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Dose-Dependent Effects of Central Leptin Gene Therapy on Genes That Regulate Body Weight and Appetite in the Hypothalamus
Harveen Dhillon,1,* Satya P. Kalra,2, and Pushpa S. Kalra1
Departments of 1Physiology and 2Neuroscience, University of Florida McKnight Brain Institute, College of Medicine, Box 100244, Gainesville, Florida 32610, USA *Current address: Beth Israel Deaconess Medical Center and Harvard Medical School, Division of Endocrinology, RN-325, 99 Brookline Avenue, Boston, Massachusetts 02215, USA

To whom correspondence and reprint requests should be addressed. Fax: (352) 294-0191. E-mail: skalra@ufbi.ufl.edu.

We have examined the dose-dependent effects and central action of intraventricular administration of a recombinant adeno-associated virus encoding rat leptin (rAAVleptin) in suppressing body weight (BW) gain in adult female rats. A low dose of rAAVleptin (5 1010 particles) suppressed weight gain (15%) without changing daily food intake (FI), but a twofold higher dose decreased BW by 30% along with a reduction in daily FI. Reduced BW was due to a loss in body adiposity because serum leptin was reduced. Serum insulin levels were decreased (96%) by only the high dose along with a slight reduction in glucose. Uncoupling protein-1 (UCP-1) mRNA expression in brown adipose tissue (BAT), reflecting energy expenditure through thermogenesis, was upregulated to the same magnitude by the two rAAVleptin doses. We analyzed by in situ hybridization the expression in the hypothalamus of genes encoding the appetite-regulating neuropeptides. Only the high dose decreased expression of neuropeptide Y (NPY), the orexigenic peptide, and increased proopiomelanocortin (POMC), precursor of the anorexigenic peptide, -MSH. Our studies show for the first time that increased availability of leptin within the hypothalamus through central leptin gene therapy dose-dependently decreases weight gain, adiposity, and serum insulin by increasing energy expenditure and decreasing FI. The decrease in FI occurs only when NPY is reduced and -MSH is increased in the hypothalamus by the high dose of rAAVleptin. Delivery of the leptin gene centrally through rAAV vectors is a viable therapeutic modality for long-term control of weight and metabolic hormones. Key Words: leptin, neuropeptide Y, melanocortin, UCP1, brown adipose tissue, insulin, glucose, rAAV, gene therapy, hypothalamus

INTRODUCTION
Leptin, a 16-kD adipocyte hormone, is a key peripheral hormonal signal from adipocytes to the hypothalamus for body weight (BW) homeostasis [1,2]. Peripheral or central administration of leptin in leptin-deficient obese Lepob/ob mice decreased BW by suppressing the daily food intake (FI) and increasing energy expenditure, but no effect was seen in obese Leprdb/db mice or fatty Zucker fa/fa rats, which lack functional leptin receptors [35]. Leptin is believed to be transported across the bloodbrain barrier to hypothalamic target sites where it acts on the appetite regulating network (ARN) to inhibit FI, and on the sympathetic nervous system (SNS) to augment energy expenditure through brown adipose tissue (BAT) thermogenesis [1,2,610]. Whether this negative relationship between

adipocytes and hypothalamic systems exists in wild-type laboratory rodents for the daily management of BW homeostasis is not clear. Leptin administration, either as a bolus or slow infusion, to normal wild-type rodents was only transiently effective at supraphysiological dosages in reducing BW and there was little or no reduction in FI [1114]. The increase in endogenous leptin levels was also found to be ineffective for preventing the age-related adiposity and weight gain in rodents [15,16]. The endogenous leptin titers are elevated in these rats but are unable to inhibit FI or to augment energy expenditure. Similarly, hyperleptinemia resulting from consumption of a highenergy diet fails to activate the compensatory hypothalamic mechanisms involved in weight homeostasis [2,1720].

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FIG. 1. Effects of the low dose (5 1010 particles) icv rAAVleptin injection on body weight (A), food intake (B), serum leptin (C), insulin (D), and glucose (E) at week 6 postinjection. The arrow denotes the time of injection. *P 0.05 versus rAAVUF5.

Among the several potential mechanisms advanced to account for this apparent ineffectiveness or resistance to leptin [1,2,7,21], we have examined the possibility that leptin insufficiency at target sites in the hypothalamus may account for the loss of control on weight gain. We recently reported that when leptin availability was enhanced locally in hypothalamic neurons following an intracerebroventricular injection (icv) of recombinant adeno-associated virus vector encoding rat leptin (rAAVleptin), the age-related weight gain in normal outbred male and female SpragueDawley rats was blocked for the entire six-month duration of the experiment [22]. Similarly, central administration of Lep (encoding leptin) by means of rAAV was recently reported to be effective in preventing weight gain in obese Lepob/ob mice [23,27]. In the rats there was a depletion of white fat deposits without decreases in daily food consumption. This diminution in fat deposition apparently resulted selectively from increased energy expenditure through BAT thermogenesis [22], an important factor in the development of obesity [1,24,25]. The blockade of age-related BW gain and fat deposition without any effect on FI was unanticipated and raised the following questions: Would a higher dose of rAAVleptin suppress both BW gain and FI? Is there a dosedependent effect of central rAAVleptin therapy? What is the mechanism of long-term control of weight and energy expenditure following central rAAVleptin gene therapy? A large body of evidence shows that leptin target neurons within the hypothalamic ARN, the orexigenic neuropeptide Y (NPY) and agouti-related peptide (AGRP)

producing neurons, and the anorexigenic -melanocyte stimulating hormone (-MSH) producing proopiomelanocortin (POMC) neurons, are located in the arcuate nucleus (ARC) and are functional antagonists [1,2,26]. We previously reported that an intravenous injection of rAAVleptin to Lepob/ob mice that raised plasma leptin levels and decreased FI and BW attenuated NPY and AGRP mRNA expression and increased POMC mRNA expression [27]. Here we investigated the dose-dependent effects of central rAAVleptin on BW, FI, and the metabolic hormones leptin and insulin in wild-type Sprague-Dawley (SD) rats. To determine the mechanism of central leptin action, we evaluated UCP-1 mRNA in BAT and mRNA expression of NPY, AGRP, and POMC in the hypothalamus.

RESULTS
Effects of rAAVLeptin on BW and FI Rats injected with rAAVUF5 showed a 15.7% weight gain from the pre-injection weights during the 6-week duration of the experiment (Fig. 1). In contrast, one rAAVleptin injection (5 1010 particles) resulted in maintenance of the preinjection weight. At week 6 postinjection, BW of the rAAVleptin treated rats was 15% lower than that of control rats, but 24-hour FI was similar to that of controls during the entire experimental period (Fig. 1B). A higher viral infection attained by two injections of rAAVleptin (total dose = 10 1010 particles) evoked

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FIG. 2. Effects of high dose (10 1010 particles) icv rAAVleptin administration on body weight (A), food intake (B), serum leptin (C), insulin (D), and glucose (E) at week 6 postinjection. The arrows denote the time of injection. **P 0.01 and *P 0.05 versus all groups.

quantitatively different effects on BW and 24-hour FI (Fig. 2). BW gain was not affected by injections of the control vector (13.6% gain from pre-injection level; Fig. 2A) as indicated by a pattern similar to that of the untreated control rats. Two rAAVleptin injections significantly decreased BW from the pre-injection level within 1 week (P 0.05) with an overall weight loss of 16.4% (initial 224.1 5.4 versus final 188.4 11.1 g) at week 6 postinjection. Compared with controls there was a 30% weight loss in rAAVleptin treated rats. Twenty-four hour FI was suppressed at one week postinjection (P 0.05) and remained low for the remainder of the experiment (average 13.6 0.1 g versus 16.6 0.4 g controls; P 0.05; Fig. 2B). The group of rats pair-fed with the rAAVleptin treated rats displayed a similar weight loss for the first four weeks; thereafter they slowly regained weight but only up to their pre-injection levels (P 0.05 versus rAAVUF5 and untreated controls at week 6). Effects of rAAVLeptin on Serum Leptin, Insulin, and Glucose Concentrations A dose-related reduction in serum leptin was also evident in rAAVleptin treated rats. Serum leptin levels were reduced by 66% in rats receiving the low dose of rAAV (P 0.05; Fig. 1C) and by 96% in response to the higher dose (P 0.01; Fig. 2C). Maintenance of weight and FI for six

weeks produced no change in serum insulin and glucose levels in the low dose rAAVleptin treated rats (Figs. 1D and 1E). In contrast, in response to the higher rAAVleptin titer (Fig. 2D), serum insulin levels were reduced by 96% (P 0.05 versus untreated and rAAVUF5 treated controls). Glucose concentrations were slightly reduced (21.2%, P 0.05 versus rAAVUF5 injected rats; Fig. 2E) but were normoglycemic (127 3.3 mg/dl). There was no change in serum leptin, insulin, or glucose levels in pair-fed rats (Figs. 2C2E). Effect of rAAVLeptin on UCP1 mRNA in BAT We found that rAAVleptin injection upregulated UCP1 mRNA in BAT compared with the respective rAAVUF5 treated controls (P 0.05; Fig. 3). However, this increase was not dose-dependent because the magnitude of increase was approximately twofold following either dose of

FIG. 3. Effects of low dose (5 1010 particles) and high dose (10 1010 particles) rAAVleptin on BAT UCP-1 mRNA expression at week 6 postinjection. *P 0.05 versus respective rAAVUF5 control. aP 0.05 versus other groups.

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

DISCUSSION
In agreement with our previous reports [22,28,29], the results of this study show that low dose rAAVleptin injected icv suppressed weight gain without affecting energy intake. This weight maintenance, accompanied by E B depletion of adipose tissue and reduced serum leptin levels, may be due primarily to augmented energy expenditure through increased thermogenesis as indicated by upregulation of UCP-1 mRNA in BAT [22]. The current findings extend these observations to show that a twofold increase in the rAAVleptin dose enhanced its effectiveness with a steady reduction in body weight along with a significant decrease in FI. The low and high doses of rAAVleptin produced an equivalent increase, F C possibly a maximal response, in BAT UCP-1 mRNA and hence equivalent energy expenditure through thermogenesis. Therefore, it is likely that BW suppression in response to two rAAVleptin injections, unlike BW maintenance after one injection, was solely a result of the attenuated energy intake. These findings, together with previous results from Lepob/ob mice and SD rats [22,2729], support the notion that two distinct neural mechanisms, with different thresholds for responsiveness to leptin, exist in the hypothalamus. Seemingly, the neural netFIG. 4. Dose-dependent effects of icv rAAVleptin on NPY (A and D), AGRP (B and E), and POMC works that augment thermogenesis through (C and F) mRNA expression in the arcuate nucleus at week 6 postinjection. Low dose = activation of BAT UCP-1 mRNA [8,10,24,25] 5 1010 particles; high dose = 10 1010 particles. *P 0.05 versus rAAVUF5 controls and PFC (pair-fed control). and, possibly, general activity to enhance energy expenditure [5] have a relatively lower threshold to leptin than those that regulate appetite or energy intake. The sustained effects of different doses of rAAVleptin. On the other hand, as reported earlier [12], rAAVleptin injected centrally are apparently due to the pair-feeding induced a significant reduction in UCP-1 paracrine/autocrine effects of augmented leptin generated mRNA (P 0.05). locally [21,22]. Using immunocytochemical analysis of green fluorescent protein (GFP) encoded in the rAAVUF5 control Effects of rAAVLeptin on Hypothalamic vector, we have demonstrated GFP expression in neurons in Neuropeptide mRNA Expression the ARC and other hypothalamic sites after an icv injection The results of the in situ hybridization analyses of NPY, AGRP, and POMC mRNA in the hypothalamic ARC are depicted in [22,28]. RTPCR analysis of leptin mRNA revealed significant upregulation in the hypothalamus of rAAVleptin Figs. 4 and 5. Low dose rAAVleptin injection, which suptreated rats [22]. Furthermore, reduced plasma leptin levels pressed BW gain and reduced serum leptin without affecting FI, failed to modify the mRNA levels of these peptides in these rats argues against the possibility of increased peripheral contributions to hypothalamic targets. (Fig. 4). In contrast, in association with the reduction in BW, In previous studies, hyperleptinemia produced by sysFI, and serum leptin produced by the high dose rAAVleptemic injections of either rAAVleptin [30] or adenovirus tin, both NPY and POMC mRNA levels were significantly altered (Figs. 4 and 5). Whereas NPY mRNA was downregu- encoding leptin [14,31] attenuated hyperinsulinemia and lated (P 0.05), POMC mRNA was elevated over the control hyperglycemia in Lepob/ob mice and decreased blood insulin rAAVUF5 treated rats (P 0.05). A similar reduction in FI in adult rats [3234]. In our earlier study, serum leptin

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levels were suppressed rapidly but the reduction in serum insulin was seen only at six months after low dose rAAVleptin administration [22]. As reported here, however, suppression of serum insulin was apparent at six weeks after the high dose of rAAVleptin in association with extremely low circulating leptin levels. We have observed that micro-injection of rAAVleptin into the hypothalamic paraventricular nucleus, the feeding relevant site, led to an immediate decrease in serum leptin and insulin (unpublished data). This suggests that peripheral leptin by itself may not be responsible for diminished pancreatic insulin secretion. A feedback relationship between leptin and insulin secretion has been suggested by in vivo and in vitro experiments. Leptin can either stimulate or inhibit insulin release in rodents [20,3539] and insulin directly affects leptin secretion from adipocytes [37,40]. Our results advocate a central component of leptin action to inhibit serum FIG. 5. NPY, AGRP, and POMC mRNA expression in the arcuate nucleus at week 6 postinjection in insulin levels. Although it is possible rAAVUF5 (control), pair-fed, and rAAVleptin (10 1010 particles)-injected rats. that the reduction in FI and serum leptin in rats receiving the high dose rAAVleptin accounts for the suppression of serum insulin that downregulation of ARC NPY expression attenuated levels, the reduction in serum insulin levels seen in rats with appetite. unaltered FI after low dose icv rAAVleptin injection suggests The functional antagonist of NPY involved in the medithat insulin suppression may not be related to reduced FI. ation of leptins effects on appetite regulation in the ARN is Furthermore, there was no reduction in serum insulin in the melanocortin system [1,2,26]. POMC is produced by neupair-fed rats that consumed an equivalently reduced amount. rons in the ARC and is the precursor of -MSH, the anorecIn this context, it is noteworthy that the reduction in tic peptide. These ARC POMC neurons express the leptin serum insulin was associated with normal levels of serum receptor and leptin enhances POMC mRNA expression in glucose in rAAVleptin treated rats in this and a previous wild-type rodents, Lepob/ob mice [1,2], and Lepob/ob mice treated study [22]. Therefore, it is possible that rAAVleptin intravenously with rAAVleptin [27]. Upregulation of ARC injected rats develop increased sensitivity to insulin for gluPOMC mRNA after the high dose icv rAAVleptin injection cose homeostasis. However, additional investigations are suggests that suppression of FI may be the combined effect warranted to support a role of central leptin action in modof decreased NPY and increased melanocortin signalings. ulating insulin sensitivity. These simultaneous shifts in opposite directions in NPY and To identify the neural factors responsible for the dose- POMC mRNA expression are likely a direct effect of leptin dependent effects of rAAVleptin on energy consumption generated locally in the hypothalamus and not secondary to and expenditure, we examined the gene expression of a the inhibition in FI, because a similar 1314% reduction in few key hypothalamic neuropeptides implicated in weight consumption in pair-fed rats affected neither NPY nor POMC regulation [1,2]. In leptin-deficient Lepob/ob mice [27], we expression. NPY neurons apparently exert a tonic restraint reported significant modulatory effects of rAAVleptin on on melanocortin signaling because they are synaptically the hypothalamic mRNA expression of NPY, AGRP, and linked with POMC neurons in the ARC [2,44], POMC neuPOMC. NPY is a powerful stimulator of feeding [2,41,42]. rons express NPY receptors [45] and NPY injected icv A subpopulation of NPY-producing neurons in the ARC decreased POMC mRNA expression and diminished -MSH expresses the biologically active long form of the leptin release from the hypothalamus in vitro [2,46]. These lines of receptor [43] and leptin has been shown to downregulate experimental evidence are in accord with the postulated twoNPY mRNA expression in the ARC [2]. Our results show- prong action of leptin in inhibiting appetite [2]. Leptin inhibits NPY and augments POMC mRNA expression directly ing decreased NPY mRNA expression in the ARC only after the high dose of rAAVleptin, which suppressed FI, imply through leptin receptors located on each of these neuronal

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populations. The resultant diminished NPYergic signaling, in turn, further curtails the restraint on POMC neurons, thereby allowing increased melanocortin signaling [27]. AGRP is coexpressed in most NPY neurons in the ARC and, like NPY, AGRP mRNA expression is increased after fasting and decreased by leptin injection [1,47]. AGRP stimulates feeding by antagonizing the action of -MSH at MC4 receptors [1,2]. Systemic rAAVleptin injection in Lepob/ob mice decreased AGRP mRNA [27]. Our failure to discern any effect on AGRP mRNA expression in the current study was unexpected. It is possible that, under the current experimental conditions, the amount of leptin produced by the rAAVleptin was ineffective in suppressing AGRP mRNA expression but effective in downregulating NPY mRNA expression. Our results show that long-term expression of Lepob in the hypothalamus can, depending upon the dose of rAAVleptin administered, either arrest the age-related weight gain or suppress BW in SD rats maintained on ad libitum rat chow. This dose-dependent effect on BW is apparently achieved either by increased energy expenditure through themogenesis alone or coupled with reduced food consumption. Additional consequences of augmented central leptin action are a dose-dependent diminution in circulating leptin and insulin levels in the presence of normoglycemia, a response indicative of increased insulin sensitivity. Diminution of appetite in response to a high level of centrally available leptin may be due to the simultaneous decrease in orexigenic NPYergic signaling and increase in anorexigenic melanocortin signaling. Because rAAV vectors are non-pathogenic and non-immunogenic [48], delivery of leptin through rAAV is potentially an efficient therapeutic modality for long-term weight control.

For experiment 1, rats were divided into two weight-matched groups (69 rats/group) and injected icv with either rAAVUF5 (control, 5 ml) or rAAVleptin (5 1010 particles in 5 l). Body weights were recorded before the icv injection and weekly thereafter. Once per week, preweighed amounts of food were placed in the food cups at 1000 h. Twenty-four hours later, the remaining food and spillage in the cup was weighed and 24 h food consumption was calculated. For experiment 2, rats were divided into two weight-matched groups (68 rats/group) and injected twice (48 h apart) with either rAAVUF5 (control, 5 l) or rAAVleptin (5 1010 particles/5 l/injection). Body weight and 24 h food intake were recorded weekly for 6 weeks as described above. Two additional control groups of unoperated rats were included in this experiment: an untreated group (n = 6) and a group of rats pair-fed to the amount consumed by the rAAVleptin treated rats (n = 6). To avoid excessive weight loss per IACUC guidelines, all rats were sacrificed at six weeks post-injection when the rAAVleptin injected rats in experiment 2 displayed a 30% loss in BW. Rats were sacrificed between 0900 and 1200 h. Brain and BAT were rapidly dissected out and frozen at 80C. BAT UCP1 mRNA expression was assessed by dot-blot analysis. Three brains per group were processed for NPY, AGRP, and POMC mRNA by in situ hybridization. Serum from trunk blood was collected for analyses of leptin, insulin, and glucose. Leptin. Serum leptin levels were assayed in duplicate with a rat leptin RIA kit (Linco Research, Inc., St Charles, MO) with a sensitivity of 0.5 ng/ml and a range of 0.550 ng/ml. In experiment 2, serum leptin was reduced and was measured with a rat/mouse leptin RIA kit (ALPCO, Windham, NH) with a sensitivity of 6 pg/ml and 12.5800 pg/ml range of detection. Insulin. Insulin was measured with a rat insulin RIA kit (Linco Research, Inc., St. Charles, MO). The sensitivity of the assay was 0.1 ng/ml and 0.110 ng/ml range. Samples from experiment 2 were assayed using a more sensitive rat insulin RIA kit (Linco Research, Inc., St Charles, MO) with a sensitivity of 0.02 ng/ml and 0.021.0 ng/ml range of detection. Glucose. Glucose was measured colorimetrically with Trinder (Sigma). Dot-blot analysis for BAT UCP-1. The full-length cDNA clone for uncoupling protein-1 (UCP-1) was provided by Leslie Kozak (Jackson Laboratory, Bar Harbor, ME) and verified by northern analysis. Total RNA was isolated from BAT with STAT-60 (Teltest Inc., Friendswood, TX) and dot-blot hybridization analysis of UCP-1 mRNA was conducted as described [9]. To minimize variability all samples from one experiment were run on the same blot. In situ hybridization. We performed in situ hybridization for NPY, AGRP, and POMC mRNA in the brain as described [27,50]. A 396-bp complete mouse AGRP cDNA (GenBank acc. no. U89484), inserted into pBSK +/ vector, was provided by Roger Cone (Oregon Health Science University, Portland, OR). The POMC probe was constructed by cloning a 478-bp cDNA fragment (5 psn 220, 3 psn 697, GenBank acc. no. J00759) into pGEM(T vector [50]. The NPY probe was constructed using a plasmid containing a 511-bp rat NPY fragment provided by Steven L. Sabol (NIH, Bethesda, MD). Antisense riboprobes were transcribed in the presence of 35S-UTP (Amersham Life Sciences, Inc., Arlington Heights, IL) using T7 (AGRP, POMC) or T3 (NPY) RNA polymerase. Fresh frozen coronal sections (16 mm) encompassing the hypothalamus were cut in a cryostat at 20C and thaw-mounted on poly-L-lysine (Sigma) coated microscope slides in four series and stored at 80C. Sections were post-fixed in 3% paraformaldehyde and acetylated and hybridized with 35Slabeled antisense probes (1 106 cpm) at 50C for 1620 h in a humidified chamber. Slides were exposed to Kodak Biomax MR autoradiography film for 5 d, followed by dipping in Kodak NTB2 emulsion for 15 d at 4C and counterstained with 0.1% cresyl violet. Slides from control and rAAVleptin treated rats were processed together to eliminate procedural variations. We estimated the relative optical density (ROD), calculated as total target area multiplied by the integrated optical density for AGRP, NPY, and POMC, from autoradiograms with the MCID image analysis system (Imaging Research, St. Catherine, Ontario, Canada). We analyzed 12 matched sections containing the ARC from each brain. The background optical density in an area adjacent to the ARC was subtracted from the target optical density. The ROD of 12 sections in the same brain were averaged and expressed relative to the average ROD from the control group.

MATERIALS AND METHODS

Animals. Adult female SD rats (200225 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN), and housed individually in a specific pathogen-free (SPF) environment in plastic cages fitted with specially designed food cups to collect spillage. Food and water were available ad libitum and lights were on from 0500 to 1900 h. The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Construction and packaging of rAAV vectors. The rAAV vectors were constructed at the University of Florida Gene Therapy Center as described [27]. Briefly, the vector pTRCBAOb EcoRI fragment of pCRrOb (a gift from Roger H. Unger, University of Texas, Dallas, TX) containing rat leptin cDNA was subcloned into rAAV vector plasmid pAAVGEnh after deletion of the EcoRI fragment carrying -glucuronidase cDNA sequence. Vectors were packaged, purified, concentrated, and titered essentially as described [49]. The titer of rAAVCBAOb vector, hereafter referred to as rAAVleptin, was 1 1013 physical particles/ml and the ratio of physical-to-infectious particles was 100. rAAV vectors, purified using iodixanol gradient/heparin-affinity chromatography, were 99% pure. The control vector, rAAVUF5, was similarly constructed and encodes GFP [49]. The titer of the rAAVUF5 was 1 1013 physical particles/ml. Experimental design. Rats were anesthetized (ketamine 100 mg/kg BW + xylazine 15 mg/kg BW) and permanent cannulae were stereotaxically implanted in the third cerebroventricle as described [22]. Cerebrospinal fluid efflux served as an indicator of the accuracy of cannula placement. Rats were allowed 710 d to recover from surgery before use in the following two experiments.

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Statistical analyses. We used two-way ANOVA with time and treatment as variables for comparison of the weekly BW and FI data. In experiment 2, differences between the four treatment groups at each time point were compared with one way ANOVA, followed by Neuman-Keuls post-hoc test. Serum leptin, insulin, and glucose levels and BAT UCP-1 mRNA, brain NPY, POMC, and AGRP mRNA expression were compared by Students t test (experiment 1) or ANOVA and Neuman-Keuls test (experiment 2). P 0.05 was considered significant for all analyses.

ACKNOWLEDGMENTS
This research was supported by grants from the National Institutes of Health (HD DK 37273 and NS 32727). We thank Nicholas Muzyczka (Director of Gene Therapy Center) and Sergei Zolotukhin (Director of the Vector Core Laboratory, University of Florida) for preparation of rAAVleptin vectors. Presented in part at the 30th Annual Meeting of the Society for Neuroscience, New Orleans, Louisiana, Nov. 49, 2000. RECEIVED FOR PUBLICATION APRIL 12; ACCEPTED JUNE 25, 2001.

REFERENCES
1. Friedman, J. M., and Halaas, J. L. (1998). Leptin and the regulation of body weight in mammals. Nature 395: 763770. 2. Kalra, S. P., et al. (1999). Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr. Rev. 20: 68100. 3. Halaas, J. L., et al. (1995). Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269: 543546. 4. Campfield, L. A., Smith, F. J., Guisez, Y., Devos, R., and Burn, P. (1995). Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269: 546549. 5. Pelleymounter, M. A., et al. (1995). Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540543. 6. Burguera, B., et al. (2000). Obesity is associated with a decreased leptin transport across the blood-brain barrier in rats. Diabetes 49: 12191223. 7. Kastin, A. J., and Pan, W. (2000). Dynamic regulation of leptin entry into brain by the blood-brain barrier. Regul. Pept. 92: 3743. 8. Himms-Hagens, J. (1991). Neural control of brown adipose tissue theremogenesis, hypertrophy, and atrophy. Front. Neuroendocrinol. 12: 3893. 9. Scarpace, P. J., and Matheny, M. (1998). Leptin induction of UCP1 gene expression is dependent on sympathetic innervation. Am. J. Physiol. 275: E259264. 10. Bamshad, M., Song, C. K., and Bartness, T. J. (1999). CNS origins of the sympathetic nervous system outflow to brown adipose tissue. Am. J. Physiol. 276: R15691578. 11. Halaas, J. L., et al. (1997). Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc. Natl. Acad. Sci. USA 94: 88788883. 12. Harris, R. B., et al. (1998). A leptin dose-response study in obese (ob/ob) and lean (+/) mice. Endocrinology 139: 819. 13. Chen, Y., and Heiman, M. L. (2000). Chronic leptin administration promotes lipid utilization until fat mass is greatly reduced and preserves lean mass of normal female rats. Regul. Pept. 92: 113119. 14. Morsy, M. A., et al. (1998). Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse. Gene Ther. 5: 818. 15. Wolden-Hanson, T., Marck, B. T., Smith, L., and Matsumoto, A. M. (1999). Cross-sectional and longitudinal analysis of age-associated changes in body composition of male Brown Norway rats: association of serum leptin levels with peripheral adiposity. J. Gerontol. A. Biol. Sci. Med. Sci. 54: B99107. 16. Pu, S., Dube, M. G., Kalra, P. S., and Kalra, S. P. (2000). Regulation of leptin secretion: Effects of aging on daily patterns of serum leptin and food consumption. Regul. Pept. 92: 107111. 17. Van Heek, M., et al. (1997). Diet-induced obese mice develop peripheral, but not central, resistance to leptin. J. Clin. Invest. 99: 385390. 18. Lin, X., et al. (1998). The effects of a high fat diet on leptin mRNA, serum leptin and the response to leptin are not altered in a rat strain susceptible to high fat diet-induced obesity. J. Nutr. 128: 16061613. 19. Wang, Z. W., et al. (1999). Comparing the hypothalamic and extrahypothalamic actions of endogenous hyperleptinemia. Proc. Natl. Acad. Sci. USA 96: 1037310378. 20. Unger, R. H. (2000). Leptin physiology: a second look. Regul. Pept. 92: 8795. 21. Morash, B., Li, A., Murphy, P. R., Wilkinson, M., and Ur, E. (1999). Leptin gene expression in the brain and pituitary gland. Endocrinology 140: 59955998. 22. Dhillon, H., et al. (2001). Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: a long-term study. Regul. Pept. 99: 6977. 23. Lundbert, C., Jungles, S. J., and Mulligan, R. C. (2001). Direct delivery of leptin to the hypothalamus using recombinant adeno-associated virus vectors results in increased therapeu-

tic efficacy. Nat. Biotechnol. 19: 169172. 24. Lowell, B. B., et al. (1993). Development of obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature 366: 740742. 25. Klaus, S., Munzberg, H., Truloff, C., and Heldmaier, G. (1998). Physiology of transgenic mice with brown fat ablation: obesity is due to lowered body temperature. Am. J. Physiol. 274: R287293. 26. Kalra, S. P. (2001). Circumventing leptin resistance for weight control. Proc. Natl. Acad. Sci. USA 98: 42794281. 27. Dhillon, H., et al. (2000). Long-term differential modulation of genes encoding orexigenic and anorexigenic peptides by leptin delivered by rAAV vector in ob/ob mice. Relationship with body weight change. Regul. Pept. 92: 97105. 28. Dhillon, H., et al. (June 913, 1999). Long-term correction of obesity using centrally delivered rAAV encoding anorexigenic cytokines. 2nd Annual Meeting American Society for Gene Therapy, Washington, D.C., Abstract 194: 45a. 29. Dhillon, H., Zolotukhin, S., Kalra, S., and Kalra, P. (October 2318, 1999). Sexual dimorphism in the response to leptin gene therapy via a recombinant adeno-associated virus (rAAV) vector. 29th Annual Mtg. Soc. Neuroscience , Miami Beach, FL, Abstract 214.3. 30. Murphy, J. E., et al. (1997). Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin. Proc. Natl. Acad. Sci. USA 94: 1392113926. 31. Muzzin, P., Eisensmith, R. C., Copeland, K. C., and Woo, S. L. (1996) Correction of obesity and diabetes in genetically obese mice by leptin gene therapy. Proc. Natl. Acad. Sci. USA 93: 1480414808. 32. Chen, G., et al. (1996). Disappearance of body fat in normal rats induced by adenovirusmediated leptin gene therapy. Proc. Natl. Acad. Sci. USA 93: 1479514799. 33. Koyama, K., et al. (1998). Resistance to adenovirally induced hyperleptinemia in rats. Comparison of ventromedial hypothalamic lesions and mutated leptin receptors. J. Clin. Invest. 102: 728733. 34. Wang, Z. W., et al. (1999). Hyperleptinemia depletes fat from denervated fat tissue. Biochem. Biophys. Res. Commun. 260: 653657. 35. Seufert, J., Kieffer, T. J., and Habener, J. F. (1999). Leptin inhibits insulin gene transcription and reverses hyperinsulinemia in leptin-deficient ob/ob mice. Proc. Natl. Acad. Sci. USA 96: 674679. 36. Mizuno, A., Murakami, T., Otani, S., Kuwajima, M., and Shima, K. (1998). Leptin affects pancreatic endocrine functions through the sympathetic nervous system. Endocrinology 139: 38633870. 37. Kieffer, T. J., and Habener, J. F. (2000). The adipoinsular axis: effects of leptin on pancreatic B-cells. Am. J. Physiol. Endocrinol. Metab. 278: E1 E14. 38. Sivitz, W. I., Walsh, S. A., Morgan, D. A., Thomas, M. J., and Haynes, W. G. (1997). Effects of leptin on insulin sensitivity in normal rats. Endocrinology 138: 33953401. 39. Kulkarni, R. N., et al. (1997). Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice. J. Clin. Invest. 100: 27292736. 40. Zheng, D., Jones, J. P., Usala, S. J., and Dohm, G. L. (1996). Differential expression of ob mRNA in rat adipose tissues in response to insulin. Biochem. Biophys. Res. Commun. 218: 434437. 41. Clark, J. T., Kalra, P. S., Crowley, W. R., and Kalra, S. P. (1984). Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 115: 427429. 42. Kalra, S. P., Dube, M. G., Sahu, A., Phelps, C. P., and Kalra, P. S. (1991). Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. Proc. Natl. Acad. Sci. USA 88: 1093110935. 43. Mercer, J. G., et al. (1996). Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J. Neuroendocrinol. 8: 733735. 44. Horvath, T. L., Naftolin, F., Kalra, S. P., and Leranth, C. (1992). Neuropeptide-Y innervation of -endorphin-containing cells in the rat mediobasal hypothalamus: a light and electron microscopic double immunostaining analysis. Endocrinology 131: 24612467. 45. Fuxe, K., Tinner, B., Caberlotto, L., Bunnemann, B., and Agnati, L. F. (1997). NPY Y1 receptor like immunoreactivity exists in a subpopulation of -endorphin immunoreactive nerve cells in the arcuate nucleus: a double immunolabelling analysis in the rat. Neurosci. Lett. 225: 4952. 46. Garcia de Yebenes, E., Li, S., Fournier, A., St-Pierre, S., and Pelletier, G. (1995). Regulation of proopiomelanocortin gene expression by neuropeptide Y in the rat arcuate nucleus. Brain Res. 674: 112116. 47. Mizuno, T. M., and Mobbs, C. V. (1999). Hypothalamic agouti-related protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting. Endocrinology 140: 814817. 48. Samulski, R. J., Sally, M., and Muzyczka, N. (1999). Adeno-associated viral vectors. In The Development of Human Gene Therapy (T. Friedman, Ed.), pp. 131172. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 49. Hauswirth, W. W., Lewin, A. S., Zolotukhin, S., and Muzyczka, N. (2000). Production and purification of recombinant adeno-associated virus. Methods Enzymol. 316: 743761. 50. Dube, M. G., Pu, S., Kalra, S. P., and Kalra, P. S. (2000). Melanocortin signaling is decreased during neurotoxin-induced transient hyperphagia and increased body-weight gain. Peptides 21: 793801.

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