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. Author manuscript; available in PMC: 2012 Jul 1.
Published in final edited form as: J Bone Miner Res. 2011 Jul;26(7):1506–1516. doi: 10.1002/jbmr.365

Hypothalamic Leptin Gene Therapy Prevents Weight Gain without Long-term Detrimental Effects on Bone in Growing and Skeletally Mature Female Rats

UT Iwaniec 1,*, S Boghossian 2, CH Trevisiol 3, TJ Wronski 4, RT Turner 1, SP Kalra 2
PMCID: PMC3129999  NIHMSID: NIHMS302212  PMID: 21328617

Abstract

Hypothalamic leptin gene therapy normalizes the mosaic skeletal phenotype of leptin-deficient ob/ob mice. However, it is not clear whether increased hypothalamic leptin alters bone metabolism in animals already producing the hormone. The objective of this study was to evaluate the long duration effects of recombinant adeno-associated virus-rat leptin (rAAV-Lep) hypothalamic gene therapy on weight gain and bone metabolism in growing and skeletally mature leptin-replete female Sprague-Dawley rats. Rats were either unoperated or implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained on standard rat chow fed ad lib for either 5 or 10 weeks (starting at 3 months of age) or 18 weeks (starting at 9 months of age). Tibiae, femora, or lumbar vertebrae were analyzed by microcomputed tomography and/or histomorphometry. In comparison to age-matched rAAV-GFP rats, rAAV-Lep rats maintained a lower body weight for the duration of studies. At 5 weeks post-vector administration, rAAV-Lep rats had lower cancellous bone volume and bone marrow adiposity but higher osteoblast perimeter compared to non-operated controls. However, these values did not differ between the two groups at 10 weeks post-vector administration. Differences in cancellous bone volume and architecture were not detected between the rAAV-Lep and rAAV-GFP groups at either time point. Also, rAAV-Lep had no negative effects on bone in the 9-month old skeletally mature rats at 18 weeks post-vector administration. We hypothesize that the transient reductions in bone mass and bone marrow adiposity at 5 weeks post-vector administration were due to hypothalamic surgery. We conclude that increased hypothalamic leptin, sufficient to prevent weight gain, has minimal specific effects (rAAV-Lep versus rAAV-GFP) on bone metabolism in normal female rats.

Keywords: osteoporosis, μCT, obesity, histomorphometry, osteoblasts, osteoclasts

INTRODUCTION

Produced primarily by adipocytes, leptin contributes to the regulation of energy homeostasis and adaptation to starvation by functioning as a messenger in a feedback loop between adipose tissue and the hypothalamus. Circulating leptin is transported into the hypothalamus where it acts on specific leptin receptors to regulate energy homeostasis (1-6). Although blood leptin levels are generally proportional to fat mass and increase with obesity, obesity in humans is associated with a dysfunction in leptin signaling. This dysfunction may be due to hypothalamic leptin insufficiency caused by impaired leptin transport across the blood brain barrier, end organ resistance to the hormone (2,6), or counter regulation (7).

In addition to its role in energy homeostasis, leptin plays a role in reproduction (8), angiogenesis (9), hematopoiesis (10), immunity (11), gastrointestinal function (12), and bone growth and turnover (13-16). Leptin exerts its various effects through LepR (also referred to as ObR), a receptor sharing a high degree of homology to members of the class I cytokine receptor family, either indirectly through the hypothalamus or directly through receptors on various cell types (17). The distribution of leptin receptors among peripheral tissues is widespread (18) and includes mesenchymal stem cells, adipocytes, chondrocytes, and osteoblasts (19-21).

Leptin is reported to be an important regulator of bone metabolism. However, the precise actions and physiological relevance of leptin on the skeleton are uncertain; the controversies relate to whether the hormone is bone anabolic or catabolic, and whether its actions are direct or central nervous system (CNS)-mediated. Leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice exhibit a mosaic skeletal phenotype. Compared to WT mice, the leptin signaling-deficient mutant mice have a relative increase in cancellous bone volume at selected sites (22,23). However, they exhibit shorter long bones with reduced cortical bone mass and reductions in whole body bone mineral content and bone mineral density (3,14,22,24-28).

These findings have been interpreted by some investigators as evidence that leptin has site-specific bone anabolic and bone catabolic effects (13,23,29).

The putative bone anabolic actions of leptin may involve a direct pathway requiring the binding of leptin to its receptors on cartilage and bone cells. Leptin, for example, has direct effects on osteoblasts to regulate their expression of RANKL and OPG (30). Leptin increases proliferation and extracellular matrix mineralization by cultured human osteoblasts (31,32). In the growth plate, leptin enhances chondrocyte proliferation but slows their terminal differentiation and reduces chondrocyte apoptosis (33,34),

Additionally, leptin has the potential to affect bone cells via indirect pathways, including a hypothalamic relay. Hypothalamic administration of leptin was reported to decrease bone formation and cancellous bone volume in mouse lumbar vertebrae (35). These findings have been interpreted as evidence for leptin-induced CNS regulation of bone metabolism in the growing and adult skeleton. Furthermore, studies using β-adrenergic receptor agonists and antagonists suggested that the antiosteogenic actions of leptin in rodents are mediated through the well established ability of leptin to increase sympathetic tone (36,37). The CNS model advocating antiosteogenic actions of leptin has been widely embraced and research is in progress to test the efficacy of β-adrenergic receptor antagonists to increase cancellous bone mass in the adult skeleton by stimulating bone formation (38-40).

In spite of much enthusiasm for the concept of leptin as an important physiological regulator of bone metabolism, a substantial body of data remains to be explained by models advocating either direct anabolic actions of leptin on bone cells or indirect CNS-mediated antiosteogenic actions. As discussed above, the antiosteogenic actions of hypothalamic leptin are reported to be due to increased sympathetic tone. However, the dramatic skeletal phenotypes observed in leptin- and leptin receptor-deficient mice are not duplicated by sympathectomy in rats (41,42). Also, we have shown that hypothalamic leptin gene therapy alters bone architecture in growing ob/ob mice to match wild type mice by decreasing the abnormally high vertebral cancellous bone volume while increasing bone growth, serum osteocalcin levels and overall bone mass, without increasing serum leptin levels (14,43). Thus, the central actions of the hormone are sufficient to account for both the putative anabolic and antiosteogenic actions of leptin.

To date, most studies investigating the role of hypothalamic leptin on bone metabolism have been performed in leptin signaling-deficient mice. If hypothalamic leptin is an important regulator of bone metabolism, then changes in hypothalamic leptin levels should have effects on bone metabolism in animals with circulating levels of the hormone. Therefore, the objective of this study was to evaluate the effects of centrally administered recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy on bone mass and architecture in growing (3-month-old) and skeletally mature (9-month-old) leptin-replete female Sprague-Dawley rats.

MATERIALS AND METHODS

Animals

Three-month-old and 9-month-old female Sprague Dawley rats obtained from Harlan (Indianapolis, IN) were used in the experiments. The rats were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Florida (Gainesville, FL). The rats were housed individually in a temperature (21-23°C) and light (lights on 8:00 – 18:00 hours)-controlled room at the McKnight Brain Institute under specific pathogen-free conditions. Food and water were available ad libitum to all animals.

Experimental Protocols

Two separate experiments were conducted in growing 3-month-old female rats. In both experiments, treatment groups (5-6 rats/group) included: 1) non-treated control rats, 2) rAAV-GFP-treated rats, and 3) rAAV-Lep-treated rats. In the first experiment, the rats were sacrificed at 5 weeks post-vector administration. In the second experiment, they were sacrificed at 10 weeks post-vector administration. A third experiment was performed in skeletally mature 9-month-old female rats. Treatment groups (8 rats/group) included: 1) baseline control rats, 2) non-treated control rats, 3) rAAV-GFP-treated rats, and 4) rAAV-Lep-treated rats. In experiment 3, the fluorochrome calcein (15 mg/kg; Sigma Chemical, St Louis, MO) was administered 14 and 5 days prior to sacrifice to label mineralizing bone for assessment of rates of bone formation. The baseline group was sacrificed at time of vector administration and treatment groups were sacrificed at 18 weeks post-vector administration.

Construction and packaging of rAAV vectors

The rAAV-Lep and rAAV-GFP vectors were constructed and packaged as described elsewhere (44). In brief, the vector pTR-CBA-Ob EcoRI fragment of pCR-rOb containing rat leptin cDNA was subcloned into rAAV vector plasmid pAAVβGEnh after deleting the EcoRI fragment carrying the β-glucoronidase cDNA sequence (45-48). The control vector, rAAV-GFP, was similarly constructed to encode the GFP gene (46-48).

Vector administration

The rats were anesthetized with ketamine (100 mg/kg) and xylazine (15 mg/kg) and stereotaxically implanted with a permanent cannula (Plastics One, Roanoke, VA) in the 3rd cerebroventricle of the hypothalamus. After 1 week of recovery, the rats were weight-matched. In experiments 1 and 2 (3-month-old rats), the rats were injected intracerebroventricularly (icv) with 7 μL of either rAAV-Lep (4.6 × 1013 particles/mL) or rAAV-GFP (4.6 × 1013 particles/mL). In experiment 3 (9-month-old rats), they were injected icv with 5 μL of either rAAV-lep (1.44 × 1013 particles/ml) or rAAV-GFP (1.65 × 1013 particles/ml). Body weight and food intake were monitored weekly for the duration studies.

Tissue Collection and Analyses

All rats were weighed prior to sacrifice. Femora and 3rd lumbar vertebrae were excised for μCT analysis. Tibiae were excised for histomorphometry. In experiment 3, abdominal white adipose tissue (WAT) was also dissected and weighed and blood samples were collected via the abdominal aorta in anesthetized animals prior to sacrifice and serum stored frozen at −20°C for hormone analyses.

Serum Leptin

Serum leptin from experiment 3 was assayed in duplicate using the rat leptin radioimmunoassay kit from Linco Research, Inc. (St Louis, MO) according to the manufacturer’s instructions (49). The assay sensitivity was 0.5 ng/mL.

μCT analysis

In experiments 1 and 2, μCT was used for nondestructive three-dimensional evaluation of bone mass and architecture. Femora and lumbar vertebrae were scanned using a Scanco μCT40 scanner (Scanco Medical AG, Basserdorf, Switzerland) at a voxel size of 20 × 20 × 20 μm. Cortical bone was evaluated at the femoral midshaft and cancellous bone in the distal femoral metaphysis. For the femoral midshaft, 20 slices (400 μm) were evaluated and total cross-sectional tissue volume (cortical and marrow volume, mm3), cortical volume (mm3), marrow volume (mm3) and cortical thickness (μm) were measured. For the femoral metaphysis, 150 slices (3 mm) of bone were measured and the volume of interest included secondary spongiosa only. Analysis of the lumbar vertebrae included the entire region of secondary spongiosa between the cranial and caudal growth plates. Cancellous bone measurements in the femur and lumbar vertebra included cancellous bone volume/tissue volume (%), trabecular number (mm−1), trabecular thickness (μm) and trabecular separation (μm) (50).

Histomorphometry

For histomorphometric evaluation of cancellous bone, proximal tibiae were dehydrated in a graded series of ethanol and xylene, and embedded undecalcified in modified methyl methacrylate as described (51). Longitudinal sections (5 μm thick) were cut with a vertical bed microtome (Leica 2065) and affixed to slides. One section/animal was stained according to the Von Kossa method with a tetrachrome counter stain (Polysciences, Warrington, PA) and used for assessing bone area and cell-based measurements. In experiments 1 and 2, measurements were performed in a standard sampling site, 0.5 mm distal to the growth plate, using the OsteoMeasure System (OsteoMetrics, Inc., Atlanta, GA). Measurements consisted of bone area/tissue area (%) and the derived architectural indices of trabecular number (mm−1), trabecular thickness (μm), and trabecular separation (μm). Osteoblast and osteoclast perimeters were measured and expressed as % of total bone perimeter. Bone marrow adiposity (adipocyte area/tissue area, %), adipocyte density (#/mm2), and adipocyte size (μm2) were determined as described (52). In addition, the mean width of the proximal tibia growth plate, width of the hypertrophic zone, and the proliferative + resting zones were determined as described (53) with modifications. In the present studies, measurements were performed on methyl methacrylate embedded tissue sections with growth plate cartilage cells stained using tetrachrome.

Fluorochrome-based indices of bone formation were evaluated in experiment 3 using unstained sections illuminated with ultraviolet light. Mineralizing perimeter was determined as percentage of cancellous bone perimeter covered with double fluorochrome labels + ½ single labels. Mineral apposition rate was calculated as the mean distance between two fluorochrome markers that comprise a double label divided by the number of days (9) between label administrations. Bone formation rate was calculated using a bone perimeter referent. All measured and derived data were generated according to standardized methods, derivations, and nomenclature (54).

Statistical Analysis

A one-way ANOVA followed by a Bonferroni post-hoc test was used to evaluate differences among treatment groups. If ANOVA assumptions of homogeneity of variance were not met, a Kruskal-Wallis followed by a Tamhane post hoc test was used (55). Differences were considered significant at p<0.05. All data are reported as mean ± SE.

RESULTS

Experiment 1: Effects of icv rAAV-Lep on body weight and bone in 3-month-old rats at 5 weeks post-vector administration

Body weight was lower in rAAV-Lep rats compared to non-operated control rats and rAAV-GFP rats at 5 weeks post-vector administration (Figure 1).

Figure 1.

Figure 1

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Effects of hypothalamic leptin gene therapy on body weight (Experiment 1). Three-month-old rats were either unoperated or implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP and maintained on standard rat chow fed ad lib for 5 weeks. aDifferent from non-treated control at termination of treatment, P < 0.05; bDifferent from rAAV-GFP control at termination of treatment, P < 0.05.

The effects of hypothalamic rAAV-Lep gene therapy on bone mass, architecture, and cellular endpoints at 5 weeks post-vector administration are shown in Table 1.

Table 1.

Effects of hypothalamic rAAV-Lep gene therapy on cancellous and cortical bone at 5 weeks post-vector administration (Experiment 1).

Endpoint Non-Treated rAAV-GFP rAAV-LEP ANOVA
 P<
μCT
3rd lumbar vertebra (cancellous bone)
 Bone volume/tissue volume (%) 40.2 ± 1.4 36.1 ± 1.6 31.3 ± 2.1a 0.010
 Trabecular number (mm−1) 4.1 ± 0.1 4.1 ± 0.1 4.0 ± 0.1 0.524
 Trabecular thickness (μm) 94 ± 2 87 ± 2 81 ± 2a 0.003
 Trabecular separation (μm) 234 ± 7 237 ± 8 249 ± 8 0.374
Distal femur metaphysis (cancellous bone)
 Bone volume/tissue volume (%) 27.9 ± 5.0 17.5 ± 4.0 11.4 ± 1.7a 0.021
 Trabecular number (mm−1) 4.1 ± 0.4 3.0 ± 0.5 2.4 ± 0.2a 0.016
 Trabecular thickness (μm) 96 ± 9 85 ± 7 71 ± 3a 0.048
 Trabecular separation (μm) 251 ± 25 450 ± 142 446 ± 35a 0.021
Midshaft femur (cortical bone)
 Cross-sectional volume (mm3) 3.4 ± 0.1 3.5 ± 0.2 3.5 ± 0.1 0.863
 Cortical volume (mm3) 2.0 ± 0.0 1.9 ± 0.1 1.8 ± 0.1 0.364
 Marrow volume (mm3) 1.4 ± 0.0 1.5 ± 0.1 1.6 ± 0.1 0.092
 Cortical thickness (μm) 620 ± 5 580 ± 8a 549 ± 1a 0.005
Histomorphometry
Proximal tibia metaphysis (cancellous bone)
 Bone area/tissue area (%) 24.3 ± 2.3 17.5 ± 2.5 14.9 ± 1.3a 0.016
 Trabecular number (mm−1) 4.1 ± 0.3 3.3 ± 0.4 2.9 ± 0.2a 0.023
 Trabecular thickness (μm) 58 ± 2 52 ± 1 52 ± 1 0.085
 Trabecular separation (μm) 187 ± 16 277 ± 55 302 ± 18 0.066
 Osteoclast perimeter/bone perimeter (%) 1.6 ± 0.2 1.7 ± 0.7 1.4 ± 0.3 0.890
 Osteoblast perimeter/bone perimeter (%) 5.2 ± 0.5 9.9 ± 1.2a 11.1 ± 1.9a* 0.021
 Adipocyte area/tissue area (%) 6.5 ± 1.3 0.6 ± 0.2a 0.2 ± 0.1a 0.005
 Adipocyte density (#/mm2) 151 ± 22 21 ± 8a 4 ± 2a 0.004
 Adipocyte size (μm2) 423 ± 29 461 ± 165 685 ± 231 0.677
Proximal tibia growth plate
 Growth plate thickness (μm) 142 ± 5 130 ± 3 124 ± 4a 0.018
 Hypertrophic zone thickness (μm) 63 ± 4 55 ± 3 51 ± 2a* 0.049
 Proliferating and resting zone thickness (μm) 79 ± 5 76 ± 4 73 ± 4 0.627
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Mean ± SE

a

Different from non-treated control, P<0.05

a*

P<0.1

Vertebral cancellous bone (μCT)

Bone volume/tissue volume and trabecular thickness were lower in rAAV-Lep rats compared to non-operated control rats. Significant differences among treatment groups were not detected for trabecular number or trabecular separation. Significant differences between rAAV-GFP and rAAV-Lep rats were not detected for any of the vertebral endpoints evaluated.

Distal femur cancellous bone (μCT)

Bone volume/tissue volume, trabecular number, and trabecular thickness were lower and trabecular separation was higher in the rAAV-Lep rats compared to non-operated control rats. Significant differences between rAAV-GFP and rAAV-Lep rats were not detected for any of the femoral cancellous bone endpoints measured.

Midshaft femur cortical bone (μCT)

Significant differences among treatment groups were not detected for cross-sectional volume, cortical volume, or marrow volume. Cortical thickness was lower in rAAV-Lep and rAAV-GFP rats compared to non-operated controls. Significant differences between rAAV-GFP and rAAV-Lep rats were not detected for cortical thickness.

Proximal tibia cancellous bone (histomorphometry)

Bone area/tissue area was lower in rAAV-Lep rats compared to non-operated controls; the lower value was associated with lower trabecular number. Significant differences in trabecular thickness or trabecular separation were not detected among groups. Significant differences in osteoclast perimeter/bone perimeter, an index of bone resorption, were likewise not detected among treatment groups. Osteoblast perimeter/bone perimeter, an index of bone formation, was higher in rAAV-GFP rats compared to non-operated controls, and tended to be higher (P = 0.073) in the rAAV-Lep rats compared to non-operated controls. Bone marrow adiposity (adipocyte area/tissue area and adipocyte density) was lower in the two surgery groups than in the non-operated controls. Significant differences in adipocyte size were not detected among groups. Significant differences between rAAV-GFP and rAAV-Lep rats were also not detected for any of the endpoints evaluated.

Total growth plate thickness was lower in rAAV-Lep rats compared to non-operated controls. There was also a strong tendency (P = 0.051) for hypertrophic zone thickness to be lower in the rAAV-Lep rats compared to controls. Significant differences in the thickness of the proliferative + resting zone were not detected between rAAV-Lep rats and non-operated controls. Significant differences between rAAV-Lep rats and rAAV-GFP rats were not detected for any of the growth plate endpoints evaluated. Representative micrographs illustrating differences in bone marrow adiposity and growth plate histomorphometry are shown in Figure 2.

Figure 2.

Figure 2

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Photomicrographs showing representative histology in proximal tibia at 5 weeks post-vector administration (Experiment 1) in a non-treated (unoperated) control rat (A), rAAV-GFP-treated rat (B) and AAV-Lep-treated rat (C). Please note the presence of numerous adipocytes in the representative unoperated control rat tibia and the near absence of adipocytes in the rAAV-GFP and rAAV-Lep rat tibiae. Also, please note the overall normal appearing but slightly thinner growth plate in the rAAV-Lep rats.

Experiment 2: Effects of icv rAAV-Lep on body weight and bone in 3-month-old rats at 10 weeks post-vector administration

Body weight was lower in rAAV-Lep rats compared to non-operated control rats as well as rAAV-GFP control rats at 10 weeks post-vector administration (Figure 3).

Figure 3.

Figure 3

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Effects of hypothalamic leptin gene therapy on body weight (Experiment 2). Three-month-old rats were either unoperated or implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP and maintained on standard rat chow fed ad lib for 10 weeks. aDifferent from non-treated control at termination of treatment, P < 0.05; bDifferent from rAAV-GFP control at termination of treatment, P < 0.05.

The effects of hypothalamic rAAV-Lep gene therapy on bone mass, architecture, and cellular endpoints at 10 weeks post-vector administration are shown in Table 2.

Table 2.

Effects of hypothalamic rAAV-Lep gene therapy on cancellous and cortical bone at 10 weeks post-vector administration (Experiment 2).

Endpoint Non-Treated rAAV-GFP rAAV-LEP ANOVA
 P<
μCT
3rd lumbar vertebra (cancellous bone)
 Bone volume/tissue volume (%) 44.4 ± 2.1 37.6 ± 2.6 40.9 ± 6.4 0.231
 Trabecular number (mm−1) 4.4 ± 0.1 4.5 ± 0.2 4.0 ± 0.2 0.082
 Trabecular thickness (μm) 97 ± 2 83 ± 2a 97 ± 2b 0.003
 Trabecular separation (μm) 213 ± 7 211 ± 11 237 ± 11 0.150
Distal femur metaphysis (cancellous bone)
 Bone volume/tissue volume (%) 24.7 ± 2.4 21.2 ± 2.7 21.1 ± 3.9 0.677
 Trabecular number (mm−1) 4.0 ± 0.3 3.4 ± 0.3 3.5 ± 0.4 0.389
 Trabecular thickness (μm) 88 ± 3 87 ± 4 81 ± 1 0.513
 Trabecular separation (μm) 259 ± 25 313 ± 28 306 ± 39 0.482
Midshaft femur (cortical bone)
 Cross-sectional volume (mm3) 3.5 ± 0.1 3.7 ± 0.1 3.6 ± 0.1 0.300
 Cortical volume (mm3) 2.2 ± 0.1 2.2 ± 0.0 2.2 ± 0.1 0.940
 Marrow volume (mm3) 1.3 ± 0.1 1.5 ± 0.1 1.4 ± 0.0 0.066
 Cortical thickness (μm) 673 ± 6 659 ± 10 659 ± 12 0.578
Histomorphometry
Proximal tibia metaphysis (cancellous bone)
 Bone area/tissue area (%) 18.6 ± 2.1 17.5 ± 2.8 21.1 ± 3.0 0.606
 Trabecular number (mm−1) 3.5 ± 0.3 3.5 ± 0.3 3.4 ± 0.4 0.955
 Trabecular thickness (μm) 53 ± 3 49 ± 4 61 ± 4 0.090
 Trabecular separation (μm) 240 ± 29 247 ± 28 264 ± 59 0.923
 Osteoclast perimeter/bone perimeter (%) 1.6 ± 0.2 1.5 ± 0.4 2.4 ± 1.1 0.637
 Osteoblast perimeter/bone perimeter (%) 5.8 ± 0.7 5.9 ± 0.6 6.7 ± 1.0 0.690
 Adipocyte area/tissue area (%) 3.7 ± 0.9 4.9 ± 0.6 5.0 ± 1.7 0.719
 Adipocyte density (#/mm2) 92 ± 22 123 ± 13 110 ± 27 0.653
 Adipocyte size (μm2) 383 ± 26 396 ± 24 436 ± 86 0.804
Proximal tibia growth plate
 Growth plate thickness (μm) 115 ± 16 142 ± 5 119 ± 8 0.204
 Hypertrophic zone thickness (μm) 42 ± 11 55 ± 2 38 ± 7 0.287
 Proliferating and resting zone thickness (μm) 76 ± 6 76 ± 4 81 ± 2 0.511
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Mean ± SE

a

Different from non-treated control, P<0.05.

b

Different from rAAV-GFP, P<0.05

Vertebral cancellous bone (μCT)

Significant differences among treatment groups were not detected for bone volume/tissue volume, trabecular number, or trabecular separation. Trabecular thickness was lower in rAAV-GFP rats compared to non-treated controls and rAAV-Lep rats. Significant differences between rAAV-GFP and rAAV-Lep rats were not detected for bone volume/tissue volume, trabecular number, or trabecular separation.

Distal femur cancellous bone (μCT)

Significant differences among treatment groups were not detected for any of the endpoints measured (bone volume/tissue volume, trabecular number, trabecular thickness, or trabecular separation).

Midshaft femur cortical bone (μCT)

Significant differences among treatment groups were not detected for any of the endpoints measured (cross-sectional volume, cortical volume, marrow volume, or cortical thickness).

Proximal tibia cancellous bone (histomorphometry)

Significant differences among treatment groups were not detected for any of the bone or cellular endpoints measured (bone area/tissue area, trabecular number, trabecular thickness, trabecular separation, osteoclast perimeter/bone perimeter, osteoblast perimeter/bone perimeter, adipocyte area/tissue area, adipocyte density, or adipocyte size). Furthermore, significant differences among groups were not detected for total growth plate thickness, hypertrophic zone thickness, or proliferative + resting zone thickness.

Experiment 3: Effects of icv rAAV-Lep on body weight and bone in 9-month-old rats at 18 weeks post-vector administration

Body weight was lower in rAAV-Lep rats compared to rAAV-GFP rats at 18 weeks post-vector administration (Figure 4). Abdominal WAT mass and serum leptin levels were also lower in the rAAV-Lep rats compared to non-treated control rats as well as rAAV-GFP rats.

Figure 4.

Figure 4

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Effects of hypothalamic leptin gene therapy on terminal body weight (A), white adipose tissue mass (B), and serum leptin levels (C) (Experiment 3). Nine month-old rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP and maintained on standard rat chow fed ad lib for 18 weeks. aDifferent from non-treated control at termination of treatment, P < 0.05; bDifferent from rAAV-GFP control at termination of treatment, P < 0.05.

The effects of age and hypothalamic rAAV-Lep gene therapy on bone mass and architecture and dynamic bone histomorphometry in the proximal tibia metaphysis are shown in Table 3.

Table 3.

Effects of hypothalamic rAAV-Lep gene therapy on cancellous bone histomorphometry at 18 weeks post-vector administration (Experiment 3).

Endpoint Baseline Non-Treated rAAV-GFP rAAV-LEP ANOVA
 P<
Histomorphometry
Proximal tibia metaphysis (cancellous bone)
 Bone Area/tissue area (%) 25.5 ± 1.4 18.8 ± 1.7a* 13.4 ± 1.6a 17.7 ± 2.1a 0.001
 Trabecular number (mm−1) 4.5 ± 0.6 3.2 ± 0.3a 2.6 ± 0.3a 3.4 ± 0.3a 0.001
 Trabecular thickness (μm) 57 ± 3 61 ± 3 49 ± 3.6a* 52 ± 2 0.029
 Trabecular separation (μm) 171 ± 11 289 ± 52 342 ± 57a 263 ± 32 0.046
 Mineralizing perimeter/bone perimeter (%) 6.7 ± 1.1 4.8 ± 0.7 4.7 ± 1.0 6.9 ± 1.3 0.313
 Mineral apposition rate (μm/d) 0.92 ± 0.05 0.50 ± 0.05 0.84 ± 0.06 0.86 ± 0.06 0.736
 Bone formation rate (μm2/μm/d) 24 ± 5 16 ± 3 14 ± 1 22 ± 5 0.200
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Mean ± SE

a

Different from baseline, P<0.05

a*

P<0.1.

b

Different from non-treated control, P<0.05

c

Different from rAAV-GFP, P<0.05

Proximal tibia cancellous bone histomorphometry

Bone area/tissue area tended to be lower (P = 0.064) in non-treated control rats and was lower in rAAV-GFP and rAAV-Lep rats at 18 weeks post-vector administration compared to rats sacrificed at baseline. Trabecular number was lower in all 3 treatment groups while trabecular separation was higher in only the rAAV-GFP group compared to baseline. Significant differences in trabecular thickness were not detected between any of the treatment groups and the baseline group. Significant differences between rAAV-GFP and rAAV-Lep rats were not detected for any of the static histomorphometric endpoints measured. Significant differences between baseline and treatment groups or among treatment groups at 18 weeks post-vector administration were not detected for any of the dynamic endpoints measured (mineralizing perimeter/bone perimeter, mineral apposition rate, or bone formation rate).

DISCUSSION

As expected (44-47,56-63), hypothalamic leptin gene therapy in 3-month-old rats was effective in decreasing body weight gain for the duration of studies. At 5 weeks post-vector administration, the proximal tibial growth plate was thinner, cancellous bone volume and adiposity were reduced and osteoblast perimeter was increased in rAAV-Lep-treated rats compared to non-operated control rats. However, these endpoints no longer differed between these two groups at 10 weeks post-vector administration. Differences in growth plate histology, bone mass, osteoclast or osteoblast perimeter, or bone marrow adiposity were not detected between rats administered rAAV-Lep or the control vector, rAAV-GFP, at either 5 or 10 weeks post-vector administration. Similarly, hypothalamic leptin gene therapy did not accelerate age-related bone loss and differences in bone mass or bone formation were not detected between 9-month-old rats administered rAAV-Lep or the control vector at 18 weeks post-vector administration. Taken together, these findings indicate that introduction of the leptin gene into the hypothalamus prevented weight gain but had few specific long-term effects on bone in rats capable of producing leptin.

Dietary obesity may occur as a result of leptin resistance by target cells in the hypothalamus. Alternatively, reduced leptin entry across the blood brain barrier, despite chronic systemic hyperleptinemia, may result in hypothalamic leptin insufficiency (29,47,57,58,60,61,64-70). Our current and prior studies support the latter mechanism. Central leptin gene therapy has been shown to be effective in maintaining lower body weight for at least 86 weeks (longest duration evaluated) in rats (57,60) and dramatically increasing lifespan in ob/ob mice (58). These findings indicate that leptin continues to be produced long-term and is biologically active in the hypothalamus following a single administration of the leptin transgene (56,58,62,63).

Observational studies in women suggest that leptin plays a physiological role in regulating skeletal mass. Circulating leptin levels have been reported to correlate positively 1) with bone area in premenarcheal females, 2) with bone mass in pre- and postmenopausal women, 3) with reduced levels of biochemical markers of bone turnover, and 4) with reductions in vertebral fractures in postmenopausal women (71-74). Others report no associations (75-77) or even negative associations between circulating leptin and bone mass (78). The reasons for these discrepancies are unclear. However, leptin levels are influenced by a variety of cytokines, hormones, and growth factors, including estrogen, testosterone, insulin, glucocorticoids, tumor necrosis factor-α, and interleukin 1, each of which have independent effects on bone metabolism (79-82). In addition, it is possible that hypothalamic leptin levels are more important to bone metabolism than circulating levels, weakening the association between systemic leptin and bone.

Short-duration (4-week) hypothalamic administration of human leptin by continuous infusion was reported to reduce bone formation and vertebral cancellous bone mass in ob/ob as well as WT mice (35). As a consequence, Ducy et al. (35) postulated that it is not high circulating leptin levels that lead to increased bone mass in obese individuals but a state of resistance to the biological effects of leptin, similar to insulin resistance in type II diabetes, that accounts for the putative protective effect of obesity on bone in humans. The results of the present study in growing and skeletally mature rats do not support this interpretation. Increasing hypothalamic leptin levels in normal rats decreased weight gain but did not alter bone mass or architecture over the long-term. The present results are in agreement with recent studies in which we showed that large (up to 7-fold) changes in circulating levels of leptin over the physiological range have no effect on bone formation in skeletally mature female rats (83).

Although studies in leptin signaling-deficient rodents have greatly enhanced our understanding of the functions of leptin, the inability to produce leptin (ob/ob mice) or respond to leptin (db/db mice and fa/fa rats) is rare and genetic models for leptin signaling deficiency in mice and rats and leptin signaling deficiency in humans results in severe immune system defects, hypogonadism, hyperinsulinemia, hyperparathyroidism and growth hormone resistance, conditions known to influence bone metabolism (2,6,84-86). Thus, to understand the physiological role of leptin, it is important to determine the effects of changes in leptin levels on bone metabolism in normal animals. Studies contrasting the skeletal response to leptin in ob/ob and normal WT mice suggest that leptin has a pivotal but predominantly permissive (on/off) effect on bone. Whereas pronounced skeletal abnormalities of ob/ob mice are corrected by peripheral administration of leptin, the hormone was reported to have little effect on the skeleton of WT mice (22). Thus, leptin may be required for optimal bone growth and the skeletal changes in ob/ob mice treated with leptin reflect normalization of leptin signaling (14). Our findings demonstrating a lack of an effect of hypothalamic leptin gene therapy on bone mass in normal growing and skeletally mature rats supports this hypothesis.

Interpretation of short-duration studies involving hypothalamic administration of leptin is complicated by surgery effects, and what has been interpreted in the literature as a CNS-mediated regulatory function of leptin on bone may in part represent the interaction between the administered leptin and coexisting surgery-induced inflammation. Leptin, in addition to its role in bone metabolism, is an important immune modulator (11,87). Leptin deficiency has been shown to confer resistance to inflammation-driven bone loss (88,89). Implantation of cannulae into the hypothalamus involves highly invasive surgery, including penetration of the calvarium and brain tissue. This surgery results in inflammation which may be responsible for the transient changes observed in the present study at 5 weeks post-vector administration. Inclusion of a sham-operated group to control for surgery, while essential, may not always adequately control for inflammation-driven changes (89). This is because the skeletal response would be determined by the combined effects of two factors, the level of leptin and surgery-induced inflammation. As a consequence, animals with high leptin levels would be expected to exhibit more surgery-induced bone loss than leptin-deplete animals. It is notable that in the present study only the rAAV-Lep rats differed significantly from non-operated controls at 5 weeks post-vector administration and that bone mass and growth plate measurements in both rAAV-GFP and rAAV-Lep intervention groups returned to normal by 10 weeks post-surgical intervention. Our long-duration (10-week and 18-week) hypothalamic leptin gene therapy studies are much less impacted by transient surgical effects and do not support a major regulatory role for leptin in bone metabolism in leptin-replete rodents. This absence of skeletal response is reproducible. A similar hypothalamic leptin gene therapy-induced decrease in body weight gain, WAT, and serum leptin with no significant effect on bone mass and architecture was observed at 10 weeks post-vector administration in a separate experiment in ovariectomized rats (data not shown).

Severe caloric restriction is associated with decreased peripheral fat depots but an increase in bone marrow adiposity (90). Hypothalamic leptin administration resulted in rapid (within 4-5 days) decreases in peripheral fat as well as bone marrow fat in ob/ob mice (91,92). In the present study, we observed a dramatic decrease in bone marrow adiposity at 5 weeks post-vector administration but this was associated with the surgery; no significant leptin-specific effect was noted. Nevertheless, by 10 weeks post-surgery bone marrow adiposity returned to the levels observed in untreated rats in both the rAAV-Lep and rAAV-GFP groups, indicating that increased hypothalamic leptin gene expression, while consistently decreasing peripheral white adipose tissue, has no long-term effect on bone marrow adiposity in growing leptin-replete rats.

The present studies investigating the skeletal response to leptin differ markedly from studies in leptin-deficient ob/ob mice showing hypothalamic leptin to be antiosteogenic (35,93). Our studies were performed in leptin-replete rats, were of longer duration, involved administration of rat leptin into the hypothalamus via gene therapy, evaluated cortical as well as cancellous skeletal sites, and importantly, included a non-operated control group to assess potential adverse effects of surgery. We cannot rule out the possibility that species differences or differences in the method used to deliver leptin into the hypothalamus are responsible for the observed inter-study differences in the skeletal response to hypothalamic leptin. Species differences in the skeletal response to leptin have been reported. In contrast to mice, reduced leptin signaling is not associated with high bone mass in rats (85,94) or in humans (86,95,96). However, similar effects on weight are observed in mice and rats whether the gene for leptin or the leptin peptide is delivered into the hypothalamus. Additionally, we reported profound effects of hypothalamic leptin gene therapy on cancellous bone mass in lumbar vertebrae as well as total bone mass and femoral length of growing leptin-deficient ob/ob mice (14). Specifically, hypothalamic leptin gene therapy in ob/ob mice resulted in a decrease in cancellous bone mass from abnormally high to normal and an increase in femoral length and mass from abnormally low to normal.

In conclusion, our results in growing and skeletally mature female rats support previous studies in rodents that hypothalamic leptin gene therapy confers long-term benefit for weight control. However, our findings in the leptin-replete animals are not in agreement with the hypothesis that increased hypothalamic leptin confers either beneficial or detrimental effects on bone metabolism. The studies provide further support for the concept that leptin acts as a permissive (on/off) factor to ensure adequate energy availability to support bone growth.

Acknowledgements

This work was supported by grants from the National Institute of Health to U.T. Iwaniec (AR 054609), R.T. Turner (AA 011140), and S.P. Kalra (DK 37273 and 27372) and a grant from the Department of Defense (W81XWH-04-1-0701) to U.T. Iwaniec.

Footnotes

Current address: S. Boghossian, Ph.D., Center for Advanced Nutrition, Utah State University, Logan, UT 84322

Conflict of Interest: The authors have no conflicts of interest.

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