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*Genes and Gene Therapy
*Nutrition
*Obesity
Endocrinology Vol. 144, No. 5 2016-2024
Copyright © 2003 by The Endocrine Society

Arcuate Nucleus-Specific Leptin Receptor Gene Therapy Attenuates the Obesity Phenotype of Koletsky (fak/fak) Rats

Gregory J. Morton, Kevin D. Niswender, Christopher J. Rhodes, Martin G. Myers, Jr., James E. Blevins, Denis G. Baskin and Michael W. Schwartz

Department of Medicine (G.J.M., K.D.N., M.W.S.), Harborview Medical Center and University of Washington, Seattle, Washington 98104; Pacific Northwest Research Institute and Department of Pharmacology (C.J.R.), University of Washington, Seattle, Washington 98112; Research Division (M.G.M.), Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215; Veterans Affairs Puget Sound Health Care System (J.E.B., D.G.B.), Seattle, Washington 98108; and Departments of Medicine and Biological Structure (D.G.B.), University of Washington, Seattle, Washington 98104

Address all correspondence and requests for reprints to: Professor Michael Schwartz, Department of Medicine, Harborview Medical Center, University of Washington, 325 Ninth Avenue, Box 359757, Seattle, Washington 98104. E-mail: mschwart{at}u.washington.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin signaling in the hypothalamic arcuate nucleus (ARC) is hypothesized to play an important role in energy homeostasis. To investigate whether leptin signaling limited to this brain area is sufficient to reduce food intake and body weight, we used adenoviral gene therapy to express the signaling isoform of the leptin receptor, leprb, in the ARC of leptin receptor-deficient Koletsky (fak/fak) rats. Successful expression of adenovirus containing leprb (Ad-leprb) selectively in the ARC was documented by in situ hybridization. Using real-time PCR, we further demonstrated that bilateral microinjection of Ad-leprb into the ARC restored low hypothalamic levels of leprb mRNA to values approximating those of wild-type (Fak/Fak) controls. Restored leptin receptor expression in the ARC reduced both mean daily food intake (by 13%) and body weight gain (by 33%) and increased hypothalamic proopiomelanocortin mRNA by 65% while decreasing neuropeptide Y mRNA levels by 30%, relative to fak/fak rats injected with a control adenovirus (Ad-lacZ) (P < 0.05 for each comparison). In contrast, Ad-leprb delivery to either the lateral hypothalamic area of fak/fak rats or to the ARC of wild-type Fak/Fak rats had no effect on any of these parameters. These findings collectively support the hypothesis that leptin receptor signaling in the ARC is sufficient to mediate major effects of leptin on long-term energy homeostasis. Adenoviral gene therapy is thus a viable strategy with which to study the physiological importance of specific molecules acting in discrete brain areas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECENT PROGRESS IN the treatment of diseases such as cystic fibrosis demonstrates that gene therapy using viral vectors can effectively restore tissue-specific expression of proteins missing because of an autosomal recessive mutation (1, 2, 3). Although this strategy has yet to be successfully applied to genetic diseases affecting the central nervous system (CNS), it has potential to both confer therapeutic benefit and shed light on the role played by a particular protein in a specific brain region. The obesity phenotype arising from genetic deficiency of leptin receptors lends itself to a brain region-specific gene therapy approach because the brain is the primary target of leptin action in the body and discrete brain areas have been identified as potentially important mediators of leptin signal transduction. We therefore sought to determine whether the obese phenotype that results from leptin receptor (Lepr) gene mutation in rodents can be ameliorated by using adenoviral gene therapy to reconstitute leptin receptors selectively in a brain region implicated as a key target for leptin action.

Several observations support the hypothesis that the adipocyte hormone, leptin, is an adiposity-negative feedback signal. Leptin circulates at levels proportionate to body fat content (4) and enters the CNS in proportion to its plasma level (5). Leptin also potently reduces food intake and body weight (6, 7, 8, 9) by activating leptin receptors expressed in brain areas known to be involved in food intake control, such as the arcuate nucleus (ARC; Ref. 10). Evidence that leptin signaling is essential for normal energy homeostasis stems from observations that genetic deficiency of either leptin (in ob/ob mice; Ref. 11) or Lepr (in db/db mice; Ref. 12, 13, 14) causes severe obesity and leptin administration reverses obesity in ob/ob, but not db/db, mice (9). Similarly, Lepr mutation causes a severe obesity phenotype in both the Zucker (fa/fa; Ref. 15) and Koletsky (fak/fak) rat models. The latter animals have a nonsense mutation (T2289A->Tyr763Stop) in the extracellular domain of Lepr, resulting in absence of all forms of Lepr protein and reduced leprb mRNA stability (16, 17). The clinical importance of intact leptin signaling is highlighted by the severe obesity that occurs in humans with homozygous mutation of either leptin (18) or Lepr (19).

The Lepr contains a single transmembrane domain and is a member of the class I cytokine receptor family (14). Of five known Lepr splice variants, only the long or signaling form of the leptin receptor, Leprb, has been shown to be essential for normal energy homeostasis. Unlike the other leptin receptor isoforms, Leprb contains a 302-amino acid cytoplasmic domain that includes motifs for binding of intracellular signaling molecules such as Janus kinase (20, 21, 22). After binding to the activated leptin receptor, Janus kinase 2 phosphorylates signal transducer and activator of transcription proteins (23) that then dimerize and translocate to the nucleus, in which they activate a specific program of gene transcription. Lepr signaling is also coupled to the intracellular insulin receptor substrate-phosphatidylinositol 3-OH kinase pathway (24, 25).

Among the various brain areas that express leprb, the ARC is implicated as playing an especially important role. Contained within the ARC are two subsets of neurons that are regulated by leptin and exert potent, opposing effects on food intake and body weight. These include neurons containing proopiomelanocortin (POMC), which inhibit food intake (26, 27) and are stimulated by leptin (28, 29) and those containing both neuropeptide Y (NPY) and agouti-related peptide neurons, which stimulate food intake (30, 31, 32) and are inhibited by leptin (33, 34). Both neuronal subsets express leprb (10, 35, 36), and leptin microinjection into the vicinity of the ARC reduces food intake and body weight in rats (37). Although these observations suggest a key role for ARC neurons as mediators of leptin action in vivo, whether leptin signaling in the ARC alone is sufficient to mediate biological responses to leptin is unknown.

To critically test this hypothesis, we developed an adenoviral gene therapy approach with which to express leptin receptors in discrete brain areas. Specifically, we employed a stereotaxic microinjection technique to deliver adenovirus expressing either leprb (Ad-leprb) or a control reporter gene (Ad-lacZ) into the ARC of Lepr-deficient fak/fak rats and thereby determine the effects on food intake and body weight of leptin signaling limited to this brain area. Results from this experiment were compared with those obtained when Ad-leprb was microinjected into the lateral hypothalamic area (LHA), a brain area implicated in energy homeostasis that does not express high levels of Leprb (10, 35). Our findings suggest that leptin signaling limited to the ARC, but not the LHA, is sufficient to mediate leptin effects on energy homeostasis and adenoviral gene therapy is a useful approach with which to restore expression of CNS proteins missing because of genetic mutation in a brain region-specific manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Male lean (Fak/Fak) and obese (fak/fak) Koletsky rats (Vassar College, Poughkeepsie, NY) were generated from serial backcrosses (NIO equivalent) of the fak mutation (also known as Koletsky, faf, f, or cp) to the inbred rat strain, LA/N. Male Wistar rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA). All animals were housed individually in microisolator polycarbonate cages under SPF conditions, provided with ad libitum access to standard laboratory chow (PMI Nutrition International Inc., Brentwood, MO) and water and maintained in a temperature-controlled room with a 12-h light, 12-h dark cycle. All procedures were performed in accordance with National Institutes of Health Guidelines for the Care and Use of Animals and approved by the Animal Care Committee at the University of Washington.

Generation of recombinant adenovirus
The Ad-lacZ adenovirus was generated and purified as previously described (38). The human leprb (hleprb) cDNA in pcDNA3 was subcloned into the adenoviral transfer vector pACCMV using HindIII and XbaI. The recombined hleprb adenovirus was generated by cotransfection of pACCMV hleprb with the adenoviral backbone vector pBHG11 in 293 cells; the resulting recombinant virus was purified as described (Ref. 39 and Fig. 1Go).



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  		Figure 1. Schematic representation of the hleprb adenoviral construct. The hleprb cDNA was subcloned into the adenoviral transfer vector pACCMV. Cotransfection of pACCMVhleprb and the adenoviral backbone vector pBHG11 into 293 cells resulted in recombination between homologous adenoviral sequences in the two vectors and delivers hleprb cDNA to the resulting recombinant adenovirus.

 
Adenovirus microinjection
Animals were placed in a stereotaxic frame (Harvard/American Scientific Institution, Holliston, MA) under ketamine/xylazine anesthesia. The ARC was targeted bilaterally using a dual-guide, 30-g injector cannula system (Plastics One, Roanoke, VA) directed to stereotaxic coordinates 3.3 mm posterior to bregma and 10.5 mm below the surface of the skull. Microinjection into the LHA was performed by placing a single guide and 30-g injector cannula (Plastics One) into each LHA of each animal at stereotaxic coordinates 1.9 mm posterior to bregma ± 1.6 mm lateral to the midline and 8.9 mm below the skull. Adenovirus expressing either leprb (2.4 x 1012 pfu/ml) or lacZ (1.7 x 1012 pfu/ml) was administered to rats via a syringe injector pump (WPI, Sarasota, FL) at a rate of 100 nl/min for 5 min (500 nl/injection site). The entire injector system and cannulae were removed 5 min after the injections were completed. Food intake and body weight were measured daily for 16 d following adenovirus microinjection and were analyzed beginning on d 3 to allow time for postoperative recovery and for adenoviral genes to be expressed.

Blood and tissue collection and processing
On d 16 after adenoviral injection, Koletsky animals were placed under ketamine/xylazine anesthesia and perfused with 4% paraformaldehyde in 0.1 M PBS. Perfused brains were removed, postfixed in the same solution overnight at 4 C, embedded in 25% sucrose solution for 48 h at 4 C, snap-frozen in isopentane cooled with liquid nitrogen, and stored at -80 C for subsequent histochemical analysis. In a separate group of Koletsky rats and for studies using Wistar rats, a wedge of mediobasal hypothalamus (defined caudally by the mamillary bodies; rostrally by the optic chiasm; laterally by the optic tract; and superiorly by the apex of the hypothalamic third ventricle) was rapidly dissected following euthanasia and frozen for subsequent analysis by RT-PCR. Blood was obtained by cardiac puncture and collected into chilled heparin-containing tubes, separated into plasma, and stored at -20 C. Plasma glucose was measured using the glucose oxidase method (Beckman Instruments, Fullerton, CA), and plasma insulin concentrations were determined by RIA (Linco, St. Louis, MO).

Histochemical analyses
ß-Galactosidase (ß-gal) staining.
Coronal cryostat sections (14 µm) of rat brains were thaw mounted on slides, washed for 2 min in ice-cold PBS, fixed for 5 min in ice-cold 0.02% glutaraldehyde and 3.8% formaldehyde, and incubated in substrate according to standard methods (40). Cells expressing ß-gal (+) were identified by their blue-staining cell bodies.

In situ hybridization (ISH).
Brains were sectioned in a coronal plane at 14 µm on a cryostat; thaw mounted on ribonuclease-free slides; and treated with 4% paraformaldehyde, acetic anhydride, ethanol, and chloroform as described (28). For each animal, five slides (10 brain sections) were selected for hybridization with each riboprobe, and care was taken to ensure that slides for each assay were taken from comparable brain areas of each animal. Sections for Pomc mRNA quantitation were selected from an area of the ARC rostral to the ventromedial nucleus but caudal to the optic chiasm. Sections for Npy hybridization contained the ventromedial nucleus and corresponded to the midregion of the ARC. For hybridization to leprb mRNA, sections were sampled at regular intervals from areas extending from the midregion to the rostral portion of the ARC. All brain slices for within-group comparisons were prepared for hybridization concurrently and used in the same assay. Riboprobes from a cDNA template for NPY, POMC, or leprb (based on nucleotides 2899–3251) were transcribed in the presence of 33P-UTP (Amersham Biosciences, Piscataway, NJ). Unincorporated label was separated using a QIAquick nucleotide removal kit (QIAGEN, Santa Clarita, CA). The hybridization signal was detected and quantitated using the Cyclone phosphor imager system (Packard Instrument Company, Meriden, CT). For each section, the background hybridization signal was subtracted from the selected target. The mean value for each animal was determined from 8–10 anatomically matched sections per animal, per riboprobe, to produce an index of mRNA levels.

RT-PCR.
Total RNA was extracted from tissue using RNAzol B according to manufacturers’ instructions (Tel-Test, Inc., Friendswood, TX). RNA was quantitated by spectrophotometry at 260 nm, and 1 µg RNA was reverse transcribed with 10 U avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI). PCR was performed on a LightCycler (Roche Molecular Biochemicals, Indianapolis, IN) using a 50-ng sample of cDNA template added to the commercially available LightCycler PCR master mix (FastStart DNA Master SYBR Green I, Roche Molecular Biochemicals). Primers that were designed to span an exon/intron boundary were optimized for mRNA encoding leprb, Npy, Pomc, melanin-concentrating hormone (Mch), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The primer sequences are listed: Leprb forward, 5'-tgttttgggacgatgttcca-3'; Leprb reverse, 5'-aaagatgctcaaatgtttcaggc-3'; Npy forward, 5'-accaggcagagatatggcaaga-3'; Npy reverse, 5'-ggacattttctgtgctttctctcatta-3'; Pomc forward, 5'-cgctcctactctatggagcactt-3'; Pomc reverse, 5'-tcacctaccagctccctcttg-3'; Mch forward, 5'-ccagctgagaatggagttcaga-3'; Mch reverse, 5'-gtcggtagactcttcccagcat-3'; Gapdh forward, 5'-aacgaccccttcattgac-3'; Gapdh reverse, 5'-tccacgacatactcagcac-3'. Expression levels of each hypothalamic neuropeptide mRNA were normalized to Gapdh mRNA content and expressed as a percent of Ad-lacZ-treated animals. Nontemplate controls were incorporated into each PCR run.

Statistical analysis
All results are expressed as mean ± SEM. Statistical analyses were performed using Statistical Package for the Social Sciences (version 10.1, SPSS, Inc., Fullerton, CA). A one-way ANOVA with a least significant differences post hoc test was used to compare means between multiple groups, and a two-sample unpaired t test was used for two-group comparisons. In all instances, probability values of less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histochemical analysis and time course of adenoviral gene expression in vivo
As a first step to determine whether our adenovirus microinjection technique successfully targets expression of encoded proteins to specific brain regions, we used ß-gal staining to detect lacZ gene expression following Ad-lacZ microinjection into the ARC of normal rats. As shown in Fig. 2AGo, ß-gal activity was concentrated in cells within the ARC following microinjection of Ad-lacZ into this brain area, with no detectable expression in other brain areas, including adjacent hypothalamic structures, midbrain, and hindbrain. Similarly, following microinjection of Ad-lacZ into the LHA, ß-gal expression was localized to the LHA and was not detected in other hypothalamic or brain areas (Fig. 2BGo). By comparison, injection of Ad-lacZ into the third ventricle resulted in a wide distribution of ß-gal-expressing cells, including choroid plexus and ependymal cells from regions throughout the neuraxis (data not shown).



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  		Figure 2. Detection of adenovirally expressed genes. Localized histochemical detection of the activity of ß-gal, the protein product encoded by lacZ, following microinjection (Ad-lacZ; 1.7 x 1012 pfu/ml in 0.5 µl) into the ARC (A) and LHA (B) of fak/fak rats. 3V, Third ventricle; ME, median eminence.

 
ISH was used to detect leprb mRNA in the ARC following microinjection of the Ad-leprb vector into this brain area. Because endogenous leprb mRNA is not detected in the hypothalamus of fak/fak rats under standard assay conditions, virally encoded leprb mRNA was readily detected in the ARC of these animals after bilateral ARC microinjection of Ad-leprb but not after injection of Ad-lacZ (Fig. 3Go, A–D). These results indicate that the ISH signal detected in the rats that received Ad-leprb was due to expression of the viral transgene.



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  		Figure 3. Regionally specific leprb mRNA expression in rat brain. ISH was used to detect leprb mRNA in the brain 16 d after microinjection of the control reporter gene Ad-lacZ into the ARC of fak/fak (A) and Fak/Fak (B) rats, respectively, or following microinjection of Ad-leprb directed to the ARC (C) or LHA (D) of fak/fak rats. E, Regional distribution of leprb mRNA by ISH across serial sections throughout the ARC 16 d after Ad-leprb microinjection directed to the ARC of fak/fak rats.

 
Analysis of leprb mRNA by ISH across serial brain sections revealed leprb gene expression to be present over a region of the ARC extending approximately 1.0 mm (Fig. 3EGo). In two of six animals, leprb mRNA was also detected along the cannula tract, just dorsal to the ARC, but was not otherwise evident outside the ARC. Brain region-specific expression of leprb mRNA was also detected in the LHA of fak/fak rats following Ad-leprb microinjection into this brain area, with no expression evident in the ARC or other medial hypothalamic nuclei (Fig. 3DGo).

To evaluate the time course of Ad-leprb mRNA expression following ARC microinjection, leprb mRNA levels were determined by RT-PCR in extracts of mediobasal hypothalamus obtained from wild-type Wistar rats either 1 or 2 wk after adenovirus microinjection into the ARC (n = 5–6 per group). One week after injection, mediobasal hypothalamic leprb mRNA content was increased more than 3-fold in Ad-leprb-treated, compared with Ad-lacZ-treated rats, and remained elevated by 2.4-fold at 2 wk (P < 0.05 for each comparison). Thus, adenovirus-mediated delivery of the gene encoding leprb to the ARC increased mediobasal hypothalamic leprb mRNA content for at least 2 wk.

Response to ARC delivery of Ad-leprb in obese and lean Koletsky rats
To test the hypothesis that leptin receptor signaling in the ARC alone is sufficient to attenuate the severe obesity phenotype of rats with global leptin receptor deficiency, fak/fak rats were microinjected directly into the ARC with either Ad-leprb (n = 6) or Ad-lacZ (n = 5). Relative to animals receiving Ad-lacZ microinjections, daily food intake was significantly reduced in Ad-leprb-treated animals on d 8, 9, 11, 15, and 16 following adenovirus injection (Fig. 4BGo). Mean daily food intake for the entire study period (d 3–16) was also significantly reduced (by 13%; 29.4 ± 1.5 vs. 33.6 ± 1.1 g/d; P < 0.05), as was cumulative food intake (382 ± 19 vs. 438 ± 14 g; P < 0.05) in Ad-leprb-treated, compared with Ad-lacZ-treated, rats. This reduction of food intake was accompanied by a 33% reduction of body weight gain in Ad-leprb-treated, compared with Ad-lacZ-treated, fak/fak rats (40.6 ± 5.3 vs. 60.2 ± 4.2 g; P < 0.05; weight at baseline: 517 ± 14 g for Ad-leprb vs. 510 ± 2 g for Ad-lacZ; Fig. 4AGo).



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  		Figure 4. Leptin signaling limited to the ARC is sufficient to affect energy homeostasis. Effect on body weight gain (A and C) and mean daily food intake (B and D) following ARC microinjection of either Ad-lacZ ({square}) or Ad-leprb ({blacksquare}) in fak/fak rats or Ad-lacZ ({circ}) or Ad-leprb ({bullet}) in Fak/Fak rats (n = 5–6 per group). Body weight gain (E) and mean daily food intake (F) following bilateral microinjection into the LHA of either Ad-lacZ ({diamond}) or Ad-leprb ({diamondsuit}) in fak/fak rats is shown. Adenovirus injections were performed on d 0. *, P < 0.05 vs. Ad-lacZ-treated group.

 
If the weight-reducing effects of Ad-leprb injection into the ARC of fak/fak rats were due to a nonspecific mechanism unrelated to leptin signaling per se, a similar outcome would be expected following Ad-leprb injection into the ARC of genetically normal rats. To investigate this possibility, either Ad-leprb (n = 6) or Ad-lacZ (n = 5) was microinjected directly into the ARC of wild-type, lean Fak/Fak rats. Unlike the response of obese fak/fak rats, neither mean daily food intake (21.2 ± 0.4 g/d for Ad-leprb vs. 20.9 ± 0.5 g/d for Ad-lacZ; P = NS) nor cumulative food intake (275 ± 5 g for Ad-leprb vs. 272 ± 6 g for Ad-lacZ; P = NS) were significantly altered by Ad-leprb treatment. Body weight gain was also unaffected by Ad-leprb microinjection into the ARC of normal rats (18.5 ± 3.6 g for Ad-leprb vs. 14.3 ± 3.4 g for Ad-lacZ; P = NS; weight at baseline: 341 ± 6 g for Ad-leprb vs. 336 ± 4 g for Ad-lacZ; Fig. 4Go, C and D).

Based on differences in food intake and rate of body weight gain between lean Fak/Fak and obese fak/fak rats treated with control virus (Ad-lacZ), we calculated the extent to which Ad-leprb gene therapy directed to the ARC reversed the phenotype of hyperphagia and excessive weight gain that results from global leprb deficiency. This analysis revealed that the effects of the fak/fak genotype to increase food intake and body weight gain were attenuated by 35% and 42%, respectively, following Ad-leprb microinjection into the ARC. Despite these beneficial effects, fak/fak rats receiving Ad-leprb microinjection into the ARC had plasma glucose (220 ± 2 vs. 210 ± 7 mg/dl; P = NS) and insulin levels (1166 ± 330 vs. 1422 ± 36 pmol; P = NS) that were comparable to Ad-lacZ-treated controls.

Response to LHA delivery of Ad-leprb in obese Koletsky rats
To investigate whether the weight-reducing effects of Ad-leprb microinjection are specific to the ARC, we microinjected either Ad-leprb or Ad-lacZ into the LHA of a separate group of fak/fak rats. Unlike leptin receptor gene therapy directed to the ARC, this intervention did not significantly reduce mean daily food intake (32.4 ± 1.3 vs. 34.5 ± 0.9 g/d; P = NS) or body weight gain, compared with treatment with Ad-lacZ (59.9 ± 1.3 vs. 70.1 ± 5.8 g; P = NS; weight at baseline: 490 ± 13 g for Ad-leprb vs. 491 ± 12 g for Ad-lacZ; Fig. 4Go, E and F).

Hypothalamic neuropeptide and leprb mRNA levels in Ad-leprb-treated obese Koletsky rats
As a first step to investigate mechanisms mediating the reduction of food intake and attenuation of body weight gain following Ad-leprb microinjection into the ARC, but not the LHA, of fak/fak rats, we used ISH to measure hypothalamic expression of Npy and Pomc mRNA levels. As predicted, levels of Pomc mRNA in the ARC were significantly increased in fak/fak rats receiving Ad-leprb into this brain area, compared with those receiving Ad-lacZ microinjection (165% ± 5% vs. 100% ± 17%; P < 0.05, n = 5–6 group). As expected, Npy mRNA levels measured by ISH were lower in Ad-leprb- than Ad-lacZ-treated fak/fak rats (53% ± 8% vs. 100% ± 33%), although this difference was not statistically significant (P = 0.08; Fig. 5Go, A and C). In contrast, neither Pomc mRNA levels (68% ± 5% for Ad-leprb vs. 100% ± 22% for Ad-lacZ; P = NS) nor Npy mRNA levels (118% ± 18% for Ad-leprb vs. 100% ± 17% for Ad-lacZ; P = NS) were significantly affected in fak/fak rats following Ad-leprb delivery to the LHA (Fig. 5Go, B and D).



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  		Figure 5. Hypothalamic neuropeptide mRNA levels determined by ISH. Levels of Pomc mRNA in the rostral ARC (A and B) and Npy mRNA (C and D) in the midregion of ARC following reconstitution of leprb in either the ARC or LHA of fak/fak rats are shown. *, P < 0.05.

 
To further characterize the hypothalamic and feeding effects of Ad-leprb treatment, we subjected a second group of fak/fak rats to leprb gene therapy. As before, we found that Ad-leprb microinjection into the ARC was associated with reduced food intake (21.2 ± 1.7 vs. 24.9 ± 2.2 g/d; P < 0.05) and body weight (-42.4 ± 9.1 vs. -15.0 ± 7.3 g over 14 d; P < 0.05), compared with treatment with Ad-lacZ (weight at baseline: 836 ± 18 g for Ad-leprb vs. 848 ± 15 g for Ad-lacZ; Fig 6Go, A and B). Relative to wild-type Fak/Fak rats receiving Ad-lacZ, the effect of the fak/fak genotype to increase food intake was reversed by 72% following Ad-leprb treatment. To clarify the extent to which leprb gene therapy restores normal levels of hypothalamic leprb mRNA levels in fak/fak rats, we used real-time PCR to measure leprb mRNA content following ARC-directed Ad-leprb gene therapy. As expected, leprb mRNA levels were markedly reduced in the mediobasal hypothalamus of Ad-lacZ-treated fak/fak rats, compared with Ad-lacZ-treated Fak/Fak rats, and this deficit was restored approximately to normal values by Ad-leprb treatment (Fig. 6CGo).



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  		Figure 6. PCR analysis of the effects of Ad-leprb treatment of fak/fak rats. Effect of Ad-lacZ and Ad-leprb treatment in fak/fak rats, compared with Ad-lacZ-treated Fak/Fak rats on body weight (A) and mean daily food intake (B). On d 14 following adenoviral microinjection, a wedge of mediobasal hypothalamus was removed for analysis. Mediobasal hypothalamic mRNA levels of leprb (C) and Npy (D), relative to Gapdh mRNA content as determined by real-time PCR. Data are expressed as a percent of Ad-lacZ-treated Fak/Fak rats (n = 5–6; each group). *, P < 0.05

 
PCR-based analysis of hypothalamic neuropeptide gene expression from these animals confirmed and extended results obtained by ISH. As expected, hypothalamic Npy mRNA levels were increased (by 63%; P < 0.05) and Pomc mRNA levels decreased (by 49%; P < 0.05) in Ad-lacZ-treated fak/fak, compared with Fak/Fak, rats. Relative to fak/fak rats receiving Ad-lacZ, Ad-leprb treatment lowered hypothalamic Npy mRNA levels significantly (by 30%; P < 0.05; Fig. 6DGo) but increased hypothalamic Pomc mRNA content (by 40%; P < 0.05), consistent with data obtained using ISH (Fig. 5Go). By comparison, expression of MCH (an orexigenic neuropeptide expressed in the LHA) was affected by neither leptin receptor deficiency nor Ad-leprb treatment (100% ± 14% for Ad-lacZ-treated Fak/Fak rats; 116% ± 8% for Ad-lacZ-treated fak/fak rats; 134% ± 15% for Ad-leprb-treated fak/fak rats; P = NS for all comparisons).

Nonspecific effects of intrahypothalamic adenovirus microinjection
To determine whether intrahypothalamic adenovirus microinjection can, in and of itself, reduce food intake or body weight, we compared the effects of ARC microinjection of Ad-lacZ with that of vehicle alone (without adenovirus particles) in wild-type Fak/Fak rats. Relative to injection of vehicle, Ad-lacZ administration failed to induce significant changes of mean daily food intake (21.5 ± 0.4 g/d for vehicle vs. 22.0 ± 1.3 g/d for Ad-lacZ; P = NS), cumulative food intake (279 ± 6 g for vehicle vs. 286 ± 17 g for Ad-lacZ; P = NS) or body weight gain (17.7 ± 1.7 g for vehicle vs. 22.1 ± 4.3 g for Ad-lacZ; P = NS) over a 16-d period. Adenovirus injection into the ARC, therefore, does not, in and of itself, appear to affect food intake or body weight in normal animals.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To investigate the contribution made by the ARC to leptin action in vivo, we used an adenoviral gene therapy approach to express leptin receptors selectively in this brain area. Unlike leptin receptor gene therapy directed to the LHA, expression of leptin receptors in the ARC of obese Koletsky (fak/fak) rats reduced their food intake and body weight gain over a 2-wk period, relative to injection of a control virus. Because these effects were not observed following delivery of Ad-leprb to the ARC of normal rats, the weight-reducing effects of leptin receptor gene therapy in leptin receptor mutants cannot be attributed to a nonspecific disruption of ARC function. This conclusion is strengthened by our finding that leptin receptor gene therapy directed to the ARC increased Pomc and decreased Npy gene expression, effects that are unlikely to have arisen as nonspecific consequences of adenovirus administration. These findings provide direct evidence that energy homeostasis is significantly influenced by leptin signaling that is limited to the ARC. Furthermore, they imply an important role for deficient leptin signaling in this brain area in the pathogenesis of hyperphagia and obesity that results from global leptin receptor deficiency.

A practical advantage conferred by the leptin receptor mutation in fak/fak rats is that it reduces leprb mRNA stability and consequently lowers its content to levels that are undetectable under standard ISH conditions. Consequently, the hypothalamic distribution of leprb mRNA derived from Ad-leprb could readily be evaluated in these animals. Using ISH, we found this leprb mRNA to be concentrated in, and largely limited to, the ARC of fak/fak rats that received Ad-leprb microinjection into this area. In some animals, leprb mRNA was additionally detected along the cannula tract, just dorsal to the ARC, but this pattern of expression did not alter any outcome measure. Because adenoviral gene products were not detected in other brain areas following microinjection into the ARC, we concluded that leprb expression in the ARC accounts for the observed effects of Ad-leprb microinjection on food intake and body weight of fak/fak rats.

To determine the extent to which adenoviral gene therapy corrected leptin receptor deficiency in the ARC of Koletsky rats, we used real-time PCR to compare hypothalamic leprb mRNA content of fak/fak animals treated with either Ad-leprb or Ad-lacZ to wild-type control values. Our finding that Ad-leprb treatment restored leprb mRNA content to values comparable with normal animals suggests that the effects on energy homeostasis of this intervention were due to a reconstitution of intact leptin signaling in this brain area. Because obesity was only partially reversed by leptin receptor gene therapy directed to the ARC, an important role for leptin action in other brain areas in the control of energy homeostasis can be inferred (41, 42). The contribution of the ARC to leptin action in vivo is likely to have been underestimated by our data, however, because adenoviral gene delivery is unlikely to have targeted all of the ARC neurons that normally express leprb.

The magnitude of food intake suppression (~15% reduction of mean daily intake) conferred by ARC-directed leptin receptor gene therapy was less than is often observed following pharmacological leptin administration to rodents (6, 7, 8, 9) and is likely insufficient to fully explain the loss of weight in our studies. These observations raise several important issues. First, restored leptin signaling to the ARC of fak/fak rats may have increased energy expenditure, in addition to its effects on food intake, and studies to investigate this hypothesis are warranted. Second, our findings suggest that unlike leptin administration, it may not be possible to achieve a pharmacological increase of leptin signaling solely by increasing leptin receptor content. This conclusion stems in part from our finding that injection of Ad-leprb into the ARC of normal rats does not reduce food intake or body weight, despite increases in hypothalamic leptin receptor content of 2- to 3-fold. As with many hormone systems, therefore, cellular leptin signal transduction may depend more on the concentration of ligand than on that of the receptor, so long as normal levels of receptor expression are present. Based on these considerations, it is perhaps not surprising that the food intake response to leptin receptor gene therapy is less than that reported following pharmacological administration of leptin. Despite these considerations, however, the effect of ARC-directed leptin receptor gene therapy is substantial when evaluated according to the extent to which hyperphagia of fak/fak rats was reversed. In our first study, we found that this intervention reversed the effect of the fak mutation on food intake by 35%; in the second study, the effect was even greater, reflecting a reversal of more than 70% of the hyperphagic phenotype of fak/fak rats.

As a first step to evaluate the relative importance of the ARC in comparison with other hypothalamic subnuclei in mediating leptin signaling, we introduced leptin receptors into the LHA in a separate group of fak/fak rats. Although neurons critical to stimulation of feeding behavior (e.g. MCH and orexin-producing neurons) (43, 44) are concentrated in this brain area, leptin receptors are not (10, 35). Because LHA neurons can stimulate food intake and are regulated by changes in nutritional state, introduction of leptin receptors in this brain area might be expected to affect energy homeostasis. Unlike the response to Ad-leprb microinjection into the ARC, however, expression of leptin receptor in the LHA of obese Koletsky rats did not significantly alter food intake or body weight gain. This finding suggests that in leptin receptor-deficient animals, neuronal leptin signaling is effective only when receptor expression is directed to brain areas that normally mediate leptin effects, such as the ARC.

To investigate the mechanisms responsible for reduced food intake and body weight gain following delivery of Ad-leprb into the ARC of fak/fak rats, we measured hypothalamic expression of neuropeptides produced by ARC neurons that, in normal animals, are implicated in leptin action. Both NPY- and POMC-expressing neurons in the ARC coexpress leptin receptor mRNA, are regulated by leptin, and participate in energy homeostasis (10, 28, 34, 36, 45). Moreover, mutations affecting either leptin or its receptor result in the combination of reduced hypothalamic expression of POMC and increased expression of NPY, responses that promote excessive food intake and weight gain (28, 29, 33, 34). Our finding that leptin receptor gene therapy directed to the ARC of fak/fak rats increased local concentrations of Pomc mRNA suggests that increased melanocortin signaling in the hypothalamus may have contributed to its weight-reducing effects. Similarly, hypothalamic Npy mRNA levels in the ARC were lower following Ad-leprb delivery to this brain area, suggesting that leptin signaling had been restored to both NPY and POMC neurons by this intervention. By comparison, there was no effect on Mch gene expression of Ad-leprb gene therapy directed to the ARC; conversely, leptin receptor gene therapy directed to the LHA had no detectable effect on Npy or Pomc mRNA levels.

Several key questions about the role played by specific brain areas in the response to leptin await further study. Leptin exerts diverse CNS effects that influence autonomic function, the reproductive axis and glucose homeostasis, and the role played by the ARC, relative to that of other brain areas, was not addressed in our studies. Our findings suggest that adenoviral gene therapy will be a useful tool with which to further dissect the contributions made by discrete brain areas to leptin’s many central effects.

In conclusion, we report that adenovirally mediated expression of the signaling isoform of the leptin receptor is sufficient to reduce food intake and body weight gain when directed to the ARC, but not the LHA, of leptin receptor-deficient Koletsky (fak/fak) rats. These findings constitute direct evidence that leprb signaling in the ARC is required for normal energy homeostasis and provide proof-of-principal endorsing viral gene therapy as a useful approach with which to study the contribution of discrete brain areas to the physiologic actions of specific molecules.


   Acknowledgments
 
The authors appreciate the excellent technical assistance provided by Hong Nguyen and Loan Nguyen.


   Footnotes
 
This work was supported by NIH Grants DK-52989, NS-32273, and DK-12829 (to M.W.S.), DK-56731 (to M.G.M.), DK-55269 (to C.J.R.), and DK-17047 (to D.G.B.). G.J.M. is supported by a mentor-based fellowship from the American Diabetes Association.

Abbreviations: ARC, Arcuate nucleus; ß-gal, ß-galactosidase; CNS, central nervous system; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hleprb, human leprb; ISH, in situ hybridization; LHA, lateral hypothalamic area; Lepr, leptin receptor; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin.

Received December 9, 2002.

Accepted for publication January 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Flotte TR, Afione SA, Conrad C, McGrath SA, Solow R, Oka H, Zeitlin PL, Guggino WB, Carter BJ 1993 Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc Natl Acad Sci USA 90:10613–10617[Abstract/Free Full Text]
  2. Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Paakko PK, Gilardi P, Stratford-Perricaudet LD, Perricaudet M, Jallet S, Pavirani A, Lecocq J-P, Crystal RG 1991 Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science 252:431–434[Abstract/Free Full Text]
  3. Rosenfeld MA, Yoshimura K, Trapnell BC, Yoneyama K, Rosenthal ER, Dalemans W, Fukayama M, Bargon J, Stier LE, Stratford-Perricaudet L 1992 In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 68:143–155[CrossRef][Medline]
  4. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, Caro JF 1996 Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 334:292–295[Abstract/Free Full Text]
  5. Schwartz MW, Peskind E, Raskind M, Boyko EJ, Porte Jr D 1996 Cerebrospinal fluid leptin levels: relationship to plasma levels and to adiposity in humans. Nat Med 2:589–593[CrossRef][Medline]
  6. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P 1995 Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269:546–549[Abstract/Free Full Text]
  7. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM 1995 Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269:543–546[Abstract/Free Full Text]
  8. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540–543[Abstract/Free Full Text]
  9. Weigle DS, Bukowski TR, Foster DC, Holderman S, Kramer JM, Lasser G, Lofton-Day CE, Prunkard DE, Raymond C, Kuijper JL 1995 Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J Clin Invest 96:2065–2070
  10. Baskin DG, Breininger JF, Schwartz MW 1999 Leptin receptor mRNA identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat hypothalamus. Diabetes 48:828–833[Abstract]
  11. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM 1994 Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432[CrossRef][Medline]
  12. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP 1996 Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84:491–495[CrossRef][Medline]
  13. Lee G-H, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI, Friedman JM 1996 Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632–635[CrossRef][Medline]
  14. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Morlarty A, Moore KJ, Smutko JS, Mays GG, Woolf EA, Monroe CA, Tepper RI 1995 Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271[CrossRef][Medline]
  15. Chua SC, White DW, Wu-Peng S, Liu S-M, Okada N, Kershaw EE, Chung WK, Power-Kehoe L, Chua M, Tartaglia LA, Leibel RL 1996 Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr). Diabetes 45:1141–1143[Abstract]
  16. Takaya K, Ogawa Y, Hiraoka J, Hosoda K, Yamori Y, Nakao K, Koletsky RJ 1996 Nonsense mutation of leptin receptor in the obese spontaneously hypertensive Koletsky rat. Nat Genet 14:130–131[CrossRef][Medline]
  17. Wu-Peng XS, Chua Jr SC, Okada N, Liu SM, Nicolson M, Leibel RL 1997 Phenotype of the obese Koletsky (f) rat due to Tyr763Stop mutation in the extracellular domain of the leptin receptor (Lepr): evidence for deficient plasma-to-CSF transport of leptin in both the Zucker and Koletsky obese rat. Diabetes 46:513–518[Abstract]
  18. Montague CT, Farooqi IS, Whitehead JP, Soos MA, Rau H, Wareham NJ, Sewter CP, Digby JE, Mohammed SN, Hurst JA, Cheetham CH, Earley AR, Barnett AH, Prins JB, O’Rahilly S 1997 Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 387:903–908[CrossRef][Medline]
  19. Clement K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, Cassuto D, Gourmelen M, Dina C, Chambaz J, Lacorte JM, Basdevant A, Bougneres P, Lebouc Y, Froguel P, Guy-Grand B 1998 A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392:398–401[CrossRef][Medline]
  20. Bjorbaek C, Uotani S, da Silva B, Flier JS 1997 Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem 272:32686–32695[Abstract/Free Full Text]
  21. Ghilardi N, Skoda RC 1997 The leptin receptor activates janus kinase 2 and signals for proliferation in a factor-dependent cell line. Mol Endocrinol 11:393–399[Abstract/Free Full Text]
  22. Fei H, Okano HJ, Li C, Lee GH, Zhao C, Darnell R, Friedman JM 1997 Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues. Proc Natl Acad Sci USA 94:7001–7005[Abstract/Free Full Text]
  23. Vaisse C, Halaas JL, Horvath CM, Darnell Jr JE, Stoffel M, Friedman JM 1996 Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet 14:95–97[CrossRef][Medline]
  24. Niswender KD, Morton GJ, Stearns WH, Rhodes CJ, Myers Jr MG, Schwartz MW 2001 Intracellular signalling. Key enzyme in leptin-induced anorexia. Nature 413:794–795[CrossRef]
  25. Zhao AZ, Huan JN, Gupta S, Pal R, Sahu A 2002 A phosphatidylinositol 3-kinase phosphodiesterase 3B-cyclic AMP pathway in hypothalamic action of leptin on feeding. Nat Neurosci 5:727–728[Medline]
  26. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD 1997 Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385:165–168[CrossRef][Medline]
  27. Giraudo SQ, Billington CJ, Levine AS 1998 Feeding effects of hypothalamic injection of melanocortin 4 receptor ligands. Brain Res 809:302–306[CrossRef][Medline]
  28. Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG 1997 Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46:2119–2123[Abstract]
  29. Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV 1998 Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47:294–297[Abstract]
  30. Stanley BG, Kyrkouli SE, Lampert S, Leibowitz SF 1986 Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity. Peptides 7:1189–1192[CrossRef][Medline]
  31. Rossi M, Kim MS, Morgan DG, Small CJ, Edwards CM, Sunter D, Abusnana S, Goldstone AP, Russell SH, Stanley SA, Smith DM, Yagaloff K, Ghatei MA, Bloom SR 1998 A C-terminal fragment of agouti-related protein increases feeding and antagonizes the effect of {alpha}-melanocyte stimulating hormone in vivo. Endocrinology 139:4428–4431[Abstract/Free Full Text]
  32. Hagan MM, Rushing PA, Pritchard LM, Schwartz MW, Strack AM, Van Der Ploeg LH, Woods SC, Seeley RJ 2000 Long-term orexigenic effects of AgRP (83–132) involve mechanisms other than melanocortin receptor blockade. Am J Physiol Regul Integr Comp Physiol 279:R47–R52
  33. Schwartz MW, Baskin DG, Bukowski TR, Kuijper JL, Foster D, Lasser G, Prunkard DE, Porte Jr D, Woods SC, Seeley RJ, Weigle DS 1996 Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes 45:531–535[Abstract]
  34. Mercer JG, Moar KM, Rayner DV, Trayhurn P, Hoggard N 1997 Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold-exposed lean mice. FEBS Lett 402:185–188[CrossRef][Medline]
  35. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P 1996 Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett 387:113–116[CrossRef][Medline]
  36. Cheung CC, Clifton DK, Steiner RA 1997 Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus. Endocrinology 138:4489–4492[Abstract/Free Full Text]
  37. Satoh N, Ogawa Y, Katsuura G, Hayase M, Tsuji T, Imagawa K, Yoshimasa Y, Nishi S, Hosoda K, Nakao K 1997 The arcuate nucleus as a primary site of satiety effect of leptin in rats. Neurosci Lett 224:149–152[CrossRef][Medline]
  38. Becker TC, Noel RJ, Coats WS, Gomez-Foix AM, Alam T, Gerard RD, Newgard CB 1994 Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol 43:161–189
  39. Skelly RH, Wicksteed B, Antinozzi PA, Rhodes CJ 2001 Glycerol-stimulated proinsulin biosynthesis in isolated pancreatic rat islets via adenoviral-induced expression of glycerol kinase is mediated via mitochondrial metabolism. Diabetes 50:1791–1798[Abstract/Free Full Text]
  40. Mercer EH, Hoyle GW, Kapur RP, Brinster RL, Palmiter RD 1991 The dopamine beta-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice. Neuron 7:703–716[CrossRef][Medline]
  41. Grill HJ, Schwartz MW, Kaplan JM, Foxhall JS, Breininger J, Baskin DG 2002 Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake. Endocrinology 143:239–246[Abstract/Free Full Text]
  42. Hosoi T, Kawagishi T, Okuma Y, Tanaka J, Nomura Y 2002 Brain stem is a direct target for leptin’s action in the central nervous system. Endocrinology 143:3498–3504[Abstract/Free Full Text]
  43. Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, Mathes WF, Przypek J, Kanarek R, Maratos-Flier E 1996 A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380:243–246[CrossRef][Medline]
  44. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richarson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DH, Yanagisawa M 1998 Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92:573–585[CrossRef][Medline]
  45. Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, Horvath TL, Cone RD, Low MJ 2001 Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411:480–484[CrossRef][Medline]



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