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

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, lepr^b, in the ARC of leptin receptor-deficient Koletsky (fa^k/fa^k) rats. Successful expression of adenovirus containing lepr^b (Ad-lepr^b) selectively in the ARC was documented by in situ hybridization. Using real-time PCR, we further demonstrated that bilateral microinjection of Ad-lepr^b into the ARC restored low hypothalamic levels of lepr^b mRNA to values approximating those of wild-type (Fa^k/Fa^k) 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 fa^k/fa^k rats injected with a control adenovirus (Ad-lacZ) (P < 0.05 for each comparison). In contrast, Ad-lepr^b delivery to either the lateral hypothalamic area of fa^k/fa^k rats or to the ARC of wild-type Fa^k/Fa^k 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 (fa^k/fa^k) rat models. The latter animals have a nonsense mutation (T2289ATyr763Stop) in the extracellular domain of Lepr, resulting in absence of all forms of Lepr protein and reduced lepr^b 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, Lepr^b, has been shown to be essential for normal energy homeostasis. Unlike the other leptin receptor isoforms, Lepr^b 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 lepr^b, 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 lepr^b (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 lepr^b (Ad-lepr^b) or a control reporter gene (Ad-lacZ) into the ARC of Lepr-deficient fa^k/fa^k 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-lepr^b was microinjected into the lateral hypothalamic area (LHA), a brain area implicated in energy homeostasis that does not express high levels of Lepr^b (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

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Experimental animals
Male lean (Fa^k/Fa^k) and obese (fa^k/fa^k) Koletsky rats (Vassar College, Poughkeepsie, NY) were generated from serial backcrosses (NIO equivalent) of the fa^k mutation (also known as Koletsky, fa^f, 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 lepr^b (hlepr^b) cDNA in pcDNA3 was subcloned into the adenoviral transfer vector pACCMV using HindIII and XbaI. The recombined hlepr^b adenovirus was generated by cotransfection of pACCMV hlepr^b with the adenoviral backbone vector pBHG11 in 293 cells; the resulting recombinant virus was purified as described (Ref. 39 and Fig. 1).

View larger version (14K): [in this window] [in a new window]   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.

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 lepr^b 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 lepr^b (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 lepr^b, Npy, Pomc, melanin-concentrating hormone (Mch), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The primer sequences are listed: Lepr^b forward, 5'-tgttttgggacgatgttcca-3'; Lepr^b 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

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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. 2A, 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. 2B). 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).

View larger version (117K): [in this window] [in a new window]   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.

View larger version (66K): [in this window] [in a new window]   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.

To evaluate the time course of Ad-lepr^b mRNA expression following ARC microinjection, lepr^b 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 lepr^b mRNA content was increased more than 3-fold in Ad-lepr^b-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 lepr^b to the ARC increased mediobasal hypothalamic lepr^b mRNA content for at least 2 wk.

Response to ARC delivery of Ad-lepr^b 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, fa^k/fa^k rats were microinjected directly into the ARC with either Ad-lepr^b (n = 6) or Ad-lacZ (n = 5). Relative to animals receiving Ad-lacZ microinjections, daily food intake was significantly reduced in Ad-lepr^b-treated animals on d 8, 9, 11, 15, and 16 following adenovirus injection (Fig. 4B). 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-lepr^b-treated, compared with Ad-lacZ-treated, rats. This reduction of food intake was accompanied by a 33% reduction of body weight gain in Ad-lepr^b-treated, compared with Ad-lacZ-treated, fa^k/fa^k rats (40.6 ± 5.3 vs. 60.2 ± 4.2 g; P < 0.05; weight at baseline: 517 ± 14 g for Ad-lepr^b vs. 510 ± 2 g for Ad-lacZ; Fig. 4A).

View larger version (30K): [in this window] [in a new window]   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 () or Ad-leprb () in fak/fak rats or Ad-lacZ () or Ad-leprb () 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 () or Ad-leprb () in fak/fak rats is shown. Adenovirus injections were performed on d 0. *, P < 0.05 vs. Ad-lacZ-treated group.

Based on differences in food intake and rate of body weight gain between lean Fa^k/Fa^k and obese fa^k/fa^k rats treated with control virus (Ad-lacZ), we calculated the extent to which Ad-lepr^b gene therapy directed to the ARC reversed the phenotype of hyperphagia and excessive weight gain that results from global lepr^b deficiency. This analysis revealed that the effects of the fa^k/fa^k genotype to increase food intake and body weight gain were attenuated by 35% and 42%, respectively, following Ad-lepr^b microinjection into the ARC. Despite these beneficial effects, fa^k/fa^k rats receiving Ad-lepr^b 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-lepr^b in obese Koletsky rats
To investigate whether the weight-reducing effects of Ad-lepr^b microinjection are specific to the ARC, we microinjected either Ad-lepr^b or Ad-lacZ into the LHA of a separate group of fa^k/fa^k 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-lepr^b vs. 491 ± 12 g for Ad-lacZ; Fig. 4, E and F).

Hypothalamic neuropeptide and lepr^b mRNA levels in Ad-lepr^b-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-lepr^b microinjection into the ARC, but not the LHA, of fa^k/fa^k 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 fa^k/fa^k rats receiving Ad-lepr^b 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-lepr^b- than Ad-lacZ-treated fa^k/fa^k rats (53% ± 8% vs. 100% ± 33%), although this difference was not statistically significant (P = 0.08; Fig. 5, A and C). In contrast, neither Pomc mRNA levels (68% ± 5% for Ad-lepr^b vs. 100% ± 22% for Ad-lacZ; P = NS) nor Npy mRNA levels (118% ± 18% for Ad-lepr^b vs. 100% ± 17% for Ad-lacZ; P = NS) were significantly affected in fa^k/fa^k rats following Ad-lepr^b delivery to the LHA (Fig. 5, B and D).

View larger version (21K): [in this window] [in a new window]   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.

View larger version (30K): [in this window] [in a new window]   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

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 Fa^k/Fa^k 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 (fa^k/fa^k) 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-lepr^b 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 fa^k/fa^k rats is that it reduces lepr^b mRNA stability and consequently lowers its content to levels that are undetectable under standard ISH conditions. Consequently, the hypothalamic distribution of lepr^b mRNA derived from Ad-lepr^b could readily be evaluated in these animals. Using ISH, we found this lepr^b mRNA to be concentrated in, and largely limited to, the ARC of fa^k/fa^k rats that received Ad-lepr^b microinjection into this area. In some animals, lepr^b 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 lepr^b expression in the ARC accounts for the observed effects of Ad-lepr^b microinjection on food intake and body weight of fa^k/fa^k 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 lepr^b mRNA content of fa^k/fa^k animals treated with either Ad-lepr^b or Ad-lacZ to wild-type control values. Our finding that Ad-lepr^b treatment restored lepr^b 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 lepr^b.

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 fa^k/fa^k 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-lepr^b 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 fa^k/fa^k rats was reversed. In our first study, we found that this intervention reversed the effect of the fa^k 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 fa^k/fa^k 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 fa^k/fa^k 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-lepr^b 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-lepr^b into the ARC of fa^k/fa^k 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 fa^k/fa^k 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-lepr^b 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-lepr^b 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 (fa^k/fa^k) rats. These findings constitute direct evidence that lepr^b 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; hlepr^b, human lepr^b; 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

 

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