinjected rats was confined to neurons and fibers at the desired site of microinjection. In addition, several studies have shown little spread after rAAV microinjections as well as a lack of retrograde transport from the site of microinjection, lending support to the notion that rAAV vectors are transduced at the site of microinjection without spreading or traveling [6,45]. Previous similar microinjection studies using rAAV also indicate that rAAVGFP immunohistochemistry approximates the area of the CNS transduced by any rAAV vector, irrespective of the properties of the transgene [1,2,36]. Therefore, the observed effects of rAAV vector microinjection on energy homeostasis and reproduction are most likely due to the actions of endogenous leptin at sites in the CNS where the human leptin receptor was transferred. The major effects of leptin receptor installation at each of the five CNS microinjection sites are summarized in Table 3. Leptin receptor installation improved energy homeostasis and reproductive function; however, the beneficial effects were not uniformly observed at every microinjection site nor were these effects equivalent. Overall, the PVN was a generally ineffective microinjection site while the ARC was the most effective. The lack of leptin action in rats expressing the leptin receptor in the PVN was unexpected. We cannot Table 3 Summary of effects of rAAV-OB-Rb microinjection in Koletsky rats ARC

VMH

PVN

MPOA

DVC

Energy homeostasis Body weight Food intake UCP-1 mRNA Leptin Insulin Glucose Ghrelin

↓ ↓ ↑ ↓ ↓ ↓ ↔

↓ ↔ ↑ ↓ ↔ ↓ ↔

↔ ↔ ↔ ↔ ↔ ↔ ↓

↓ ↔ ↑ ↔ ↑ ↓ ↔

↓ ↔ ↑ ↔ ↓ ↓ ↔

Reproduction Estrous cycles Ovarian weight Uterine weight Progesterone Estradiol Ovarian histology

↓ ↔ ↓ ↓ ↔ ↔

↔ ↔ ↔ ↓ ↔ ↔

↔ ↔ ↔ ↔ ↔ ↔

↓ ↑ ↔ ↓ ↔ ↔

↔ ↔ ↔ ↔ ↔ ↔

Hypothalamic neuropeptides LHRH ↑ NPY mRNA ↓ NPY peptide ↓ POMC ↔ ␣-MSH ↑ AGRP ↔ CART ↔

↔ ↔ ↔ ↑ ↔ ↔ ↔

↔ ↔ ↔ ↔ ↔ ↔ ↔

↑ ↔ ↓ ↔ ↔ ↔ ↔

↔ ↔ ↓ ↔ ↔ ↔ ↔

↓: decreased, ↔: no change, ↑: increased.

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rule out the possibility that in the current study, the amount of leptin receptor expressed in the PVN injected group may have been insufficient. The notion that the ARC may be the primary site of leptin action is supported by several studies [1,2,24,32]. In addition, others have shown that diet-induced obesity causes specific reductions in leptin sensitivity in the ARC and not other sites in the CNS, including the PVN, VMH and DVC [34]. Therefore, it is not surprising that leptin receptor expression in the ARC stimulated both energy expenditure and reproductive axis function, and inhibited food intake. The rate of body weight gain was reduced by rAAV-OB-Rb microinjection in the hypothalamic ARC, VMH, and MPOA as well as the DVC in the hindbrain. This was accompanied by a decrease in food intake only with microinjection in the ARC. Therefore, the reduction in body weight gain caused by rAAV-OB-Rb microinjection at the other CNS sites was likely due to increased energy expenditure [12]. Indeed, we detected increases in BAT UCP-1 mRNA expression in rats microinjected with rAAV-OB-Rb in every site where body weight gain was reduced. Leptin receptor installation also had a variety of effects on metabolic hormones. The impact on body weight gain following leptin receptor installation in the ARC and VMH was accompanied by reduced serum leptin concentrations. Obese Koletsky rats also are diabetic [26], and interestingly rAAVOB-Rb microinjection at all sites except the PVN attenuated hyperglycemia. Additionally, rAAV-OB-Rb in the ARC and DVC attenuated hyperinsulinemia. The present study suggests that leptin may regulate insulin secretion by acting primarily in the ARC and hindbrain. Normalization of glucose despite the reduction in insulin secretion suggests that leptin’s influence on insulin secretion is distinct from its effects on insulin sensitivity. These data suggest that through action at receptors in the ARC, VMH, and DVC leptin increases insulin sensitivity thereby attenuating hyperglycemia. In addition to metabolic pathologies, leptin receptor deficient Koletsky rats are infertile [26]. Leptin normally increases LHRH concentrations in the hypothalamus and LH secretion from the pituitary thus stimulating the reproductive axis [47]. As reported earlier [25], LHRH concentrations were decreased in the hypothalami of obese Koletsky rats. rAAV-OB-Rb microinjection in the ARC or MPOA significantly increased hypothalamic LHRH concentrations to the range observed in lean control rats. Because leptin receptors have not been localized to LHRH producing neurons, leptin likely stimulates LHRH production by modulating the expression of other neuropeptides, such as NPY. At persistent high levels, NPY inhibits LHRH production, and blockade of NPY activity with antiserum in Zucker rats stimulated reproductive behavior [19,23,29]. In the two groups (ARC and MPOA) where leptin receptor installation increased hypothalamic LHRH, hypothalamic NPY peptide concentrations were concurrently reduced. This supports the concept that reduced NPY in the hypothalamus reduced LHRH neuronal inhibition, thereby increasing hypothalamic LHRH.

Estrous cycle length in obese Koletsky rats is prolonged as compared with that in lean counterparts [25,26]. Leptin receptor installation in the ARC or MPOA normalized estrous cycle length. This confirms our recent results of normalization of estrous cyclicity in obese Zucker rats following leptin receptor installation in the ARC [24] and indicates that leptin action in the ARC and MPOA stimulates the reproductive axis. The mechanism of leptin action on energy balance and reproductive function involves modulation of hypothalamic neuropeptides. Leptin reduces NPY production in the hypothalamus to influence a variety of physiological functions [4,8,19,23,43]. NPY mRNA and peptide concentrations were both reduced with leptin receptor installation in the ARC, where NPY-producing neurons are located. This reduction in levels of a potent orexigenic peptide may account for the observed reduction in food intake seen only with leptin receptor installation in the ARC. Although hypothalamic NPY mRNA was not reduced with rAAV-OB-Rb microinjection in the DVC, NPY levels were reduced in the hypothalami of these rats. It is possible that as in the ARC, leptin action on the newly installed leptin receptors in the hindbrain reduced NPY synthesis in catecholaminergic neurons, thus reducing the contribution of the brainstem to hypothalamic stores of NPY [21]. In addition to its orexigenic activity, NPY also inhibits SNS activity to BAT [4,8]. The observed reduction in NPY peptide seen with leptin receptor installation in the ARC, MPOA, and DVC most likely curtailed the inhibition of sympathetic activity to the BAT as evident from the increases in UCP-1 mRNA in these groups. Interestingly, leptin receptor installation in the VMH increased UCP-1 mRNA without altering NPY concentrations suggesting that other neuropeptides, such as the melanocortins, may act along with leptin to stimulate BAT UCP-1 mRNA expression [17,28]. This is supported by our observations of a significant increase in POMC mRNA in the VMH microinjected groups. Leptin reportedly increases melanocortin signaling in the hypothalamus to reduce food intake and increase energy expenditure [10,17]. With the exception of the VMH injected group, we did not detect an increase in POMC mRNA expression. Although leptin receptor installation in the ARC did not increase POMC mRNA, concentrations of ␣-MSH, derived from POMC, were significantly increased. It is likely that differential processing of the POMC precursor protein resulted in increased ␣-MSH with possible reductions in other POMC derived peptides. Another member of the melanocortin pathway, AGRP, the melanocortin-3/4 receptor antagonist, is inhibited by leptin and may be involved in leptin regulation of food intake [11]. However, in rats with leptin receptor mutations, such as the fatty Zucker rat and the obese Koletsky rat, AGRP mRNA expression is not increased [24,25,27]. We observed only a small increase in AGRP mRNA in obese compared with lean rats, and leptin receptor installation did not alter hypothalamic AGRP mRNA expression. Therefore, although

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the melanocortin pathway may play an important role in the ability of leptin to regulate food intake it most likely acts through regulation of ␣-MSH, and not AGRP, in this model system. Another anorexigenic neuropeptide, CART, reportedly stimulated by leptin, decreases food intake [28,37]. CART mRNA levels are reduced in obese Koletsky rats compared to lean rats, but leptin receptor installation had no effect on hypothalamic CART mRNA levels, regardless of the site of microinjection [25]. It is likely that the effect of leptin on energy balance either does not involve regulation of CART, or requires leptin receptors in locations other than those tested in order to affect CART expression.

5. Conclusion In summary, this series of experiments delineates some of the CNS areas of leptin action, and supports the hypothesis that different neuronal populations may mediate different aspects of leptin physiology. Data suggest that leptin receptor expression in the ARC is required for leptin action on both the reproductive axis and energy balance. The data also show that other sites in the central nervous system are essential parts of the leptin pathway as well. With the exception of the PVN, leptin receptor installation in all other sites tested stimulated UCP-1 expression, presumably by decreasing NPY induced inhibition of sympathetic activity to BAT [4]. The one exception, the VMH injected group, had increased energy expenditure without changes in NPY, which was most likely attributable to the stimulation of melanocortin signaling [17]. It appears that leptin regulation of NPY signaling is a crucial regulatory factor in the maintenance of energy balance and reproductive function. Reductions in NPY peptide contributed to the stimulation of energy expenditure, as well as increased hypothalamic LHRH that stimulated the reproductive axis function [4,9,29]. Reduced food intake was seen only when both NPY decreased and ␣-MSH increased. Therefore, both actions may be necessary to decrease food intake in these obese Koletsky rats. Thus, these experiments with rAAV vector mediated site-specific leptin receptor installation demonstrate that different neuronal populations in the hypothalamus and brainstem are key mediators of different aspects of leptin action. In addition, these studies support the idea that the ARC is a crucial site of leptin action and that NPY is a critical factor in the central nervous system for leptin regulation of energy balance, metabolic hormones, and the reproductive axis.

Acknowledgements These studies were supported by a grant from the NIH NS 32727 and a fellowship from the American Heart Association 011028B. The expertise of Dr. S. Zolotukhin in preparation of the viral vectors is acknowledged. Word processing assis-

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