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Endocrinology Vol. 143, No. 11 4409-4421
Copyright © 2002 by The Endocrine Society

ARTICLE

Evidence for the Existence of Distinct Central Appetite, Energy Expenditure, and Ghrelin Stimulation Pathways as Revealed by Hypothalamic Site-Specific Leptin Gene Therapy

M. Bagnasco, M. G. Dube, P. S. Kalra and S. P. Kalra

Departments of Neuroscience (M.B., S.P.K.) and Physiology and Functional Genomics (M.G.D., P.S.K.), McKnight Brain Institute, University of Florida, Gainesville, Florida 32610-0244

Address all correspondence and requests for reprints to: Satya P. Kalra, Ph.D., Department of Neuroscience, College of Medicine, University of Florida McKnight Brain Institute, P.O. Box 100244, Gainesville, Florida 32610. E-mail: skalra{at}ufbi.ufl.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Effects of rAAV-lep...
 References
 
To identify the specific hypothalamic sites in which leptin acts to decrease energy intake and/or increase energy expenditure, recombinant adeno-associated virus vector-encoding leptin was microinjected bilaterally into one of four hypothalamic sites in female rats. Leptin transgene expression in the ventromedial nucleus and paraventricular nucleus induced comparable decreases in daily food intake (FI; 18–20%) and body weight (BW; 26–29%), accompanied by drastic reductions in serum leptin (81–97%), insulin (92–93%), free fatty acids (35–36%), and normoglycemia. Leptin transgene expression in the arcuate nucleus (ARC) decreased BW gain (21%) and FI (11%) to a lesser range, but the metabolic hormones were suppressed to the same extent. Leptin transgene expression in the medial preoptic area (MPOA) decreased BW and metabolic hormones without decreasing FI. Finally, leptin transgene expression in all four sites augmented serum ghrelin and thermogenic energy expenditure, as shown by uncoupling protein-1 mRNA expression in brown adipose tissue. Proopiomelanocortin gene expression in the ARC was up-regulated by leptin expression in all four sites, but neuropeptide Y gene expression in the ARC was suppressed by leptin transgene expression in the ARC but not in the MPOA. Thus, whereas leptin expression in the paraventricular nucleus, ventromedial nucleus, or ARC suppresses adiposity and insulin by decreasing energy intake and increasing energy expenditure, in the MPOA it suppresses these variables by increasing energy expenditure alone.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Effects of rAAV-lep...
 References
 
LEPTIN PRODUCED BY adipocytes and hypothalamus (1, 2, 3, 4) controls the daily management of body weight (BW) homeostasis by restraining food intake (FI) and enhancing energy expenditure (5, 6, 7). A loss of leptin control on these two central mechanisms invariably results in uncontrolled energy intake leading to obesity and attendant metabolic disorders such as hyperleptinemia, hyperinsulinemia, and type 2 diabetes (5, 6, 7, 8). Numerous studies now suggest that despite the presence of elevated circulating leptin levels, the progressive age-related and environmentally based increase in adiposity is due to leptin insufficiency in the hypothalamus rendered by defective transport of peripheral leptin across the blood brain barrier and/or suboptimal production of leptin locally in the hypothalamus (9, 10, 11, 12, 13, 14).

Gene delivery in vivo to the central nervous system has been facilitated by the development of a nonimmunogenic and nonpathogenic recombinant adeno-associated virus (rAAV) vector (15, 16). The rAAV has advantages over other viral vector systems because of availability of stable, high-titer vector for long-term expression of target genes in nondividing cells (15, 16, 17). Consequently, leptin gene therapy offers a novel way to reinstate the hypothalamic leptin insufficiency responsible for the age-related and environmentally based abnormal weight gain and adiposity.

We recently developed a rAAV-vector encoding the leptin transgene (rAAV-lep) (18). A single intracerebroventricular (icv) injection of this vector inhibited weight gain and adiposity for long periods in rats of both sexes maintained either on regular rat chow or high-fat diet (10, 11, 19, 20). Interestingly, in association with suppressed weights, these rats displayed drastic reductions in serum leptin, insulin, and free fatty acids (FFAs) along with normoglycemia. In addition, icv rAAV-lep augmented thermogenic energy expenditure alone or along with decreased FI (10, 11 19, 20, 21, 22).

The physiologically active long form of the leptin receptor is expressed in various hypothalamic sites, and leptin administration to rodents activates c-Fos protein in groups of neurons in multiple hypothalamic sites (5, 23, 24, 25, 26, 27), suggesting that receptive elements in these sites play a role in regulating energy balance. Experimental results showed that microinjection of leptin into several hypothalamic sites decreased food intake (28, 29). These sites include the arcuate nucleus (ARC)-paraventricular nucleus (PVN) axis in which leptin receptors are expressed in neurons expressing the orexigenic peptides, neuropeptide Y (NPY), and agouti- related peptide (AgrP) and in proopiomelanocortin (POMC) neurons producing the anorexigenic peptide, {alpha}-MSH (5, 6, 30, 31).

We have now extended our icv leptin gene therapy studies to ascertain whether intracranial delivery of rAAV-lep in distinct hypothalamic sites would transduce leptin-transgene. Because the evidence outlined above suggests multiple sites of leptin action in weight control, another goal of these studies was to determine whether leptin transgene expression is one or more than one hypothalamic site that will reproduce the effects of icv rAAV-lep administration on age-related weight gain and metabolic hormones. In view of the fact that leptin administration inhibits FI and increases energy expenditure, it was of interest to identify the site(s), if any, in which leptin expression would selectively modulate either energy intake or energy expenditure and whether peptidergic pathways in the ARC-PVN axis are affected by the site-specific leptin transgene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Effects of rAAV-lep...
 References
 
Animals
Adult female Sprague Dawley rats (225–250 g) purchased from Harlan (Harlan, Inc., Indianapolis, IN) were housed individually in air-conditioned rooms (22–24 C) under controlled light/dark cycle (lights on 0500–1900 h) and fed standard rat chow and water ad libitum. The animal protocols were approved by the Institutional Animal Care and use Committee (IACUC).

Construction and packaging of rAAV vectors
The vector pTR-CBA-Ob EcoRI fragment of pCR-rOb (a gift from Dr. Roger H. Unger, Southwestern Medical School, Dallas, TX) containing rat leptin cDNA was subcloned into rAAV vector plasmid pAAVßGEnh after deleting the EcoRI fragment carrying ß-glucuronidase cDNA sequence (10, 17, 18). Vectors were packaged, purified, concentrated, and titered as described earlier (10, 17, 18). The titer of rAAV-CBA-Ob (hereafter referred to as rAAV-lep) vectors used in this study was 1 x 1013 physical particles/ml with a ratio of physical-to-infectious particles less than 100. The rAAV vector, purified using iodixanol gradient/heparin-affinity chromatography, was 99% pure as judged by polyacrylamide gel/silver-stained gel electrophoresis (not shown). The control vector, rAAV-UF5, was similarly constructed to encode the green fluorescence protein (GFP) gene (10, 17, 18).

Surgical procedures
To evaluate the effects of microinjection of rAAV-lep on BW, FI, and blood metabolic hormones, rats were anesthetized with an ip injection of ketamine/xylazine (ketamine 100 mg/kg BW + xylazine 15 mg/kg BW) and microinjected in the hypothalamic nuclei with the aid of a rat brain atlas (32) in the following two experiments.

Experiment 1.
Weight-matched rats (six to eight rats per group) were placed in a stereotaxic frame fitted with a Kopf microinjector and microinjected bilaterally either with rAAV-lep or rAAV-UF5 (1013 particles/ml) in the PVN (0.3 µl/injection, 3 x 109 particles/nuclei, at 1.8 mm behind the bregma, 0.6 mm lateral to the sagittal sinus, and 7.5 mm below the dura) or in the ventromedial nucleus (VMN, 0.5 µl/injection, 5 x 109 particles/nuclei, at 2.7 mm behind the bregma, 0.5 mm lateral to the sagittal sinus, and 9.0 mm below the dura). The vector was delivered slowly over a 2- to 3-min period, and the needle was left in place for an additional 15 min to prevent diffusion along the needle track (33). FI and BW were monitored biweekly for 45 d. An additional control group consisting of weight-matched untreated rats (n = 8) and a group of rats (n = 8) pair fed to the amount consumed by rats receiving rAAV-lep treatment were monitored concurrently. Rats were killed by decapitation and trunk blood was collected for analysis. The data collected from all experimental and control groups of rats were analyzed. Brains were dissected out and processed for analysis of leptin mRNA in neural sites and for in situ hybridization of neuropeptide mRNA. Brown adipose tissue (BAT) was dissected and kept frozen until analysis of uncoupling protein-1 (UCP1)-mRNA.

Experiment 2.
In this experiment, a comparison of various metabolic variables before and after the intrahypothalamic microinjection was undertaken. Rats were divided into three weight-matched groups (untreated, rAAV-UF5, and rAAV-lep, six to eight rats per group). Rats were anesthetized with ketamine + Xylazine, a blood sample (1 ml) was withdrawn from the jugular vein, and plasma was stored frozen for analysis. Rats were then microinjected bilaterally either rAAV-lep or rAAV-UF5 (1013 particles/ml) in the ARC (0.3 µl/injection, 3 x 109 particle/nuclei, at 2.7 mm behind the bregma, 0.2 mm lateral to the sagittal sinus, and 9.8 mm below the dura) or in the medial preoptic area (MPOA) (0.3 µl/injection, 3 x 109 particle/nuclei, at 0.3 mm behind the bregma, 0.6 mm lateral to the sagittal sinus, and 7.8 mm below the dura). Vector solutions were microinjected slowly as described for experiment 1. An additional untreated group and pair-fed (PF) group were added. FI and BW were monitored biweekly for 45 d and blood samples and neural tissues were collected for analysis as described above.

Immunohistochemistry for GFP
Brains of rats microinjected with rAAV-UF5 in the four sites were processed for immunocytochemical localization of GFP as described earlier (10, 11, 19). To verify site location, rAAV-GFP was injected into either PVN or VMN of a separate group of rats (three rats/site). For rAAV-GFP localization in MPOA and ARC, brains of three rats/site from experiment 2 were processed. Animals were anesthetized with an ip injection of sodium pentobarbital (100 mg/kg) and perfused intracardially with saline (0.9% NaCl) followed by cold 4% paraformaldehyde in PBS. Brains were removed and kept in paraformaldehyde at 4 C and then transferred to 20% followed by 30% sucrose solutions. Brains were sectioned (39–42 µm thick) starting at approximately 1.0 mm anterior to the site of injection and caudally through the entire hypothalamus past the mammillary body. Immunohistochemistry for GFP was performed on floating sections as described earlier using a GFP polyclonal antibody (CLONTECH Laboratories, Inc., Palo Alto, CA) (10, 11, 19).

RT-PCR for leptin mRNA expression
Leptin mRNA expression was analyzed with RT-PCR to confirm leptin mRNA overexpression in the PVN and VMN of rats receiving rAAV-lep in these sites (10, 11, 19). Briefly, neural tissue encompassing either the PVN or VMN was grossly dissected from the brains of untreated, rAAV-UF5 or rAAV-lep-microinjected rats (three rats/group) and analyzed for leptin mRNA by RT-PCR as previously described (10, 11).

RIAs
Serum leptin, insulin, and ghrelin levels were assayed in duplicate with the help of RIA kits (leptin, Alpco Diagnostic, Windham, NH; insulin, Linco Research, Inc., St. Charles MO; ghrelin, Phoenix Pharmaceuticals, Inc., Belmont, CA) according to the manufacturer’s instructions. Experimental and control samples from each site were analyzed for leptin, insulin, and ghrelin in individual assays. The sensitivity ranges were 6 pg/ml, 0.02 ng/ml, and 0.01 ng/ml and intraassay coefficients of variance were 5.0%, 5.5%, and 4.7% for leptin, insulin, and ghrelin, respectively. Serum glucose was measured using a glucometer (Glucometer Elite XL, Bayer Corp., Pittsburgh, PA) with sensitivity of 20 mg/dl and a range of detection between 20 and 600 mg/dl. Serum FFA levels were measured using an Acyl-CoA synthetase-acyl-CoA oxidase method (NEFA C, Wako Chemical, Inc., Richmond, VA) based on a quantitative, colorimetric, enzymatic reaction read at 550 nm. The sensitivity of the assay, expressed as absorptivity, is 52 liters/mEq-cm and intraassay coefficient of variance was 1.1%.

In situ hybridization (ISH)
ISH hybridization for NPY, AgrP, and POMC mRNA analysis in the brain was performed as previously described (11, 18, 19). Briefly, the probes used in this procedure were constructed as follows. The NPY probe was constructed using plasmid containing a 511-bp rat NPY fragment provided by Steven L. Sabol (NIH, Bethesda, MD). A 396-bp complete mouse AgrP cDNA (GenBank accession no. U89484), inserted into pBSK +/- vector, was kindly provided by Dr. R. Cone (Oregon Health Science University, Portland, OR). The rat POMC cDNA was prepared as described earlier (11, 18, 19). Antisense riboprobes were transcribed in the presence of 35S-UTP (Amersham Life Science, Inc., Arlington Heights, IL) using T7 RNA polymerase. Fresh-frozen coronal sections (16 µm) encompassing the hypothalamus were cut in a cryostat at -20 C and thaw mounted at -80 C until analysis for NPY, AgrP, and POMC mRNA. Processing of the brain sections for ISH was performed in two phases. In the first phase, brain sections from rats microinjected with vectors in PVN and VMN were processed for analysis of AgrP, POMC, and NPY mRNA. Because of an unfortunate power outage, analysis of NPY mRNA could not be undertaken in brain sections of these microinjected rats. In the second phase, brains from rats microinjected with the vector in MPOA and ARC were sectioned and processed for analysis of AgrP, POMC, and NPY mRNA.

For semiquantitative analysis, the relative OD (ROD), calculated as total target area multiplied by the integrated OD for NPY, AgrP, and POMC, was estimated from autoradiograms with the MCID image analysis system (Imaging Research, Inc., St. Catherine, Ontario, Canada). Twelve matched sections from each brain were analyzed (three rats per group). The background OD in an area adjacent to each target was subtracted from the target. The ROD of 12 sections in the same brain was added and expressed relative to the ROD from the control group.

Dot-blot analysis of UCP-1 mRNA
UCP-1 mRNA expression in BAT was measured as described (10, 11, 18). The UCP-1 probe (UCP1 cDNA was provided by Dr. L. Kozac) was random prime labeled using Prime-A-Gene kit (Promega Corp., Madison, WI) according to manufacturer’s instructions. The labeled probe was purified by filtering through a Nick column (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Total RNA was isolated from BAT using an RNA isolation kit (STAT-60, Tel-Test Inc., Friendswood, TX); the integrity of the isolated RNA was verified using 1% agarose gels stained with ethidium bromide and quantified by spectrophotometric absorption at 260 and 280 nm. The blots were exposed to a phosphor-imaging screen for 48 h. The latent image on the phosphor imager screen was scanned using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and analyzed by ImageQuant software (Molecular Dynamics). Intensities were calculated per microgram total RNA for each animal. Samples from control and treated animals were applied on the same blot to minimize variability. All samples from one experiment were run on the same blot.

Statistical analysis
BW and FI, leptin, insulin, glucose, FFAs, ghrelin levels, and UCP-1 mRNA were analyzed using one-way ANOVA measurement followed post hoc by Bonferroni’s multiple comparison test and two-way ANOVA or t test, as appropriate. The P value was set at less than 0.05 for all analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Effects of rAAV-lep...
 References
 
The capability of intracranial microinjection of rAAV vector to transduce target genes in hypothalamic sites was verified in two ways. GFP gene expression in brain sections of rAAV-UF5-microinjected rats was examined by immunohistochemical localization. As shown in Fig. 1Go, 45 d after injection GFP immunoreactivity was observed discretely at the sites of injections in the PVN, VMN, ARC, and MPOA nuclei (Fig. 1Go, A–D), mostly in cells displaying neuronlike morphology and in axon and dendrites (Fig. 1Go, A–D, inset). GFP-positive cells were seen bilaterally and uniformly around injection sites.



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  		Figure 1. Localization of GFP by immunohistochemistry in hypothalamic sites microinjected with rAAV-UF5 (low power, x4; high-power inset, x10). A, PVN; B, VMN; C, ARC; D, MPOA; V, third ventricle; AC, anterior commissure.

 
That this site targeted vector infection results in increased leptin transgene expression was confirmed by RT-PCR analysis of leptin mRNA in neural tissue containing the injection sites. As shown in Fig. 2Go, although detectable in neural tissues from control rats, leptin mRNA expression was significantly higher in PVN and VMN of rats injected with rAAV-lep in these nuclei (P < 0.05).



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  		Figure 2. Effects of rAAV-lep microinjection either in the PVN (left) or VMN (right) on leptin mRNA expression, as assessed by RT-PCR, in the hypothalamic fragment containing the PVN or VMN. *, P < 0.001 vs. rAAV-UF5 and untreated groups.

 

   Effects of rAAV-lep microinjection in hypothalamic sites on BW, FI, serum leptin, insulin, and metabolic variables
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Effects of rAAV-lep...
References
 
PVN.
Whereas the control, untreated, and rAAV-UF5-microinjected rats slowly gained BW during the 45-d experimental period, rAAV-lep-microinjected rats displayed a steady decline in BW starting on d 4 (P < 0.05) to reach near nadir by d 25 after injection (P < 0.05, Fig. 3AGo). On d 45 post injection, BW of rAAV-lep-injected rats was approximately 26% below that of control groups (P < 0.05). FI decreased by approximately 18% and there was drastic suppression of serum leptin (97%), insulin (93%), and FFA (35%, Fig. 3CGo, inset), compared with control groups (P < 0.05). Serum glucose levels were reduced by about 20% from control values, but these levels were still in the normoglycemic range. In contrast, serum ghrelin levels were increased in rAAV- lep-injected rats by 50% over those found in control groups (Fig. 3FGo, P < 0.05).



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  		Figure 3. Effects of rAAV-leptin microinjection (arrow, A) in the PVN on BW (A); FI (B); and serum leptin (C), FFA (inset, C), insulin (D), glucose (E), and ghrelin (F) levels. *, P < 0.05 vs. untreated controls and rAAV-UF-5.

 
Rats allowed to consume food to the range of rAAV- lep-treated rats (PF group) showed an initial decrease in BW, followed by a slow return to the control range. Forty-five days of PF resulted in significant decreases in serum leptin (45%) and glucose (32%, P < 0.05), but these decreases were relatively smaller than in rAAV-lep-injected rats. In contrast to the rAAV-lep-treated rats, PF rats displayed no significant changes in serum insulin, FFA, and ghrelin levels (Fig. 3CGo, inset, and D–F).

VMN.
Results of rAAV-lep microinjection into the VMN were similar to those observed after PVN injections with the exception of slightly greater decreases in BW (29%) and FI (20%) (Fig.4Go , A and B vs. Fig. 3Go, A and B). The onset of BW decrease on d 4 after injection and the quantitative decrease of serum leptin (81%), insulin (92%), FFA (36%), and glucose (29%) were similar to those in PVN-microinjected rats (Fig. 4Go, C–E). There was also a comparable increase in serum ghrelin levels from control range (55%, Fig. 4FGo). PF prevented the slow increase in BW as seen in control groups and maintained weight during the experiment to result in reduction in serum leptin by 50% from that in rAAV-UF5 controls (Fig. 4CGo). However, serum FFA, insulin, glucose, and ghrelin levels were essentially unchanged (Fig.4CGo, inset, and D–F).



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  		Figure 4. Effects of rAAV-leptin microinjection (arrow, A) in the VMN on BW (A); FI (B); and serum leptin (C), FFA (inset, C), insulin (D), glucose (E), and ghrelin (F) levels. *, P < 0.05 vs. rAAV-UF-5 and untreated control.

 
ARC.
The rAAV-lep injection in the ARC elicited a decrease in BW from initial values beginning d 4 (P < 0.05) to reach maximal 21% suppression at d 45 post injection (P < 0.05, Fig. 5AGo). FI decreased by 11%, compared with rAAV-UF5 control group of rats (Fig. 5BGo, P < 0.05). Although the weight and FI suppression responses were somewhat smaller than those seen after rAAV-lep injection in the PVN and VMN (Figs. 3Go and 4Go), reductions in serum leptin (89%), insulin (94%), FFA (52%), and glucose (32%) levels were quantitatively similar (Fig. 5Go, C–E). Ghrelin levels were significantly elevated in response to rAAV-lep injection in the ARC (3.7-fold, Fig. 5FGo, P < 0.05). It is noteworthy that in this group it was possible to show that the time-related increase in serum insulin seen in the two control groups of rats was not only blocked by rAAV-lep treatment but levels were also significantly lower than the preinjection values (P < 0.05).



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  		Figure 5. Effects of rAAV-leptin microinjection (arrow, A) in the ARC on BW (A); FI (B); and serum leptin (C), FFA (inset, C), insulin (D), glucose (E), and ghrelin (F) levels. *, P < 0.05 vs. rAAV-UF-5 and untreated controls on d 45; a, P < 0.05 vs. d 0 levels.

 
PF blocked the slow weight gain seen in control rats, thereby resulting in maintenance of weight below controls but higher than that of rAAV-lep group (Fig. 5Go, A and B, P < 0.05). Serum leptin and ghrelin levels were slightly but significantly increased from the d 0 values (Fig. 5Go, C–F).

MPOA.
The effects of rAAV-lep injection in the MPOA were somewhat different. The rAAV-lep injection in the MPOA reduced weight. At d 45 post injection, BW was only 18% lower than in rAAV-UF5 control rats, and energy consumption was not reduced (Fig.6Go, A and B). This moderate weight reduction with normal daily intake started on d 4 after injection (P < 0.05) and evoked decreases in serum leptin (90%), insulin (84%), FFA (53%), and glucose (26%) and elevations in serum ghrelin levels (3-fold, P < 0.05) in magnitudes similar (Fig. 6Go, C–F) to those observed in the ARC rAAV-lep-injected rats (Fig. 5Go). The age-related increase in insulin (Fig. 6DGo) was blocked by rAAV-lep treatment, as also seen in ARC-injected rats (Fig. 5DGo).



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  		Figure 6. Effects of rAAV-leptin microinjection (arrow, A) in the MPOA on BW (A); FI (B); and serum leptin (C), FFA (inset, C), insulin (D), glucose (E), and ghrelin (F) levels. *, P < 0.05 vs. rAAV-UF-5 and untreated controls on d 45; a, P < 0.05 vs. d 0 levels.

 
Effects of rAAV-lep microinjection in various hypothalamic sites on BAT UCP1 mRNA
The rAAV-lep microinjection in each of the four neural sites resulted in up-regulation of UCP1 mRNA expression, compared with the range seen in the respective untreated or rAAV-UF5-treated control groups (P < 0.05, Fig. 7Go). Decreased FI imposed by PF, on the other hand, decreased BAT UCP1 mRNA expression (significant only in the PVN study, P < 0.05).



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  		Figure 7. The effects of rAAV-leptin microinjection in four hypothalamic sites on UCP-1 mRNA expression in the BAT. *, P < 0.05 vs. all other groups; **, P < 0.05 vs. untreated control.

 
Effects of rAAV-lep microinjection in various hypothalamic sites on POMC, AgrP, and NPY mRNA expression in the ARC
PVN and VMN.
We observed that rAAV-lep microinjection in the PVN and VMN significantly augmented POMC mRNA expression in the ARC over that in the two control groups (P < 0.05, Fig. 8Go). Interestingly, POMC mRNA in the ARC was not affected in PF rats. On the other hand, AgrP mRNA in the ARC was unaffected by rAAV-lep injection either in the PVN or VMN, but PF significantly increased the ARC AgrP mRNA expression, compared with control and rAAV-lep-injected groups (P < 0.05, Fig.8Go).



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  		Figure 8. Effects of rAAV-leptin microinjection in the PVN and VMN on POMC mRNA (A) and AgrP mRNA (B) expression in the ARC. *, P < 0.05 vs. other groups; **, P < 0.05 vs. controls.

 
ARC and MPOA.
Microinjection of rAAV-lep in the ARC also increased ARC POMC mRNA expression over that seen in two control groups (P < 0.05, Fig 9AGo). AgrP mRNA in the PF group was also augmented over controls and rAAV- lep-injected rats (Fig. 9BGo). Interestingly, ARC NPY mRNA of rats microinjected with rAAV-lep in the ARC was significantly decreased (P < 0.05), compared with controls and PF rats (Fig. 9CGo). The rAAV-lep microinjections in the MPOA (Fig. 9Go), as observed after microinjection in three other sites (Fig. 8Go), enhanced POMC mRNA without affecting AgrP mRNA in the ARC (Fig. 9Go, D and E). In contrast to a decrease in ARC NPY mRNA expression in rats receiving rAAV-lep in the ARC (Fig. 9CGo), ARC NPY mRNA expression in response to rAAV-lep microinjection in the MPOA was not affected, a response associated with unabated food intake (Fig. 6BGo).



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  		Figure 9. Effects of rAAV-leptin microinjection in the ARC and MPOA on POMC mRNA (A and B), AgrP mRNA (B and E), and NPY mRNA (C and F) expression in the ARC. *, P < 0.05 vs. other groups.

 
Discussion
The objectives of these investigations were 2-fold: first, to determine whether microinjection of rAAV vector-encoding leptin gene directly into hypothalamic sites can affect weight homeostasis and second, to identify the leptin transgene-expressing sites capable of modifying weight homeostasis on a long-term basis in a manner similar to that observed after icv rAAV-lep injection (10, 11). The results of the current studies show that enhanced leptin transgene expression in four discrete sites suppressed the time-related weight gain, first by immediately decreasing weight from the preinjection range to a nadir and then sustaining the reduced weight through the remaining period of observation. A similar bimodal pattern of weight suppression for extended periods was observed in adult and prepubertal rats of both sexes after icv injection of 17–20 times higher doses of rAAV-lep (10, 11, 19). Because GFP expression in rats receiving icv rAAV-GFP was visualized in several sites along the midline neuroaxis, from the anterior commissures to regions past the mammillary bodies, the identity of hypothalamic site or sites participating in reducing weight gain could not be ascertained (10, 11, 19).

The current microinjection study shows that enhanced leptin transgene expression in any one of the four hypothalamic sites is capable of suppressing weight in a bimodal pattern. The existence of leptin-responsive sites in an extensive topography in the diencephalon was unexpected in view of the current focus on the ARC-PVN axis as the primary neuroanatomical substrate mediating leptin feedback in weight regulation (5, 6, 34). However, several investigations that assessed the hypothalamic site(s) of leptin action lend credence to a more extensive neuroanatomic substrate (5). Leptin receptors are localized in multiple hypothalamic nuclei (5, 26, 27) and leptin administration activates neuronal c-Fos in multiple hypothalamic sites (23, 24), including the four sites examined in our current study. Microinjection of leptin in the PVN, VMN, and ARC decreased FI and BW (28, 29). Further, disruption of signaling by neural ablation (5, 35, 36, 37) or by neurotoxins in discrete sites (38) implicates the VMN, PVN, and ARC in weight homeostasis either directly or indirectly by engaging distinct neural pathways in the vicinity.

Consequently, we infer that the four sites evaluated here are, indeed, the integral components of the interconnected hypothalamic network in the leptin feedback control of weight homeostasis (5). It is well known that subgroups of neuronal populations in these sites exert a wide spectrum of effects on neuroendocrine axes, weight homeostasis, temperature, activity, and autonomic systems. Chemical identity of only a limited subpopulation in weight control in these sites has been deciphered (5). Future studies should be directed toward clarifying the chemical phenotype of neurons infected by rAAV-lep vector.

In addition to decreasing weight, enhanced leptin transgene expression selectively in the VMN, PVN, and ARC decreased food intake and augmented nonshivering thermogenesis, as shown by enhanced BAT UCP1 mRNA response in these rats (21, 22). In contrast, leptin transgene overexpression in the MPOA augmented a similar magnitude of energy expenditure without decreasing food intake. This selective increase in energy expenditure in the MPOA rAAV-lep group resulted in the smallest magnitude of weight loss, compared with the other groups receiving rAAV-lep in three caudal sites. This dichotomy of effects, attenuation of the age-related weight gain accompanied by unhindered intake, is not unprecedented. We have reported that the effects of icv rAAV-lep on FI and weight reduction were dose dependent; whereas higher doses of rAAV-lep increased energy expenditure and decreased food intake, and lower doses selectively augmented energy expenditure (10, 11, 19, 20).

We have recently observed a similar diminution in weight gain in association with increased energy expenditure without a decrease in FI in adult mice (Ueno, N., M. Dube, A. Katz, P. Kalra, and S. Kalra, unpublished data). In fact, a similar differential effect of leptin administration itself has been reported in wild-type rodents (39, 40, 41). It is possible that expression of leptin transgene in the ARC, PVN, and VMN attained a titer range capable of concomitantly increasing energy expenditure and decreasing energy intake, but the level of leptin expression in the MPOA increased to the range sufficient only to augment energy expenditure. Consequently, in conjunction with previous results (10, 11, 19, 20), one can suggest that the central effects of leptin on energy intake and expenditure are dose dependent and the energy expenditure response has a lower threshold of responsiveness to leptin feedback. Alternatively, it is also likely that leptin responsive elements in the MPOA exclusively enhance thermogenic energy expenditure. Indeed, recent evidence from tracing studies demonstrating a direct sympathetic outflow from MPOA to BAT along with the evidence of electrical stimulation of MPOA increasing BAT thermogenesis and firing rate of innervations in BAT are in line with this possibility (42, 43, 44). Also, in a preliminary assessment of rectal temperature for 3 d before death in the MPOA-injected rats, we observed a significant increase (36.6 ± 0.09 C) in rAAV-lep-injected rats over that in rAAV-UF5 controls (36.1 ± 0.11 C, P < 0.05, our unpublished data).

Apparently, blockade of the age-related weight gain and sustenance of low weight are due largely to reduced adiposity because serum leptin levels were reduced by 80–90% after rAAV-lep injection in four hypothalamic sites. Serum leptin levels are directly proportional to body fat mass (7, 45). We have previously reported that body fat mass and not lean muscle mass was suppressed in response to icv rAAV-lep injection in both wild-type lean (10, 12) and high-fat diet-induced obese rats (20). This suggests that leptin overexpression in any one of these four hypothalamic sites can sustain a drastically reduced fat mass for extended periods. Further, concomitant with marked reduction in serum leptin levels, FFA, the other metabolic index of adiposity, was also reduced by 40–52%, a finding similar to that seen after icv rAAV-lep injection (10, 11, 12, 20). Although not evaluated in this study, it is highly likely that reduced serum FFAs are also a consequence of reduced fat mass because PF rats with mildly reduced serum leptin failed to show a consistent decrease in serum FFAs. However, future analysis of lipid body composition of these four experimental groups will provide clear evidence for a relationship between fat loss and severely reduced serum leptin.

Another important new finding of the current study is that microinjection of extremely small amounts of rAAV-lep in discrete sites reduced serum insulin by 85–90% simultaneously with sustenance of normoglycemia. A similar response for extended period after icv rAAV-lep, in one case extending for up to 10 months (19), was seen in rats maintained on either normal rat chow or fed high-fat diet (10, 11, 20). Obviously, hyperinsulinemia, thought to be a consequence of insulin resistance in aging and high-fat diet- induced obesity (46, 47), was blocked by central leptin action. Our results also suggest that leptin transgene expression in hypothalamic sites facilitated insulin sensitivity at peripheral target sites. It remains to be determined whether apparent abolition of insulin resistance resulted from diminished adiposity itself (46) or whether it is a consequence of central leptin action on insulin secretion by pancreatic ß-cells (10, 11, 19, 20). We and others (35, 36, 37, 38) have shown that disruption of neural signaling in the VMN either by lesions or neurotoxins evoked hyperinsulinemia and obesity, which could not be blocked by peripheral or central leptin replacement, an observation invoking the VMN as one of the central sites exerting an inhibitory influence on pancreatic ß-cell function. We have recently observed that despite the low circulating insulin levels in rAAV-lep-treated mice, insulin secretion in response to glucose challenge was unimpaired (Ueno, N., M. Dube, A. Katz, P. Kalra, and S. Kalra, unpublished data), a finding in line with the possibility of an independent central inhibitory action of leptin on pancreatic ß-cell function.

Interestingly, we observed that regardless of the site of rAAV-lep microinjections, serum ghrelin levels were elevated by about 50% over those seen in the respective control groups. This stimulation of ghrelin secretion from the stomach and intestine (48, 49) is similar to that seen after icv administration of rAAV-lep in rats (20) and mice (Ueno, N., M. Dube, A. Katz, P. Kalra, and S. Kalra, unpublished data) maintained either on normal laboratory chow or high-fat diet. Ghrelin administration has been shown to stimulate feeding and promote adiposity (50, 51). Because fasting increases ghrelin secretion (50, 52), it has been suggested that ghrelin may be a peripheral signal to the hypothalamus to replenish energy stores through stimulation of feeding. However, despite the elevated serum ghrelin levels, food intake remained attenuated in rats expressing leptin transgene in the PVN, VMN, and ARC and was unaffected in rats expressing leptin transgene in the MPOA. Also, we observed that long-term reduced intake in PF rats failed to stimulate ghrelin secretion. Therefore, the mechanism(s) involved in stimulation of ghrelin secretion for extended periods and failure of elevated levels to stimulate feeding in rAAV-lep- microinjected rats and high-fat diet-induced obese rats after icv rAAV-lep injection (20) remain to be determined. Fasting also results in marked decrease in adipocyte leptin secretion concomitant with high ghrelin output (52). It is possible that extremely low circulating leptin levels, as in rAAV-lep- microinjected rats and not the moderately low leptin levels of PF rats, constitute a signal acting either centrally or peripherally to stimulate ghrelin secretion. If this is true, then it is highly likely that markedly diminished leptin secretion promoted ghrelin output in rats overexpressing leptin in hypothalamic sites. With respect to the inability of high circulating ghrelin levels to evoke feeding, one can suggest that central leptin exerted a profound restraining effect on feeding that could not be overcome by increased ghrelin signaling. The cellular and molecular bases of the central interplay of leptin and ghrelin in regulating ingestive behavior are the subject of future investigations.

We previously reported that administration of rAAV-lep either peripherally into ob/ob mice (18) or centrally into adult (10, 11) and prepubertal rats (19) down-regulated ARC NPY gene expression without affecting AgrP gene expression and up-regulated POMC gene expression in the ARC. This evidence supported a combined outcome of decreased orexigenic NPY and increased anorexigenic melanocortin signaling to participate in suppressing weight gain through reduced energy intake and/or increased energy expenditure. We proposed that in rats receiving icv rAAV-lep, increased leptin transgene expression induced locally in the ARC modulated the appetite regulating neuropeptidergic signals through paracrine and/or autocrine mechanisms (10, 11, 19, 20). In the current study, we observed that POMC gene expression was up-regulated by rAAV-lep microinjection in all four sites, and it was unaffected in PF rats. As seen before (10, 11, 19, 20), rAAV-lep was ineffective, but PF augmented the ARC AgrP mRNA expression.

Additionally, we observed that rAAV-lep injection in the ARC decreased NPY and increased POMC gene expression in association with a sustained decreased FI. A similar correlation with NPY and POMC gene expression and behavioral effects was observed after rAAV injection either intraventricularly (11, 19) or into the PVN and VMN. On the other hand, rAAV-lep microinjection in the MPOA decreased neither ARC NPY gene expression nor FI. Because NPY is a potent physiological orexigenic signal (5), it is highly likely that unimpeded availability and action of NPY in the ARC-PVN axis accounted for undiminished energy consumption in these rats.

Thus, with respect to the mode of action, whereas the proposal that leptin expression by autocrine/paracrine mode augmented POMC and diminished NPY gene expression locally in rats receiving rAAV-lep in the ARC may be valid (18, 19), a different mechanism is likely to operate in rats receiving rAAV-lep microinjection in the remote MPOA, PVN, and VMN. We propose that increased leptin transduced by neurons at these sites is transported axonally to POMC neurons in the ARC. Axonal transport of leptin to ARC targets is possible because neural projections from the PVN, VMN, and MPOA have been seen to innervate the ARC (5, 42, 43, 53, 54). Another possibility is intercellular communication by volume transmission involving leptin transport to the ARC through extracellular fluid (55). In any case, our results clearly demonstrate that blockade of weight gain and attenuation of FI by intracranial microinjection of rAAV-lep in the PVN and VMN and presumably in the MPOA may, in addition to activation of leptin receptor locally at these sites, also involve peptidergic pathways that emanate from the ARC and have been implicated in the hypothalamic control of appetite and energy expenditure (5, 6, 7, 34). A rigorous neuroanatomical mapping of the rAAV-lep targets in the hypothalamus is warranted.

In summary, these results demonstrate that microinjection of extremely small amounts of rAAV vector-encoding leptin into hypothalamic sites can inhibit the time-related weight gain in rats maintained on regular rat chow diet. The long-term weight gain suppression, attributable to a voluntary decreased energy intake and/or increased energy expenditure, is accompanied by suppression of adiposity and time-related hyperinsulinemia. In addition to rAAV-microinjection in the ARC in which paracrine/autocrine effects of leptin transgene overexpression may involve appetite and energy expenditure regulating neuronal cells, microinjection in distant PVN, VMN, and MPOA sites may also involve these neurons by axonal transport or volume transmission of the expressed leptin to receptive elements in the ARC and/or elsewhere in the hypothalamus. Overall, the current findings extend the results of previous investigations with icv rAAV-lep injection and demonstrate the existence of multiple leptin receptive sites in the hypothalamus. Our findings strengthen the emerging view (10, 11, 19, 20) that advocates central leptin gene therapy with a nonpathogenic and nonimmunogenic rAAV-vector (15, 16) as a viable therapeutic modality to suppress the time-related weight gain for long periods.


   Acknowledgments
 
We thank Dr. Nicholas Muzyczka, director of the Gene Therapy Center, for assistance in preparation of viral vectors and Ms. Sandra Clark for word processing assistance.


   Footnotes
 
This work was supported by NIH Grants DK-37273, HD-08634, and NS-32727.

These data were partially presented at the Meeting of the Society for Ingestive Behavior (SSIB), June 26–30, 2001, Philadelphia.

Abbreviations: AgrP, Agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; BW, body weight; FFA, free fatty acid; FI, food intake; GFP, green fluorescence protein; icv, intracerebroventricular; ISH, in situ hybridization; MPOA, medial preoptic area; NPY, neuropeptide Y; PF, pair fed; POMC, proopiomelanocortin; PVN, paraventricular nucleus; rAAV, recombinant adeno-associated virus; rAAV-lep, rAAV-vector encoding the leptin transgene; ROD, relative OD; UCP1, uncoupling protein-1; VMN, ventromedial nucleus.

Received May 13, 2002.

Accepted for publication July 24, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
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 Effects of rAAV-lep...
 References
 

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