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Keywords:

  • LEPTIN;
  • OSTEOPOROSIS;
  • BONE;
  • GENE EXPRESSION;
  • OB/OB MICE

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Both central and peripheral leptin administrations reduce body weight, food intake, and adiposity in ob/ob mice. In this study we compared effects of intracerebroventricular (ICV) and subcutaneous (SC) administration of leptin on bone metabolism in the appendicular and axial skeleton and adipose tissue gene expression and determined the effects of ICV leptin on bone marrow gene expression in ob/ob mice. In experiment 1, leptin (1.5 or 0.38 µg/d) or control was continuously injected ICV for 12 days. Gene expression analysis of femoral bone marrow stromal cells showed that expression of genes associated with osteogenesis was increased after ICV injection, whereas those associated with osteoclastogenesis, adipogenesis, and adipocyte lipid storage were decreased. In experiment 2, leptin was injected continuously ICV (0.0 or 1.5 µg/d) or SC (0.0 or 10 µg/d) for 12 days. In both experiments, regardless of mode of administration, leptin decreased body weight, food intake, and body fat and increased muscle mass, bone mineral density, bone mineral content, bone area, marrow adipocyte number, and mineral apposition rate. Serum insulin was decreased, whereas serum osteocalcin, insulin-like growth factor 1, osteoprotegerin, pyridinoline, and receptor activator of nuclear factor κB ligand concentrations were increased. In experiment 2, expression of genes in adipose tissue associated with apoptosis, lipid mobilization, insulin sensitivity, and thermogenesis was increased, whereas expression of genes associated with cell differentiation and maturation was decreased regardless of mode of administration. Thus ICV injection of leptin promotes expression of pro-osteogenic factors in bone marrow, leading to enhanced bone formation in ob/ob mice. © 2011 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Leptin, a cytokine-like hormone secreted by adipocytes, was discovered originally as the missing protein in the genetically obese ob/ob mouse.1 Leptin receptors are expressed in peripheral tissues, including skeletal muscle, bone, and cartilage, but a primary target of leptin binding is the brain, specifically the hypothalamus and hindbrain.2 Leptin's effects on bone are mediated via a central neuroendocrine signaling pathway, as well as directly on bone marrow stem cells to enhance their differentiation to osteoblasts and inhibit their differentiation to adipocytes.3–8

There are contradictory published data regarding the effects of leptin on bone mass in rodents. Some studies have described leptin-deficient ob/ob mice as having a high bone mass,4, 9, 10 whereas others11, 12 observed that leptin-deficient mice had lower bone mass than normal mice. One explanation for the discrepancies may be related to the mode of leptin administration and the type of bone investigated. Subcutaneous (SC) administration of leptin in ob/ob mice increased bone growth and indices of bone formation in the axial skeleton,11, 12 whereas intracerebroventricular (ICV) infusion of leptin in both wild-type and ob/ob mice led to rapid bone loss in the vertebrae.4 These findings suggested initially that leptin regulates bone mass through alternative pathways, one involving a direct stimulatory effect on bone growth when administered peripherally and another that is indirect, involving a hypothalamic relay that suppresses bone formation when administered centrally. However, in a study conducted by Iwaniec and colleagues in which ob/ob mice were injected in the hypothalamus with a recombinant adenovirus expressing leptin, femoral bone mass and length increased, whereas trabecular bone volume decreased.13 Furthermore, while both Hamrick and colleagues and Ducy and colleagues found increased trabecular bone volume in vertebrae of ob/ob mice, Hamrick and colleagues reported reduced cortical thickness, cortical area, and trabecular bone volume in the femur and decreased cortical bone thickness in the vertebrae of ob/ob mice.4, 8 These observations suggest not only differential responses to leptin signaling between the appendicular and axial skeletal sites but also different actions of leptin on cortical and trabecular bone.

Interestingly, leptin has been shown to reduce bone marrow adipocyte number and size and to increase marrow adipocyte apoptosis after either peripheral administration in ob/ob mice or central administration in rats.7, 12 This effect is similar to the enhancement of adipose tissue apoptosis observed in fat pads of mice and rats treated either peripherally or centrally with leptin.14–17 Elimination of adipocytes in bone marrow may have specific beneficial effects contributing to the promotion of bone growth. For example, adipocytes have been shown to produce inflammatory cytokines that increase osteoclastogenesis,18 although the specific effect of bone marrow adipocytes on local bone metabolism has not been investigated.

Because of these varied reports regarding the effects of leptin treatment and leptin deficiency on bone growth and metabolism, the objectives of this study were to determine the effects in ob/ob mice of 12-day administration of centrally administered (ICV) leptin on bone marrow gene expression and bone growth (experiment 1) and to compare the effects of peripheral and ICV leptin administration on bone growth and adipose tissue (experiment 2).

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

All experimental and surgical procedures in this study were approved by the Animal Care and Use Committee of the University of Georgia.

Animals

Female leptin-deficient (B6.V-Lepob/J; ob/ob) mice on the C57BL6 background were purchased from Jackson Laboratories, Inc. (Bar Harbor, ME, USA). The mice were individually housed in plastic shoebox cages in a room with a 12/12-hour light/dark cycle, 22 ± 1°C ambient temperature, and 50% humidity. Mice had ad libitum access to pelleted standard lab chow (LabDiet 5001, PMI Nutritional International, St. Louis, MO, USA) and water throughout the study.

All mice were surgically implanted with prefilled and primed osmotic minipumps (0.25 µL/h; Model 1002, Alzet Corp., Cupertino, CA, USA) for injection of treatment solutions. The pumps were inserted into a subcutaneous pocket. For mice assigned to receive intracerebroventricular (ICV) injections, unilateral lateral ventricular (ICV) guide cannulas (Model 3280P/spc; Plastics One, Inc., Roanoke, VA, USA) were surgically implanted and attached to the osmotic pumps, as described previously.19

All mice were allowed to recover for 2 days after surgery, during which time 6 µL of artificial cerebrospinal fluid (aCSF) or PBS was infused prior to the treatment infusion, which occurred for the next 12 days. The mice were administered intraperitoneal injections of calcein (20 mg/kg of body weight; C-0875, Sigma, St Louis, MO, USA) on the first day of treatment and again on the day before they were to be euthanized to label active bone-forming surfaces. The health of the mice was monitored and body weight (BW) and food intake (FI) were measured and recorded daily.

Mice were euthanized by decapitation using a guillotine after sedation in a CO2 chamber at the end of the twelfth day of treatment injection. Soleus and gastrocnemius (GC) muscles were collected and weighed. Trunk blood was collected for measurement of blood glucose. Serum receptor activator of nuclear factor κB ligand (RANKL) and insulin-like growth factor 1 (IGF-1) concentrations were determined using the Luminex100 and single-plex assay kits (Millipore, Bedford, MA, USA). The Mouse Bone Panel (Millipore) was used to determine the serum insulin, osteocalcin, osteoprotegerin (OPG), and pyridinoline (PYD; experiment 2 only) concentrations.

Body composition was analyzed by PIXImus densitometry (GE Lunar Corp., Madison, WI, USA). Fat mass, lean mass, percentage fat, bone mineral density (BMD), and bone mineral content (BMC) were measured for each animal. The right and left tibias and lumbar spine of each mouse were dissected free of soft tissue. The right tibia and lumbar spine (L4–L5) were fixed in 10% buffered formalin for 48 hour at room temperature and then stored in 70% ETOH for measurement of bone marrow adipocyte number (experiment 2). The left tibia and femur and lumbar spine (L2–L3) were fixed in 70% ethanol and embedded in methyl methacrylate for measurement of mineral apposition rate (MAR).

Two sources of intra-abdominal white adipose tissue, the retroperitoneal (RP) and parametrial (PM) fat pads, were collected. Interscapular brown adipose tissue (BAT), liver, and GC and soleus muscles also were collected. All tissues were weighed and then frozen in liquid nitrogen before storing at −80°C. In experiment 2, half the PM tissue samples were fixed in 4% paraformaldehyde for tissue apoptosis assay.

Experiments

Experiment 1

Treatments were randomly assigned to 18 female ob/ob mice (15 weeks old, 60.6 g initial BW) and included control (aCSF) 6 µL/d, 0.38 µg leptin/d, and 1.5 µg leptin/d administered as continuous ICV injections. Recombinant mouse leptin (R&D Systems, Minneapolis, MN, USA) was dissolved in aCSF, which consisted of 8.66 g/L of NaCl, 0.224 g/L of KCl, 0.206 g/L of CaCl2 · 2H2O, 0.163 g/L of MgCl2 · 6H2O, 0.214 g/L of Na2HPO4 · 7H2O, and 0.027 g/L of NaH2PO4 · H2O for ICV delivery.

Experiment 2

Treatments were randomly assigned to 40 female ob/ob mice (15 weeks old, 61.3 g initial BW) according to a 2 × 2 factorial design and included SC control (saline) 6 µL/d, SC leptin 10 µg/d, ICV control (aCSF) 6 µL/d, and ICV leptin 1.5 µg/d. Previous studies found that leptin produced a maximal suppression of FI and BW reduction at 10 µg/d in a dose range of 2.5 to 10 µg/d when administered SC and at 1.5 µg/d in a dose range of 0.38 to 1.5 µg/d when administered ICV (experiment 1 and ref. 16). Recombinant mouse leptin (R&D Systems) was dissolved in either aCSF for ICV delivery or 0.9% PBS solution for SC delivery.

Methods

Bone adipocyte area and mineral apposition rate

The right tibia was cut across the proximal third of the shaft, and the L4 vertebra was cut across the horizontal plane, decalcified in EDTA, embedded in paraffin, and sectioned at approximately 5 µm. Sections were stained with hematoxylin and eosin to visualize adipocytes (experiment 2). Adipocytes were counted over a 0.10-mm2 area. Adipocyte number and area were determined using ImagePro analysis software (Media Cybernetics, Inc., Bethesda, MD, USA).

The left tibia (experiments 1 and 2) and lumbar spine (L3, experiment 2) were fixed in 70% ethyl alcohol, dehydrated, embedded in methyl methacrylate, and sectioned at 30 µm across the proximal third of the shaft for the tibia and across the horizontal plane for the L3 vertebra.12 The mineral apposition rate was derived from fluorochrome interlabel distances.

TUNEL apoptosis assay (experiment 2)

Apoptosis of the PM adipose tissue was quantified using the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay. Briefly, sections of formalin-fixed and paraffin-embedded adipose tissue were dewaxed by washing in xylene and rehydrated through a gradual series of ethanol and distilled water. Proteinase K–permeabilized sections were subjected to enzymatic in situ labeling of DNA strand breaks using the TUNEL technique of the Molecules Probes TUNEL Cell Kit (Molecular Probes, Inc., Eugene, OR, USA). TUNEL+ cells were counted in nine sections per sample.

Bone marrow gene expression (experiment 1)

Bone marrow was flushed from the left femur and mixed with 1 mL of prewarmed Dulbecco's modified Eagle medium (DMEM; with 10% FBS). After quantification of the cell number, cells were seeded in 100-mm tissue culture dishes at a density of 5 × 107 per 10 mL of medium. Two hours after plating, nonadherent cells were removed by washing once with 5 mL of medium. Twenty-four hours after initial plating, the adherent bone marrow stem cells were washed twice with 1× PBS in order to remove the floating red blood cells, followed by RNA extraction using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA) following the manufacturer's instructions. One microliter of the 15-µL sample was used as an integrity check and for quantification by the Agilent 2100 bioanalyzer and RNA 6000 Nano Assay (Agilent Technologies, Santa Clara, CA, USA). After real-time reverse-transcriptase Taqman RT-PCR (Applied Biosystems, Inc., Foster City, CA, USA) was used to quantitatively measure mRNA levels of selected genes in the bone marrow stem cells, as described previously.20 Data were expressed as relative quantification (RQ), which presents the fold difference of mRNA level in treatment groups relative to the aCSF control group, and were analyzed using sequence-detection systems software. All the oligonucleotide primer and fluorogenic probe sets for Taqman real-time PCR were from ABI (Carlsbad, CA, USA) (Supplemental Table S1).

Adipose tissue gene expression (experiment 2)

Total RNA was isolated from PM fat pads using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. One hundred milligrams of frozen adipose tissue from each sample was homogenized by adding 1 mL of Trizol reagent. The samples were centrifuged at 12,000g for 15 minutes at 4°C, and the aqueous phase was removed and mixed with 0.5 mL of isopropyl alcohol. After centrifugation at 12,000g for 10 minutes at 4°C, the pellet was washed with 1 mL of 75% ethanol and then dissolved in 12 µL of RNase-free water. One microliter of the 12-µL sample was used as an integrity check and for quantification by the Agilent 2100 Bioanalyzer and RNA 6000 Nano Assay (Agilent Technologies). The conditions for RT-PCR were as described for experiment 1. All the oligonucleotide primer and fluorogenic probe sets for Taqman real-time PCR were from ABI (Supplemental Table S2).

Statistical analysis

All statistical analyses were conducted with SAS (Version 9.0, SAS Institute, Inc., Cary, NC, USA). Significance of treatment effects was determined by one- or two-way ANOVA. Significance of differences among means was determined by Fisher's LSD. Significance was established at p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Experiment 1

Food intake, body weight, body composition, and tibial MAR

Regardless of dose, there were significant effects on BW owing to leptin. By the end of the 12-day injection period, high- and low-dose ICV leptin-treated mice had lost 17.2 ± 1.9 g and 5.2 ± 1.6 g of BW, respectively (p < .0001), whereas the aCSF-injected mice gained 4.3 ± 2.7 g. The high dose of leptin decreased daily FI by 86.6%, and the low dose of leptin, by 58.2% (p < .001; Table 1).

Table 1. Food Intake, Body Weight, Weight Gain, Body Composition, and Bone MAR of ob/ob Mice Injected for 12 Days With Control (aCSF), 0.38 µg/d Leptin, or 1.5 µg/d Leptin
 aCSF (mean ± SEM)0.38 µg/d Leptin (mean ± SEM)1.5 µg/d Leptin (mean ± SEM)
  1. BW, body weight; BAT, brown adipose tissue; PM, parametrial fat pad; RP, retroperitoneal fat pad; GC, gastrocnemius muscle; Sol, soleus muscle; BMD, bone mineral density; BMC, bone mineral content; MAR, mineral apposition rate. Means without a common letter are significantly different: a,b,cp < .05; x,y,zp < .01.

Final BW (g)63.9 ± 2.3x53.1 ± 2.5y41.2 ± 1.5z
Weight gain/loss (g)4.3 ± 0.4x−5.2 ± 2.7y−17.2 ± 0.9z
Food intake (g/d)6.7 ± 0.3x2.8 ± 1.2y0.9 ± 0.1y
BAT (mg)141.5 ± 6.7a94.4 ± 8.2b85.6 ± 2.2b
PM (g)5.9 ± 0.6a5.3 ± 0.3a,b3.7 ± 0.6b
RP (mg)2.1 ± 0.1a1.9 ± 0.2a,b1.3 ± 0.3b
GC (mg)173.4 ± 7.7162.7 ± 9.9174.0 ± 5.2
Sol (mg)13.5 ± 1.2b12.6 ± 0.7b16.5 ± 0.5a
Lean, g21.9 ± 0.5b20.3 ± 0.9b16.6 ± 1.1a
% Lean34.4 ± 1.3a38.2 ± 1.3a,b40.3 ± 2.3b
Fat, g35.0 ± 1.9b30.6 ± 1.8b22.3 ± 1.0a
% Fat60.6 ± 0.560.1 ± 1.357.9 ± 1.7
BMD, g/cm20.0536 ± 0.0024a0.0624 ± 0.0009b0.0628 ± 0.0022b
BMC, g0.399 ± 0.058a0.486 ± 0.005a0.677 ± 0.076b
Area, cm27.4 ± 0.9a7.8 ± 0.1a10.7 ± 1.0b
Tibial MAR, µm/day2.4 ± 0.3a2.6 ± 0.3a4.9 ± 0.3b

Dual-energy X-ray absorptiometry (DXA) results showed that mice administered the high dose of leptin had decreased fat mass and increased percent lean mass (p < .05; Table 1). Both groups of leptin-treated mice had an increase in whole-body bone mineral density (BMD; p < .05), but only mice receiving the high dose of leptin had an increase in whole-body bone mineral content (BMC) and bone area (p < .05). The tibial MAR was higher in mice receiving the high dose of leptin compared with the control and low dose of leptin (p < .05). The soleus muscle weight was increased in mice receiving the high dose of leptin (p < .05). Muscle mass was positively correlated with BMD (r = 0.49, p = .004) and BMC (r = 0.73, p < .0001).

Serum hormone concentrations

The high dose of leptin reduced blood glucose concentrations (p < .01) and dose-dependently decreased serum insulin concentrations (p < .01; Table 2). Leptin also dose-dependently increased concentrations of osteocalcin (p < .01), OPG (p < .01), and RANKL (p < .01). Serum IGF-1 concentration was higher in mice that received the high dose of leptin compared with the other treatment groups (p < .01), and serum IGF-1 levels were positively correlated with muscle mass (r = 0.49, p = .02), BMD (r = 0.63, p = .0001), and osteocalcin concentrations (r = 0.57, p = .009).

Table 2. Serum Hormone Concentrations of ob/ob Mice Injected ICV for 12 Days With Control (aCSF), 0.38 µg/d Leptin, or 1.5 µg/d Leptin
 aCSF (mean ± SEM)0.38 µg/d Leptin (mean ± SEM)1.5 µg/d Leptin (mean ± SEM)
  1. OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor kappa-B ligand; IGF-1, insulin-like growth factor-1. Means without a common letter are significantly different: x,y,zp < .01.

OPG (pg/mL)660.0 ± 20.3x905.2 ± 19.1y1447.6 ± 47.7z
Osteocalcin (ng/mL)16.5 ± 1.1x26.4 ± 1.2y92.4 ± 2.2z
RANKL (pg/mL)103.9 ± 1.3x123.2 ± 3.5y148.3 ± 2.1z
IGF1 (ng/mL)73.9 ± 2.6x83.3 ± 0.9x99.8 ± 5.1y
Insulin (pM)961.4 ± 14.5x446.1 ± 12.0y196.3 ± 3.2z
Glucose (mg/dL)261.5 ± 261.5x219.0 ± 219.0x82.8 ± 82.8y
Bone marrow gene expression

Leptin treatment decreased expression of genes involved in osteoclastogenesis; both dose levels decreased expression of receptor activator of NFκB (Rank) (p < .05), whereas the high dose of leptin decreased expression of colony-stimulating factor 1 (Csf1) (p < .05; Fig. 1A). The high dose of leptin also increased expression of runt-related transcription factor 2 (Runx2), osterix (Sp7), and alkaline phosphatase (Ccl27), all of which are involved in osteogenesis (p < .05; Fig. 1B). The high dose of leptin decreased expression of genes involved in adipogenesis: peroxisome proliferator-activated receptor γ (Pparg), B-cell leukemia/lymphoma 2 (Bcl2), adipocyte enhancer binding protein 1 (Aebp1) delta like 1 homologue (Dlk1), GATA binding protein 3 (Gata3), and resistin (Retn) (p < .05; Fig. 1C).

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Figure 1. Gene expression in femoral bone marrow of female ob/ob mice injected ICV for 12 days with control (aCSF), 0.38 µg/d of leptin, or 1.5 µg/d of leptin. Data shown are mean ± SEM of log2(RQ) value. RQ value, which was calculated as described in “Materials and Methods,” is an indicator of mRNA expression level for each gene in each treatment group. Means within a gene that are not denoted with a common letter are different, a,bp < .05.

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Experiment 2

Body weight, food intake, tissue weights, and body composition

Leptin treatment reduced BW (p < .05), weight gain (p < .05), and FI (p < .01), but there was no difference between modes of administration (Table 3). PM fat pad mass was reduced in ICV leptin-treated mice (p < .05), whereas RP fat pad mass was reduced significantly only in SC leptin-treated mice (p < .05). GC muscle mass was increased in the ICV leptin-treated mice (p < .05).

Table 3. Food Intake, Body Weight, Weight Gain, Body Composition, and Adipose Tissue Apoptosis of ob/ob Mice Injected SC or ICV for 12 Days With Control (aCSF) or Leptin (10 µg/d SC, 1.5 µg/d ICV)
 SC controlSC leptinICV controlICV leptin
  1. BW, body weight; BAT, brown adipose tissue; PM, parametrial fat pad; RP, retroperitoneal fat pad; GC, gastrocnemius muscle; Sol, soleus muscle; BMD, bone mineral density; BMC, bone mineral content. Means without a common letter are significantly different: a,b,cp < .05; x,y,zp < .01.

Final BW (g)63.1 ± 1.4x50.8 ± 1.1y61.3 ± 1.2x47.5 ± 4.7y
Weight gain/loss (g)2.7 ± 0.5a,x−8.9 ± 0.6b,y2.5 ± 0.5a,x−12.0 ± 3.8c,y
Food intake (g)5.8 ± 0.8a3.7 ± 0.5b5.6 ± 0.6a3.3 ± 0.8b
BAT (mg)120.0 ± 12.695.1 ± 7.4121.9 ± 10.995.8 ± 12.0
PM (g)5.5 ± 0.4a,b5.0 ± 0.2b6.0 ± 0.2a4.4 ± 0.5c
RP (g)2.5 ± 0.1a1.8 ± 0.1b2.1 ± 0.1a,b1.8 ± 0.3b
GC (mg)104.8 ± 4.4a115.5 ± 3.9a91.6 ± 4.3b105.5 ± 3.2a
SOL (mg)9.5 ± 0.99.5 ± 0.47.8 ± 0.711.9 ± 3.0
Lean (g)23.3 ± 0.420.6 ± 0.222.4 ± 0.623.2 ± 1.3
% Lean41.2 ± 0.8a44.9 ± 0.7b40.1 ± 0.7a43.8 ± 1.2b
Fat (g)33.3 ± 0.9a25.4 ± 0.8b33.5 ± 1.0a30.1 ± 2.3a
% Fat58.8 ± 0.8a55.1 ± 0.7b59.9 ± 0.7a56.2 ± 1.2b
BMD, g/cm20.0492 ± 0.0012a0.0545 ± 0.001b0.0513 ± 0.0016a,b0.0553 ± 0.0009b
BMC, g0.386 ± 0.003a0.477 ± 0.011b0.390 ± 0.020a,b0.429 ± 0.020c
Area, cm27.1 ± 0.2a7.8 ± 0.2b7.1 ± 0.3a8.3 ± 0.2b
% apoptosis, PM fat pad5.0 ± 0.6a11.7 ± 3.7a,b4.2 ± 0.9a19.0 ± 4.3b

DXA results showed that both the SC and ICV leptin-treated mice had significant decreases in percent body fat (p < .05), but absolute body fat mass was decreased only in the SC leptin-treated mice (p < .05). Percent lean mass was increased in both SC and ICV leptin-treated mice (p < .05). Whole-body BMD was increased significantly only in the SC leptin-treated mice (p < .05), whereas BMC and bone area were increased significantly in both groups of leptin-treated mice (p < .05; Table 3). The muscle mass of the leptin-treated mice was positively correlated with BMD (r = 0.49, p = .004) and BMC (r = 0.73, p < .0001).

Adipose tissue apoptosis assay

ICV leptin treatment increased apoptosis in the PM fat pad (p < .05; Table 3).

Serum hormone concentrations

Both ICV and SC leptin administration decreased serum insulin concentration (p < .01; Table 4). Serum IGF-1, OPG, osteocalcin, PYD, and RANKL concentrations were increased with both ICV and SC leptin administration (p < .01).

Table 4. Serum Hormone Concentrations of ob/ob Mice Injected SC or ICV for 14 Days With Control (Saline SC; aCSF ICV) or Leptin (10 µg/d SC; 1.5 µg/d ICV)
 SC controlSC leptinICV controlICV leptin
  1. OPG, osteoprotegerin; RANKL, Receptor activator of nuclear factor kappa-B ligand; IGF-1, insulin-like growth factor-1. Means without a common letter are significantly different: a,b,cp < .05; x,y,zp < .01.

OPG (pg/mL)596.8 ± 29.7b792.4 ± 29.7a533.7 ± 29.7b802.2 ± 29.7a
Osteocalcin (ng/mL)39.1 ± 5.0x134.8 ± 13.7y31.9 ± 2.8x96.4 ± 18.1y
RANKL (pg/mL)97.3 ± 6.6x170.6 ± 5.8y104.9 ± 8.0x155.4 ± 4.6y
PYD (nmol/l)0.94 ± 0.8a2.45 ± 0.8a0.72 ± 0.8b2.58 ± 0.8a
IGF-1 (ng/mL)374.8 ± 24.3x547.8 ± 26.1y369.1 ± 27.0x493.7 ± 15.6y
Insulin (pM)754.3 ± 123.8a,x95.8 ± 8.9b,y847.0 ± 105.3a,x60.4 ± 19.3c,y
Bone marrow adipocyte number and MAR

The MARs of both tibia and vertebra in leptin-treated mice were significantly higher than in control mice (p < .05), but there was no difference between SC and ICV leptin treatments (Fig. 2A, B). Likewise, bone marrow adipocyte number was decreased in both tibia and vertebra of leptin-treated mice (p < .05), and there was no difference between SC and ICV leptin treatments (Fig. 2C, D).

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Figure 2. Tibial and vertebral mineral apposition rate (MAR) and adipocyte number from female ob/ob mice injected SC or ICV for 12 days with control (saline SC, aCSF ICV) or leptin (10 µg/d SC, 1.5 µg/d ICV). Graphs show means ± SEM. Means without a common letter are significantly different: a,bp < .05.

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Adipose tissue gene expression

Leptin treatment increased the expression of the apoptosis-related genes BCL2-associated X protein (Bax) and caspase-3 (Casp3) genes and decreased expression of Bcl2 (p < .05), with no significant differences between SC and ICV leptin administration (Fig. 3A). Both SC and ICV leptin treatments also increased the mRNA expression of the mitochondrial uncoupling proteins Ucp2 and Ucp3 (p < .05; Fig. 3B).

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Figure 3. Gene expression in parametrial adipose tissue of female ob/ob mice injected SC or ICV for 12 days with control (saline SC, aCSF ICV) or leptin (10 µg/d SC; 1.5 µg/d ICV). Data shown are mean ± SEM of log2(RQ) value. RQ value, which was calculated as described in “Materials and Methods,” is an indicator of mRNA expression level for each gene in each treatment group. Means within a gene that are not denoted with a common letter are different, a,bp < .05.

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Leptin treatment increased expression of genes for the lipolytic enzymes hormone sensitive lipase (Lipe) and lipoprotein lipase (Lpl) (p < .05) while decreasing the expression of the gene for the lipid synthesis enzyme fatty acid synthase (Fasn) (p < .05); there were no significant differences between SC and ICV administration (Fig. 3C). Expression of Pparγ, fatty acid–binding protein 4 (Fabp4), and CCAAT/enhancer binding protein α (Cebpa) was decreased in both leptin treatment groups. The products of these genes are involved in fatty acid storage and glucose metabolism, fatty acid transport, and regulation of adipogenesis, respectively. Expression of phospholipase C (protein disulfide isomerase-associated 3; Pdia3) and prohibitin (Phb/Fyb) was increased (p < .05; Fig. 3D). Phospholipase C is involved in the mitogen-activated protein kinase (MAPK) signaling cascade that is activated by leptin.21 Prohibitin functions as a membrane receptor and has been shown to modulate insulin-stimulated glucose and fatty acid oxidation in adipocytes.22

Leptin treatment decreased the mRNA level of leptin receptor (Lepr), sterol regulatory element binding factor 1 (Srebf1), which is involved in adipogenesis, and tumor necrosis factor α (Tnfa), a cytokine that regulates other adipokines, including adiponectin23 (p < .05). Leptin treatment increased expression of adiponectin (Adipoq) (p < .05), a cytokine involved in glucose and lipid metabolism. The expression of retinol-binding protein 4 (Rbp4), a mediator associated with decreased glucose transportation and impaired insulin function, was decreased, whereas the expression of solute carrier family 2 member 4 (Slc2a4, Glut4), a glucose transporter, was increased with leptin administration (p < .05). There were no significant differences between SC and ICV leptin administration.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Leptin administered either SC or ICV decreased BW and adiposity while stimulating bone growth in ob/ob mice, as indicated by the increased BMD, BMC, bone area, and MAR. The dose-dependent increases in serum OPG, osteocalcin, and RANKL levels also demonstrate the metabolic effects of leptin on bone leading to increased bone growth. Furthermore, there were no major differences in response of bone or adipose tissue between SC and ICV leptin treatments at the doses tested. The reason for the lack of differences in experiment 2 between SQ and ICV leptin treatments in most of the parameters may have been the fact that we selected doses for both routes of administration that caused near-maximal suppression of FI and BW, which are centrally mediated effects of leptin. Thus we hypothesize that the reason there were no differences in the parameters measured between SC and ICV leptin treatments is that receptors involved in leptin's effects on food intake, adipose tissue mobilization and apoptosis, physical activity, and bone metabolism were stimulated similarly. The central regulation of bone mass is complex and still incompletely understood. A number of neurotransmitters and neuropeptides are involved, including cocaine- and amphetamine-regulated transcript (CART), neuromedin U, neuropeptide Y, brain-derived serotonin, cannabinoid receptors, and efferent sympathetic pathways (reviewed in refs 24 and 25). Leptin interacts directly or indirectly with these systems, thereby accounting for some of the differential effects reported for leptin in different bone compartments.

Leptin also has been shown to have direct effects on both osteoblasts and osteoclasts in vitro, stimulating maturation and mineralization of osteoblasts, reducing RANKL secretion from bone marrow stem cells, and inhibiting osteoclastogenesis.26–28 Both central and peripheral administration of leptin have been shown to reduce bone marrow adiposity,12, 29 as we also found in this study. There is evidence that this is due in part to increased apoptosis of marrow adipocytes,7 which is likely a centrally mediated effect because leptin does not act directly on adipocytes to promote apoptosis.30 However, leptin has been shown to act directly on bone marrow stem cells to promote osteoblastogenesis and inhibit adipogenesis.31 Thus SC administration of leptin may have had additional beneficial effects on bone, but we were not able to detect them in the parameters measured in this study.

Although Ducy and colleagues4 did not report effects of ICV leptin on whole-body BMD or BMC, the apparent difference between our results and theirs may be due in part to the differences in doses administered ICV and the length of treatment. In our study, mice received 1.5 µg/d of leptin for 12 days, whereas Ducy and colleagues administered 192 ng/d for 28 days. Our results clearly showed an anabolic effect on both tibial and vertebral cortical bone, whereas they showed a decrease in vertebral trabecular bone volume. Iwaniac and colleagues13 did not report whole-body BMD or BMC in response to increased hypothalamic leptin expression, but their finding of increased femoral length and volume certainly corresponds to an anabolic effect of central leptin on bone and in that respect is similar to our results.

Lean mass regulates local bone formation by muscle-derived mechanical stimuli,32 and muscle mass is directly correlated with the amount of bone mass.33 Leptin has been shown to have marked effects on physical activity in both normal rats and ob/ob mice,16, 34, 35 and the increased physical activity in conjunction with increased muscle mass likely contributed to the increased BMD and BMC after both modes of administration of leptin. In this study we did not attempt to determine the relative contribution of the increased physical activity and muscle mass compared with any direct or indirect metabolic effect of leptin on bone, but this is an important issue that should be investigated in future studies.

Our results also show that mice treated with leptin had increased serum IGF-1 levels, which likely contributed to increased muscle and overall lean mass. It is somewhat surprising that IGF-1 levels were increased in this study because caloric restriction is known to suppress IGF-1 levels. However, Luque and colleagues36 found that peripheral administration of leptin to ob/ob mice prevented the reduction of IGF-1 levels that occurred in pair-fed controls, and Chan and colleagues37 found that r-metHuleptin administration to women under chronic energy-deficit conditions increased IGF-1 levels. Thus leptin administration may act independent of energy intake to increase IGF-1 levels under certain conditions, for example, when endogenous leptin levels are chronically reduced.

Changes in femoral bone marrow gene expression were consistent with the increased bone mass and decreased marrow adiposity. For example, femoral bone marrow expressions of both Rank and Csf1 were decreased in ICV leptin-treated mice, indicating a decrease in osteoclastogenesis in these mice. RANK is an osteoclast differentiation factor that is an essential signal for osteoclastogenesis. It is also a receptor for OPG, a secreted protein that inhibits osteoclastogenesis.38 CSF1 is produced by osteoblasts to inhibit osteoclast apoptosis and may increase bone resorption by prolonging the lifespan of osteoclasts. Dobbins and colleagues also suggested that CSF1 may be required for osteoclast differentiation and activation.39

Expression of genes associated with osteoblastogenesis (Runx2, Sp7, and Ccl27) was increased in mice that received the high dose of leptin. RUNX2 has been shown to be an osteoblast specific transcription factor and a regulator of osteoblastic differentiation.40 Sp7 is expressed in all developing bones, and Sp7 null mice were found to have a complete lack of bone formation.41 The decrease in expression of Pparg in the bone marrow cells of mice treated with the high dose of leptin is likely associated with the decrease in bone marrow adipocytes but also may have contributed to the propensity of bone marrow stem cells to differentiate into osteoblasts rather than adipocytes, thus resulting in increased bone mass and decreased marrow adipocyte number.

Decreased expression of Bcl2, Aebp1, Dlk1, Gata3, and Retn also may be related to the increased bone formation in the mice treated with the high dose of leptin, primarily through their association with the decreased marrow adipogenesis and increased osteogenesis.12, 42–44 The AEBP1 protein plays an important role in adipogenesis by modulating the differentiation of preadipocytes, and Aepb1 gene expression is increased in proliferating preadipocytes.42, 43 DLK1 is a regulator of mesenchymal cell differentiation.44 The decreased expression of Dlk1 in bone marrow of mice treated ICV with the high dose of leptin may have been associated with increased differentiation of mesenchymal stem cells to osteoblasts. The decreased expression of Gata3 and Retn in bone marrow of mice treated ICV with the high dose of leptin likely reflects the reduced population of adipocytes in bone marrow of these mice.

In experiment 2, there was no significant difference in apoptosis rate between SC and ICV leptin treatments. There also were no differences in adipose tissue gene expression between SC and ICV leptin treatments. We found similar changes in gene expression in this study as compared with our earlier study,45 with a few exceptions. These differences may be due, in part, to the source of adipose tissue used. In this study, gene expression was measured in the parametrial fat pad, which is intra-abdominal white adipose tissue, whereas in the earlier study, tissue from the inguinal fat pad, subcutaneous white adipose tissue, was used.

Apoptosis is regulated by several members of the BCL2 family, which promote or block formation of the mitochondrial outer membrane permeabilization pore (MOMPP).46, 47 BCL2 is antiapoptotic and blocks formation of the MOMPP, whereas BAX is proapoptotic and promotes formation of the MOMPP. In this study, both SC and ICV injection of leptin caused increased expression of Bax and decreased expression of Bcl2, along with increased expression of Casp3. Thus all these changes are indicative of the increased adipose tissue apoptosis observed in the leptin-treated mice.

In our previous study and this one, Ucp2 expression was increased following leptin treatment. While all UCPs are involved in uncoupling of oxidative phosphorylation from ATP synthesis in mitochondria to some extent, recent evidence suggests that primary functions of UCP-2 and UCP-3 may include reactive oxygen species scavenging and fatty acid translocation.48 Upregulation of these genes after leptin administration thus may indicate increased fatty acid transport and enhanced lipid β-oxidation.

Increased Lipe expression levels likely indicate enhanced triglyceride hydrolysis and fatty acid mobilization in adipose tissue of leptin-treated mice, whereas decreased Fas expression indicates a reduction in lipogenesis. The decreased expression of Pparg also suggests a reduction in adipocyte differentiation. The decreased Srebf1 expression in leptin-treated mice is consistent with findings that show that Srebf1 regulates several key genes related to lipid metabolism, including Fas,49 and is associated with a reduction in lipogenic activity.50

TNF-α is a cytokine that stimulates lipolysis and induces apoptosis. In this study, Tnfa expression was decreased, whereas in our previous study, it was increased. This may be due to the different sources of adipose tissue used in the two studies. Montague and colleagues show that expression of Tnfa and other genes varied among adipose tissue depots in humans.51

Expression levels of both Adipoq, an adipokine that plays an important role in modulating insulin action and inhibiting gluconeogenesis,52 and Slc2A4/Glut4 were increased in leptin-treated mice, which is consistent with the improved insulin sensitivity of the mice treated with leptin in both this study and our previous one. As expected, expression of Lep (leptin) was decreased after both SC and ICV leptin treatments. Leptin mRNA is expressed in adipose tissue of ob/ob mice, but the protein is defective because of the gene mutation, and no measurable leptin is found in the blood. The decreased expression in the adipose tissues collected at the end of the experiment reflects the decreased content of body fat that occurred with leptin treatment.

In conclusion, both ICV and SC leptin treatment of ob/ob mice stimulated bone growth and caused body weight and fat loss. ICV leptin dose-dependently enhanced osteogenesis and promoted expression of genes associated with osteogenesis while reducing marrow adipogenesis and expression of genes associated with adipogenesis. Furthermore, the decreased presence of adipocytes in bone marrow likely resulted in reduced production of inflammatory cytokines that promotes osteoclastogenesis. Since bone marrow is known to accumulate adipocytes with age,3 decreased marrow adipogenesis, along with increased indices of osteogenesis observed in mice treated with leptin, also may suggest that a reduction in marrow adipocytes has a positive effect on bone formation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

The work was supported by the Georgia Research Alliance (GRA) Eminent Scholar endowment (CAB) and a GRA Challenge Grant (CAB and JXS).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
jbmr_406_sm_SuppTab1.doc49KSupplementary Table 1
jbmr_406_sm_SuppTab2.doc47KSupplementary Table 2

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