Full text of DOI:10.1359/jbmr.2002.17.6.1034

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 6, 2002
© 2002 American Society for Bone and Mineral Research
Leptin Acts as a Growth Factor on the Chondrocytes
of Skeletal Growth Centers
GILA MAOR,1 MIRI ROCHWERGER,1 YAEL SEGEV,2 and MOSHE PHILLIP2,3
ABSTRACT
Childhood obesity frequently is associated with an increase in height velocity and acceleration of epiphyseal growth
plate maturation despite low levels of serum growth hormone (GH). In addition, obesity is associated with higher
circulating levels of leptin, a 16-kDa protein that is secreted from the adipocytes. In this study, we evaluated the
direct effect of leptin on the chondrocyte population of the skeletal growth centers in the mouse mandibular
condyle, a model of endochondral ossification. We found that chondrocytes in the growth centers contain specific
binding sites for leptin. Leptin, at a concentration of 0.5–1.0 ␮g/ml, stimulated in a dose-dependent manner the
width of the chondroprogenitor zone (up to 64%), whereas higher concentrations had an inhibitory effect. Leptin
induction of both proliferation and differentiation activities in the mandibular condyle was confirmed by our
findings of an increase in bromodeoxyuridine (BrdU) incorporation into DNA and in (acidic) Alcian blue (AB)
staining of the cartilaginous matrix. Leptin also increased the abundance of the insulin-like growth factor (IGF) I
receptor and IGF-I receptor messenger RNA (mRNA) within the chondrocytes and the progenitor cell population.
Our results indicate that leptin acts as a skeletal growth factor with a direct peripheral effect on skeletal growth
centers. Some of its effects on the growing bone may be mediated by the IGF system via regulation of IGF-I
receptor expression. We speculate that the high circulating levels of leptin in obese children might contribute to
their growth. (J Bone Miner Res 2002;17:1034–1043)
Key words: leptin, insulin-like growth factor I, insulin-like growth factor I receptor, epiphyseal growth plate,
endochondral ossification
then.(2–7) Children with exogenous obesity also usually
INTRODUCTION
show an increase in height velocity concomitant with ac-
celeration of bone epiphyseal maturation,(8) despite low
HILDREN’S GROWTH is dependent on an intact and func-
C
tioning growth hormone (GH)—insulin-like growth
plasma GH levels even during the phase of acceleration.
The decrease in GH in these cases has been attributed to
both decreased secretion by the pituitary gland and in-
creased clearance from the plasma.(9,10) The mechanism
whereby obese children continue to grow despite low levels
of GH has not been elucidated fully.
Circulating leptin levels are increased in obesity and
correlate positively with fat mass.(11) This is true for both
adults and children.(12) Studies using the ob/ob mouse
factor (IGF) I) axis. However, there are some clinical con-
ditions in which normal growth persists in the presence of
low GH serum levels. In 1962, Matson(1) was the first to
describe normal height velocity in children after craniophar-
yngioma surgery in association with marked obesity, and
similar findings in larger series have been reported since
The authors have no conflict of interest.
1Department of Morphology Science, Rappaport Faculty of Medicine, Technion, Haifa, Israel.
2Molecular Endocrine Laboratory, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
3Felsenstein Medical Research Center, Institute for Endocrinology and Diabetes, Schneider Children’s Medical Center of Israel, Petah
Tikva, Israel.
1034

LEPTIN AND ENDOCHONDRAL OSSIFICATION
1035
model have shown that leptin is the product of the ob gene, analysis system with appropriate morphometry software
which when mutated, results in obesity.(13) Leptin, a 16-kDa (Olympus Corp., Lake Success, NY, USA). The system
protein is secreted mainly by the white adipose tissue and consists of a Zeiss Universal R photomicroscope (ϫ10
regulates food intake and body weight by negative feedback objective; Zeiss, Oberkochen, Germany) fitted with a Pana-
at the hypothalamic nuclei.(14) The biological activity of sonic WV-CD50 camera (Matsushita, Mississauga, Ontario,
leptin is mediated by specific receptors that belong to class Canada). The video image is viewed on a Sony 14-in color
I cytokine receptors, which are single membrane-spanning monitor attached to an IBM-compatible PC.
receptors. Leptin receptors were first cloned by Tartaglia(15)
who showed that they exist in several variants because of
alternative splicing. In the hypothalamus, the predominant
BrdU incorporation into DNA
and the only biologically active isoform of leptin receptor is
Condyles cultured under either normal or leptin-treated
the long receptor.(16) Leptin receptors also have been iden-
conditions were incubated with BrdU for the last 18 h of
tified in various peripheral tissues mediating a variety of
incubation (as described previously). At the end of the
incubation period, the condyles were washed thoroughly
with Hanks’ buffer and processed routinely for paraffin
embedding. Detection of BrdU-labeled cells was performed
on deparaffinized 6-␮m-thick sections using the Zymed
BrdU staining kit (catalogue no. 93–3943; Zymed Labora-
activities.(16) Despite the fact that the central activity of
leptin differs between man and mice, the peripheral effect of
leptin in both species has yet to be explored fully.
In this study, we report the presence of leptin receptors in
cartilaginous skeletal growth centers of a young mouse
involved in leptin-induced skeletal growth. We evaluated
tories, Inc., San Francisco, CA, USA) according to the
the direct effect of leptin on the chondrocyte population of
manufacturer’s instructions. Briefly, after quenching the
skeletal growth centers in the mice mandibular condyle, a
endogenous peroxidase, sections were mildly trypsinized
model of endochondral ossification.
and then reacted with anti-BrdU antibody (Ab) after biotin-
ylated second Ab with streptavidin-peroxidase–conjugate
and diaminobenzidine (DAB) as a substrate; counterstaining
was performed with hematoxylin.
MATERIALS AND METHODS
Culture system
Immunohistochemistry
Mandibular condyles from 6-day-old Institute of Cancer
Research (ICR) mice were grown in an organ culture as
previously described.(17,18) The BGJb-Fitton Jackson Mod-
ification (Beth Haemek, Israel) was supplemented with 2%
fetal calf serum (FCS), 100 ␮g/ml of ascorbic acid, and
antibiotics. The tissues were incubated for 2–4 days at
37°C, 5% CO2 /95% air, and maximal humidity in the
presence of 0.5, 1.0, or 1.5 ␮g/ml of ovine leptin or under
control conditions. For the bromodeoxyuridine (BrdU) in-
corporation assay, cultures were treated with 100 ␮M of
BrdU for the last 18 h of incubation.
For IGF-I immunoinhibition, condyles were incubated in
the presence of anti–IGF-I antibodies, diluted 1/500 in the
culture medium. At the end of the incubation period, the
explants were washed thoroughly with cold Hanks’ buffer,
fixed with neutral-buffered formalin, and routinely pro-
cessed for paraffin embedding.
Deparaffinized paraffin sections were reacted for 2 h at
room temperature with one of the following specific anti-
bodies: anti-leptin receptor monoclonal Ab (MAb; cata-
logue no. sc-8391; Santa Cruz Biotechnology, Santa Cruz,
CA, USA; this Ab is mapping the protein between the aa
877–894), rabbit anti–IGF-I receptor (anti–␣-subunit; cata-
logue no. SC-712; Santa-Cruz Biotechnology), mouse anti-
cartilage proteoglycans (MAB 2010; Chemicon Interna-
tional, Inc., Temecula, CA, USA), mouse anti-collagen type
II (catalogue no. MAB 8887; Santa-Cruz Biotechnology), or
mouse antiproliferating cell nuclear antigen (PCNA; cata-
logue no. 08–0110; Zymed Laboratories, Inc.). This was
followed by incubation with an appropriate biotinylated
second Ab, with streptavidin-peroxidase conjugate, and
aminoethyl carbazole (AEC) as a substrate (Histostain-SP
kit; Zymed Laboratories, Inc.); counterstaining was done
with hematoxylin.
Alcian blue ϩ hematoxylin and eosin staining
Paraffin sections (6 ␮m) were deparaffinized in xylene,
hydrated in graduated ethanols, and pretreated with 3%
acetic acid for 3 minutes. Then, sections were stained with
1% Alcian blue (AB) at pH 2.5 for 30 minutes, thoroughly
Sodium dodecyl sulfate–polyacrylamide gel
electrophoresis and Western analysis
Tissue lysates prepared from pools of 3-day cultured
rinsed with tap water, and counterstained with hematoxylin mandibular condyles treated with 0.5 ␮g/ml of leptin, non-
and eosin (H&E).
treated, and from murine brains were separated by reducing
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) and electrotransferred to nitrocellulose mem-
brane. Blots were incubated with anti-leptin receptor MAb’s
Morphometric studies
AB ϩ H&E–stained sections were used in the morpho- or rabbit ant–IGF-I receptors Ab’s and detected by rabbit
metric studies. The length of the various cellular layers and anti–mouse-horseradish peroxidase (HRP; Sigma-Aldrich,
the number of BrdUϩ nuclei and the relative area occupied St. Louis, MO, USA) and goat anti–rabbit-HRP conjugates
by them were determined with an Olympus Cue-2 image (Upstate Biotechnology, Lake Placid, NY, USA) and re-

1036
MAOR ET AL.
FIG. 1. Immunohistochemical localization of the leptin receptor within the 6-day-old murine-derived skeletal growth centers. Deparaffinized
sections of mandibular condyle were reacted with rabbit anti-leptin receptor and specific binding was detected with the avidin-biotin-peroxidase
system. (A) Leptin receptors were localized in the chondrocytic population of the mandibular condyle (magnification ϫ190). (B) Higher
magnification revealed that leptin receptors were especially located in the chondrocyte (yc) population of the mandibular condyle (magnification
ϫ240).
vealed by chemiluminescence reagent (Perkin Elmer, Shel- iments with at least three condyles cultured under each
ton, CA, USA).
culture condition in each experiment and at least three
measurements of each condyle. Results are shown as
mean Ϯ SD. Statistical significance was determined with
analysis of variance (ANOVA) followed by Bonferroni
pairwise t-test analysis.
In situ hybridization
For this study, 6-␮m paraffin sections were loaded on
precleaned poly-L-lysine–coated slides, deparaffinized with
xylene, hydrated with graduated ethanols, and treated with
3% H2O2 in methanol to neutralize endogenous peroxidase.
Sections then were treated for 15 minutes with 12.5 ␮g/ml of
proteinase K, rinsed with 2 mg/ml of glycine, and acetylated in
0.5% acetic anhydride in 0.1 M of Tris at pH 8.0. Thereafter,
sections were postfixed with 4% paraformaldehyde/phosphate
buffered saline (PBS); prehybridization for 10Ј in 2ϫ SSC
was followed by 1 h in hybridization buffer: 50% form-
amide, 0.5 mg/ml of salmon sperm DNA, 4ϫ SSC, and 1ϫ
Denhardt. Hybridization was done overnight (18 h) at 42°C
and maximal humidity was achieved with a 5-ng/␮l digoxi-
genin (DIG)-labeled probe (see the following section). At
the end of the incubation period, slides were rinsed in SSC
under increasingly stringent conditions and then with 0.1 M
of Tris and 0.15 M of NaCl at pH 7.5. Hybrids were
detected using anti-DIG Ab’s conjugated with peroxidase
(Roche Diagnostics GmbH, Mannheim, Germany) and AEC
as a substrate, and counterstained with hematoxylin.
RESULTS
Mandibular condyle contains leptin receptors
Mandibular condyles derived from 6-day-old ICR mice
were fixed and routinely processed for paraffin embedding
as described previously. Deparaffinized sections were re-
acted with anti-leptin receptor Ab and positive staining was
detected using relevant biotinylated second Ab (Zymed
detection kit) as described previously. Staining was positive
as shown in Fig. 1. The mandibular condyle contains spe-
cific receptors for leptin (Fig. 1A), and these are especially
abundant in the chondrocytes (Fig. 1B) but also are present
in the hypertrophic cells.
Mandibular chondrocytes express heavy-type isoform
of leptin receptors
Lysates derived from 6-day-old mandibular condyles
treated and nontreated with 0.5 ␮g/ml of leptin as well as
from murine brain were separated on SDS-PAGE and elec-
trotransferred onto nitrocellulose membrane. Western anal-
ysis with anti-leptin receptor Ab (Fig. 2A) reveals a major
band that corresponds to 148 kDa and a minor one of ϳ80
kDa. Both are identical to those appearing in brain lysates.
Leptin (1.0 ␮g/ml) up-regulates the levels of its own recep-
tors; however, higher concentrations (1.5 ␮g/ml) of leptin
do not further increase the level of leptin receptors. Densi-
tometry has shown that brain tissue contains 130% more
leptin receptors than does the mandibular condyle. This
DIG-labeled antisense RNA probes for in situ
hybridization
We used probes for mouse IGF-I receptor (IGF-IR) cloned
in pBluescript SKϩ ampϩ, for mouse IGF-I cloned in pGEM3
ampϩ (386 base pairs [bp]). After linearization, antisense
RNA was transcribed with the (Sp6/T7) DIG-RNA labeling
kit (Boehringer Mannheim) according to the manufacturer’s
instructions.
Statistics
Each point in the morphometric studies represents a mean probably is caused by the low cellularity of the cartilage
of measurements performed in three to five different exper- tissue in comparison to the brain.

LEPTIN AND ENDOCHONDRAL OSSIFICATION
1037
0.5–1.0 ␮g/ml had a dose-dependent stimulatory effect on
the size of the proliferative zone of the condyle (17% and
23% increase over the control, respectively). ANOVA anal-
ysis has shown that the difference between the groups is
highly significant (p Ͻ 0/001). The stimulatory effect of
leptin was significantly reduced by 1.5 ␮g/ml of leptin (p Ͻ
0/001 compared with 0.5 ␮g/ml or 1.0 ␮g/ml of leptin,
pairwise comparison according to Bonferroni).
Leptin stimulates the differentiation of chondrocytes
within the mandibular condyle
Normal skeletal growth depends on the coupling of pro-
liferation and differentiation activities within the skeletal
growth centers. The effect of leptin on differentiation was
studied with acidic (pH 2.8) AB staining of the cartilage,
which indicates a highly sulfated proteoglycan-containing
matrix. Figures 4A and 4B depict the increase in AB stain-
ing after a 4-day culture in the presence of 1.0 ␮g/ml of
leptin (note the positive staining within the chondroprogeni-
tor zone in Fig. 4B).
Thereafter, using a more direct approach, we examined
the production of chondroitin sulfate by these cells. The
chondroitin sulfate was localized by immunohistochemistry
staining using specific anticartilage proteoglycan Ab’s. As
shown in Fig. 5, 1.0 ␮g/ml of leptin (Fig. 5B) significantly
increased the content of chondroitin sulfate within the chon-
drocytes compared with that of control (Fig. 5A), whereas
1.5 ␮g/ml had only a mild stimulatory effect (Fig. 5C).
Parallel sections reacted with normal rabbit serum served as
a negative control (data not shown).
FIG. 2. SDS-PAGE analysis of leptin receptors in control mandibular
chondrocytes, treated with 1.0 ␮g/ml of leptin, 1.5 ␮g/ml of leptin, and
brain tissue, blots were incubated with anti-leptin receptors MAb’s that
recognize the intracellular fragment, aa 877–894. (A) The 148-kDa
isoform appears in both the brain and the mandibular condyle. Densi-
tometry (B) reveals that the level of leptin receptors in the brain is
130% higher than that of the condyle. The 1.0-␮g/ml of leptin slightly
increases the level of leptin receptors.
Leptin stimulates proliferation activity in the cultured
mandibular condyle
Mandibular condyles were cultured in the presence of
various concentrations of leptin (0.25, 0.5, 1, or 1.5 ␮g/ml)
for several days with the addition of BrdU for the last 18 h
of incubation. At the end of the incubation period, condyles
were processed routinely for paraffin embedding and depar-
Leptin increases the overall size of the
mandibular condyle
The effect of leptin on the condylar growth was estimated
affinized sections were detected for BrdU-containing DNA morphometrically. Condyles cultured for 4 days in the pres-
using the Zymed BrdU detection kit (based on specific ence of either 0.5 ␮g/ml or 1.0 ␮g/ml leptin were processed
anti-BrdU Ab).
routinely for paraffin embedding, and H&E-stained sections
As shown in Fig. 3, the level of BrdU–containing nuclei were used for morphometric analysis. Results shown in Fig.
increased markedly over the untreated control after 2 days 6 reveal that treatment with 0.5 ␮g/ml leptin for 4 days
of incubation starting with 0.5 ␮g/ml of leptin (Fig. 3C vs. increases the overall size of the condyle by 8% (p Ͻ 0.05).
Fig. 3A). A lower dose of leptin (Ϫ0.25 ␮g/ml) was still However, the 1.5-␮g/ml of leptin decreases significantly
ineffective in stimulating proliferating activity (Fig. 3B).
Morphometric analysis revealed a 125% increase of the
BrdUϩ cells; this figure rose by an additional 34% with 1.0
␮g/ml of leptin. However, in the presence of 1.5 ␮g/ml of
leptin, the BrdUϩ cells increased by only 15% over the
control (Fig. 3D). This increase in proliferation activity was
(p Ͻ 0.001) the overall condylar size.
Leptin increases IGF-I-receptors in the
cultured condyle
One of the most important local regulatory factors in the
reflected by the enhancement of the chondroprogenitor development of endochondral ossification is IGF-I. We
zone. We applied ANOVA to the four groups (control group studied the effect of leptin on the expression of IGF-I
and the three different leptin doses) and found that the receptors at both the protein and the messenger RNA
difference between the groups is highly significant (p Ͻ (mRNA) levels using immunohistochemical and in situ
0.001). Bonferroni analysis of paired t-tests reveals signif- hybridization techniques, respectively. Results are depicted
icant differences between control and 0.5 ␮g/ml of leptin in Fig. 7. After 3 days of incubation, 1.0 ␮g/ml of leptin
(p Ͻ 0.001) and between control and 1.0 ␮g/ml of leptin (Fig. 7B) markedly increased the production of IGF-I re-
(p Ͻ 0.001). No significant difference was found between ceptors in the entire chondrocytic population as compared
the control and the 1.5-␮g/ml leptin-treated group. Figure with the control (Fig. 7A); 1.5 ␮g/ml had only a mild
3E shows the results of a dose-response study performed on stimulatory effect (Fig. 7C). In situ hybridization, using a
4-day leptin-treated cultures. Leptin at a concentration of specific DIG-labeled riboprobe for IGF-I receptor yielded

1038
MAOR ET AL.
FIG. 3. The effect of different leptin concentrations on cell proliferation of cultured mandibular condyles. Condyles were incubated alone or in
the presence of 0.25, 0.5, 1.0, or 1.5 ␮g/ml of leptin for 2 days. BrdU (100 ␮M) was added for the last 18 h of incubation. Sections were studied
for the presence of BrdU-labeled nuclei using anti-BrdU Ab’s. ANOVA analysis has shown that the difference between the groups is highly
significant (p Ͻ 0.001). (A) Compared with controls, incubation in the presence of 0.5 ␮g/ml of leptin for 2 days markedly increased the
BrdU-containing cell population (C) within the proliferating zone (cp); (B) 0.25 ␮g/ml is ineffective (magnification ϫ240). (D) Morphometric
analysis showed a 125% (p Ͻ 0.001 pairwise comparison according to Bonferroni) and 159% (p Ͻ 0.001, pairwise comparison according to
Bonferroni) increase in the relative BrdUϩ area induced by 0.5 ␮g/ml and 1.0 ␮g/ml of leptin, respectively. Leptin, in a dose of 1.5 ␮g/ml, was
not significantly different from that in the control. (E) Morphometric studies performed on condyles after 4 days of culture showed that 0.5 ␮g/ml
and 1.0 ␮g/ml of leptin induced an increase of 17% and 23% in the width of the chondroprogenitor zone. The stimulatory effect of leptin was
significantly reduced by 1.5 ␮g/ml of leptin (p Ͻ 0.001 compared with 0.5 ␮g/ml or 1.0 ␮g/ml leptin; ANOVA).
FIG. 4. Effect of leptin on
metachromatic staining of the
cartilaginous matrix of the man-
dibular condyle. Condyles cul-
tured for (A) 3 days alone or (B)
in the presence of 1.0 ␮g/ml of
leptin were stained in acidic (pH
2.5) AB. Leptin treatment in-
creased both the metachromatic-
stained area and the intensity of
the staining (magnification ϫ240).
positive-stained cells (red) distributed among the chondro- dose (1.5 ␮g/ml; Fig. 7F) was less potent. This indicates that
progenitor and chondrocytic cells of the 1.0-␮g/ml leptin- leptin induced a dose-dependent up-regulation of IGF-I
treated condyle (Fig. 7E) but only a very faint positive receptors in the chondrocytic population of the mandibular
reaction in the control (Fig. 7D). Again, the higher leptin condyle is at the level of gene regulation. Parallel sections

LEPTIN AND ENDOCHONDRAL OSSIFICATION
1039
FIG. 5. Synthesis of chondroitin
sulfate in the leptin-treated man-
dibular condyles. Chondroitin sul-
fate was localized in (A) untreated,
(B) 1.0 ␮g/ml of leptin-treated, or
(C) 1.5 ␮g/ml of leptin-treated
3-day cultured condyles using spe-
cific antichondroitin sulfate MAb.
The positive reaction appears in
red; nuclei are counterstained
with hematoxylin blue (magnifi-
cation ϫ240).
in the presence or absence of 1.0 ␮g/ml of leptin. Levels of
IGF-I receptor were assessed immunohistochemically. Re-
sults, shown in Fig. 8, reveal that levels of IGF-I receptors
are already increased after 2 days of leptin treatment (Fig.
9B) as compared with the untreated control (Fig. 9A).
Maximal stimulatory effect appears on the third day of
treatment (Fig. 9C); after 4 days of treatment (Fig. 9D), the
level of IGF-I receptors is similar to that of the control.
Immunoinhibition of IGF-I blunted the stimulative
activity of leptin on the condylar growth
To verify the foregoing assumption that leptin-induced
condylar growth is mediated by IGF-I system, we studied
the effect of immunoneutralization of IGF-I on leptin activ-
ity. Cultures were incubated for 3 days in the presence of
anti–IGF-I Ab diluted 1/500 with or without 0.5–1.0 ␮g/ml
of leptin. The effects of leptin in the presence of IGF-I
immunoinhibition on proliferation and differentiation activ-
ities are shown in Figs. 10 and 11, respectively.
Proliferation was studied by detection of the expression
of PCNA immunohistochemically. Results shown in Fig. 10
depict that the inductive effects of 1.0 ␮g/ml of leptin (Fig.
10B) in comparison with the untreated control (Fig. 10A)
are blunted completely in the presence of anti–IGF-I (Fig.
10C). Figure 11 shows the effects of leptin on collagen type
FIG. 6. The effect of leptin on the overall condylar growth. Mor-
phometry of H&E-stained sections derived from 4-day cultured con-
dyles treated with 0.5, 1.0, or 1.5 ␮g/ml of leptin or nontreated.
ANOVA analysis has shown that the difference between the four
groups is highly significant (p Ͻ 0.001); 0.5 ␮g/ml of leptin increased
the overall length of the condyle by 8%; 1.5 ␮g/ml of leptin decreased
significantly (p Ͻ 0.001; ANOVA) the overall condylar size.
incubated with DIG-labeled sense IGF-I receptor riboprobe II synthesis in the presence (Fig. 11C) and absence (Fig.
served as a negative control (data not shown).
11B) of anti–IGF-I antibodies. Leptin seems to increase
Quantification of leptin’s effect on the IGF-I receptors collagen type II expression within the chondroblastic zone
levels (Fig. 8) was estimated by Western analysis of tissue (cb, Fig. 11B) as compared with the control culture (Fig.
lysates derived from 3-day cultures of control, 0.5 ␮g/ml 11A). Immunoinhibition of IGF-I (Fig. 11C) decreases col-
and 1.0 ␮g/ml leptin-treated condyles (Fig. 8 A). Densitom- lagen type II secretion within the chondroblastic zone but
etry of the bands (Fig. 8B) reveals an increase of 63% and left some positive reaction within the hypertrophic zone.
79% by 0.5 ␮g/ml and 1.0 ␮g/ml leptin, respectively.
DISCUSSION
Time-course effect of leptin on the levels of
IGF-I receptors
Leptin, a small polypeptide hormone secreted primarily
Because IGF-I is considered to be a regulator of early by the adipocytes, controls body weight after its binding to
endochondral ossification functions such as proliferation a specific receptor located in the hypothalamus. However,
and differentiation, we examined the effect of leptin on the recent studies have shown that in addition to its effects on
changes in the levels of IGF-I receptors throughout the the central nervous system, leptin acts through high-affinity
culture period. Cultures were incubated for 2, 3, and 4 days leptin receptors on cells in peripheral tissues.(19) Leptin has

1040
MAOR ET AL.
FIG. 7. Leptin’s effect on the
IGF-I receptor and IGF-I receptor
mRNA abundance in the mandib-
ular condyle. Condyles cultured
(A and D) under control condi-
tions, (B and E) with 1.0 ␮g/ml
of leptin, or (C and F) with
1.5␮g/ml of leptin for 3 days
were paraffin embedded. (A–C)
Deparaffinized sections were as-
sayed for the IGF-I receptor
abundance by immunohistochem-
istry using specific rabbit anti–
IGF-I receptor Ab. The positive
cells are stained red (arrows).
(D–F) In situ hybridization tech-
nique, using DIG-labeled anti-
sense probe for IGF-I receptors
was used for assessing the IGF-I
receptor mRNA abundance. The
positive cells are stained in purple
(arrows).
know whether this isoform is identical to the long leptin
receptor b (ObR-b) isoform of the brain. However, it has
been shown that several peripheral activities of leptin prob-
ably are not mediated by the long form of the leptin recep-
tor(26) principal found in the brain. We have also found that
leptin stimulates, in a dose-dependent manner, the width of
the proliferating zone. The increment in BrdU incorporation
into DNA and the increase in the expression of chondroitin
sulfate within the cartilaginous matrix confirms that leptin
induces both proliferation and differentiation activities in
the mandibular condyle, which is reflected ultimately in an
increase in the overall size of the treated condyle.
Leptin concentrations used in this study are rather high
(0.5–1.5 ␮g/ml) in comparison with physiological levels.
Nonetheless, this range of concentrations is quite common
in other in vitro studies,(25,27,28) probably because of the fact
that part of the leptin activity is blocked by the serum
component of the medium used in in vitro studies. Using
this range of doses, we found a dose-dependent effect of
leptin on the condylar growth. In most cases, 0.5 ␮g/ml and
1.0 ␮g/ml of leptin were stimulatory and 1.5 ␮g/ml was less
effective. Because no down-regulation of leptin receptors
has been observed in the presence of 1.5 ␮g/ml of leptin, it
seems more likely that the reduction of the stimulatory
effect under higher doses of leptin probably is caused by
cross-reactivity of leptin with other cytokine type I recep-
tors that have catabolic effects on the condylar develop-
ment, for example, interleukin (IL)-6.
FIG. 8. Quantification of the effect of leptin on the levels of IGF-I
receptors in the mandibular condyle. Densitometry (B) of western
analysis (A) of 3-day-old control and 0.5 ␮g/ml or 1.0 ␮g/ml of
leptin-treated cultures reveals an increase of 63% and 79% by 0.5
␮g/ml and 1.0 ␮g/ml of leptin, respectively.
been reported to act on hematopoietic precursor cells,(20,21)
adipocytes,(22) cultured hepatocytes,(23) and pancreatic islet
cells(24) and on human marrow stromal cells enhancing
osteoblast differentiation and inhibiting adipocyte differen-
tiation.(25)
In this study, we show that chondrocytes in the skeletal
growth centers contain specific binding sites for leptin. We
found a 148-kDa isoform of leptin receptor in the chondro-
Childhood obesity is associated with accelerated growth.
Childhood growth usually is dependent on an intact and
functioning GH–IGF-I axis. However, measurements per-
formed both during sleep and over 24 h have shown that GH
secretion in children with obesity is reduced under physio-
logical conditions.(29,30) In addition, obese individuals are
cytes, which also appeared in the brain. We still do not characterized by abnormally low GH responses to pharma-

LEPTIN AND ENDOCHONDRAL OSSIFICATION
1041
FIG. 9. The effect of leptin on
the levels of IGF-I receptors
throughout the culture period:
time-course study. Condylar cul-
tures incubated under control
conditions (A) or with 1.0 ␮g/ml
of leptin for (B) 2, (C) 3, and (D)
4 days were processed routinely
for paraffin embedding. Deparaf-
finized sections were stained with
rabbit anti–IGF-I receptor Ab.
Maximum stimulatory effect ap-
pears after (C) 3 days of treatment.
FIG. 10. Effect of IGF-I immu-
noinhibition on leptin-induced
proliferation activity in the man-
dibular condyle. Condyles cul-
tured for (A) 3 days under con-
trol, (B) 1.0 ␮g/ml of leptin, or
(C) 1.0 ␮g/ml of leptin ϩ anti–
IGF-I Ab (1/500) conditions were
processed routinely for paraffin
embedding. Deparaffinized sec-
tions were stained with mouse
anti-PCNA antibodies and coun-
terstained with hematoxylin. Pos-
itively stained nuclei are red (ar-
rows; magnification ϫ240).
cologic stimulation with insulin, clonidine, arginine, lactinemia,(5,40,42) induced bioactive but nonimmunore-
levodopa, glucagon, GH-releasing hormone, and opiate active GH molecules,(43) and an increase in free IGF-I
peptides,(31–34) much like children with GH deficiency. In- levels.(37) The possibility that children with obesity may
terestingly, evaluations of serum IGF-I levels in obese chil- have an as yet unidentified “circulating factor” that stim-
dren have yielded inconsistent findings, with some stud- ulates growth independent of the presence of GH also has
ies reporting a normal level(35) and others an increase(36) been suggested.(44) The results of this study indicate that
or decrease.(37) The mechanism whereby obese children leptin might have a direct GH-independent effect on the
continue to grow despite the low levels of GH is not bone growth centers.
known. Several mechanisms have been postulated to
Like in adults, serum leptin concentrations in children
explain the “growth without GH” in obesity, such as are directly proportional to the level of adiposity.(12) In
obesity-induced hyperinsulinemia,(5,38–41) hyperpro- humans, obesity is associated with central resistance to

1042
MAOR ET AL.
FIG. 11. Effect of IGF-I immunoinhibition on leptin-induced differentiation activity in the mandibular condyle. Condyles cultured for (A) 3 days
under control, (B) 1.0 ␮g/ml of leptin, or (C) 1.0 ␮g/ml of leptin ϩ anti–IGF-I Ab (1/500) conditions were processed routinely for paraffin
embedding. Deparaffinized sections were stained with mouse anti-type II collagen antibodies and counterstained with hematoxylin. (B) Leptin
increases the positive staining within the matrix of the chondroblastic zone (cb), and (C) anti–IGF-I blunts this effect leaving only remnants of
staining within the hypertrophic zone (hc; magnification ϫ240).
circulating leptin. A differential sensitivity between the
center (hypothalamus) and periphery (epiphyseal growth
plate) to the effect of leptin might serve as an attractive
explanation for the accelerated growth in children with
obesity.
ACKNOWLEDGMENTS
We appreciate the technical assistance of Irena Reiter and
Oren Moran.
In this study, no attempt was made to study the cellular
signaling of leptin in the growing chondrocytes and the
involvement of the activation of JAK-STSAT system in the
process.(16) We found that leptin increased the immunohis-
tochemically positive staining of IGF-I receptor within the
chondrocytes and the progenitor cells. This direct in vitro
effect of leptin on the activity of the IGF system was
independent of changes in GH levels. The increment in the
abundance of IGF-I receptor suggests that the IGF system
might be involved, at least partially, in mediating the effect
of leptin on the growth center of the mandibular condyle.
The principal role played by the IGF-I system in mediating
the stimulatory effect of leptin on the condylar growth was
indicated further by the blocking the IGF-I in the leptin-
treated culture. Immunoinhibition of IGF-I completely abol-
ished the effect of leptin on both proliferation, studied by
the production of PCNA, and differentiation, studied by the
excretion of type II collagen. Further studies are needed to
verify the direct interaction between leptin, IGF, and the
binding proteins and receptors in the bony growth centers.
This study was conducted to investigate the peripheral ef-
fect of leptin on the mice mandibular condyle. The appli-
cability of leptin’s peripheral activity in mice to human
physiology should be studied further, especially in light of
the known difference in leptin’s central activity between
mice and man.
REFERENCES
1. Matson DD 1964 Craniopharyngioma. Clin Neurosurg 10:
116–129.
2. Thomsett MJ, Conte FA, Kaplan SL, Grumbach MM 1980
Endocrine and neurologic outcome in childhood craniophar-
yngioma: Review of effect of treatment in 42 patients. J Pe-
diatr 97:728–735.
3. Carmel PW, Antunes JL, Chang CH 1982 Craniopharyngio-
mas in children. Neurosurgery 11:382–389.
4. Lyen KR, Grant DB 1982 Endocrine function, morbidity, and
mortality after surgery for craniopharyngioma. Arch Dis Child
57:837–841.
5. Bucher H, Zapf J, Torresani T, Prader A, Froesch ER, Illig R
1983 Insulin-like growth factors I and II, prolactin and insulin
in 19 growth hormone-deficient children with excessive, nor-
mal, or decreased longitudinal growth after operation for cra-
niopharyngioma. N Engl J Med 309:1142–1146.
6. Stahnke N, Grubel G, Lagenstein I, Willig RP 1984 Long-term
follow-up of children with craniopharyngioma. Eur J Pediatr
142:179–185.
7. Sorva R 1988 Children with craniopharyngioma: Early growth
failure and rapid postoperative weight gain. Acta Pediatr
Scand 77:587–592.
8. Vignolo M, Naselli A, Di Battista E, Mostert M, Aicardi G
1988 Growth and development in simple obesity. Eur J Pediatr
147:242–244.
9. Vanderschueren-Lodeweyckx M 1993 The effect of simple
obesity on growth and growth hormone. Horm Res 40:23–30.
10. Veldhuis JD, Iranmanesh A, Ho KKY, Waters MJ, Johnson
ML, Lizarralde G 1991 Dual effects in pulsatile growth hor-
mone secretion and clearance subserve the hyposomatotropism
of obesity in man. J Clin Endocrinol Metab 72:51–59.
11. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Ste-
phens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ,
Bauer TL, Caro JF 1996 Serum immunoreactive-leptin con-
centrations in normal-weight and obese humans. N Engl J Med
334:292–295.
In summary, leptin apparently acts as a skeletal growth
factor with a direct peripheral effect on the skeletal growth
centers. Some of the effect leptin had on the growing bone
may be mediated by the IGF system through an increase in
local IGF-I receptor production. We speculate that the high
circulating levels of leptin in obese children contribute to
their accelerated growth.

LEPTIN AND ENDOCHONDRAL OSSIFICATION
1043
12. Hassink SG, Sheslow DV, de Lancey E, Opentanova I, Con- 30. Meistas MT, Foster GV, Margolis S, Kowarski AA 1982
sidine RV, Caro JF 1996 Serum leptin in children with obesity:
Relationship to gender and development. Pediatrics 98:201–
203.
Integrated concentrations of growth hormone, insulin,
c-peptide and prolactin in human obesity. Metabolism 31:
1224–1228.
13. Zhang Y, Proenca R, Maffei M, Barone M, Lori L, Friedman 31. Csizmadi I, Brazeau P, Serri O 1989 Effect of dietary restric-
JM 1994 Positional cloning of the mouse obese gene and its
human homologue. Nature 372:425–432.
14. Cinti S, Frederich RC, Zingarreti MC, Dematteis R, Flier JS,
tion and repeated growth hormone-releasing factor injections
on growth hormone response to growth hormone-releasing
factor in obese subjects. Metabolism 38:1016–1021.
Lowell BB 1997 Immunohistochemical localization of leptin 32. Davies RR, Turner SJ, Cook D, Alberti KG, Johnston DG
and uncoupling protein in white and brown adipose tissue.
Endocrinology 138:797–804.
15. Tartaglia LA, Demsbski M, Weng X, Deng N, Culpepper J,
1985 The response of obese subjects to continuous infusion of
human pancreatic growth hormone-releasing factor 1–44. Clin
Endocrinol (Oxf) 23:521–525.
Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, 33. Kopelman PG, Noonan K, Goulton R, Forrest AJ 1985 Im-
Muir C, Sanker S, Moriarty A, Moore KJ, Smutko JS, Mays
GG, Woolf EA, Monroe CA, Tepper RI 1995 Identification
and expression cloning of a Leptin Receptor, OB-R. Cell
83:1263–1271.
paired growth hormone response to growth hormone releasing
factor and insulin-hypoglycemia in obesity. Clin Endocrinol
(Oxf) 23:87–94.
34. Singh SK, Agrawal JK, Rai M, Gupta SS 1991 Growth hor-
mone response to clonidine in obese children. Indian Pediatr
28:737–740.
16. Houseknecht KL, Baile CA, Matteri RL, Spurlock ME 1998
The biology of Leptin: A review. Journal of Animal Science
76:1405–1420.
35. Park MJ, Kim HS, Kang JH, Kim DH, Chung CY 1999 Serum
levels of insulin-like growth factor (IGF)-I, free IGF-I, IGF
binding protein (IGFBP)-1, IGFBP-3 and insulin in obese
children. J Pediatr Endocrinol Metab 12:139–144.
36. Van Vliet G, Bosson D, Rummens E, Robyn C, Wolter R 1986
Evidence against growth hormone-releasing factor deficiency
in children with idiopathic obesity. Acta Endocrinol 279:403–
410.
37. Attia N, Tamborlane WV, Heptulla R, Maggs D, Grozman A,
Sherwin RS, Caprio S 1998 The metabolic syndrome and
insulin-like growth factor I regulation in adolescent obesity.
J Clin Endocrinol Metab 83:1467–1471.
17. Maor G, Segev Y, Phillip M 1999 Testosterone stimulates
insulin-like growth factor-I and insulin-like growth factor-I-
receptor gene expression in the mandibular condyle—a model
of endochondral ossification. Endocrinology 140:1901–1910.
18. Maor G, Hochberg Z, Silbermann M 1993 Insulin-like growth
factor I accelerates proliferation and differentiation of cartilage
progenitor cells in cultures of neonatal mandibular condyles.
Acta Endocrinol (Copenh) 128:56–64.
19. Flier JS 1997 Leptin expression and action: New experimental
paradigms. Proc Natl Acad Sci USA 94:4242–4245.
20. Bennett BD, Solar GP, Yuan JQ, Mathias J, Thomas GR,
Matthews W 1996 A role for leptin and its cognate receptor in 38. Costin G, Kogut MD, Phillips LS, Daughaday WH 1976
hematopoiesis. Curr Biol 6:1170–1180.
21. Gainsford T, Willson TA, Metcalf D, Handman E, Mcfarlane
Craniopharyngioma: The role of insulin in promoting postop-
erative growth. J Clin Endocrinol Metab 42:370–379.
C, Ng A, Nicola NA, Alexander WS, Hilton DJ 1996 Leptin 39. Holmes LB, Frantz AG, Rabkin MT, Soeldner JS, Crawford
can induce proliferation, differentiation, and functional activa-
tion of hematopoietic cells. Proc Natl Acad Sci USA 93:
14564–14568.
JD 1968 Normal growth with subnormal growth hormone
levels. N Engl J Med 279:559–566.
40. Kenny FM, Iturzaeta NF, Mintz D, Drash A, Garces LY, Susen
A, Askari HA 1968 Iatrogenic hypopituitarism in craniophar-
yngiomas: Unexplained catch-up growth in three children.
J Pediatr 72:766–775.
22. Bai Y, Zhang S, Kim KS, Lee JK, Kim KH 1996 Obese gene
expression alters the ability of 30A5 preadipocytes to respond
to lipogenic hormones. J Biol Chem 271:13939–13942.
23. Cohen B, Novick D, Rubinstein M 1996 Modulation of insulin 41. Silverman LA, Grumbach MM, Conte FA 1993 Growth and
activities by leptin. Science 274:1185–1188.
24. Shimabukuro M, Koyama K, Chen G, Wang MY, Trieu F, Lee
Y, Newgard CB, Unger RH 1997 Direct antidiabetic effect of
acromegaloid features in a hypophysectomized (HY) male:
The role of prandial hyperinsulinism (HI) [abstract]. Pediatr
Res 33(Suppl):565.
leptin through triglyceride depletion of tissues. Proc Natl Acad 42. Finkelstein JW, Kream J, Ludam A, Hellman L 1972 Sulfation
Sci USA 94:4637–4641.
25. Thomas T, Gori F, Khosla S, Jensen MD, Burguera B, Riggs
BL 1999 Leptin acts on human stromal marrow cells to en-
factor (somatomedin): An explanation for continued growth in
the absence of immunoassayable growth hormone in patients
with hypothalamic tumors. J Clin Endocrinol Metab 35:13–17.
hance differentiation to osteoblasts and to inhibit differentia- 43. Bistritzer T, Lovchik JC, Chalew SA, Kowarski AA 1988
tion to adipocytes. Endocrinology 140:1630–1638.
26. Kitawaki J, Kusuki I, Koshiba H, Tsukamoto K, Honjo H 1999
Growth without growth hormone: The “invisible” GH syn-
drome. Lancet 1:321–323.
Leptin directly stimulates aromatase activity in human lutein- 44. Geffner ME 1996 The growth without growth hormone syn-
ized granulosa cells. Mol Hum Reprod 5:708–713.
27. Ranganathan S, Ciaraldi TP, Henry RR, Mudaliar S, Kern PH
1998 Lack of effect of leptin on glucose transport, lipoprotein
lipase, and insulin action in adipose and muscle cells. Endo-
crinology 139:2509–2513.
drome. Endocrinol Metab Clin North Am 25:649–663.
Address reprint requests to:
Moshe Phillip, M.D.
28. Tena-sempere M, Pinilla L, Gonzalez LC, Dieguez C, Casan-
ueva FF, Aguilar E 1999 Leptin inhibits testosterone secretion
from adult rat testis in vitro. Journal of Endocrinology 161:
211–218.
29. Grasso P, White DW, Tartaglia LA, Leinung MC, Lee DW
1999 Inhibitory effects of Leptin-Related synthetic peptide
116–130 on food intake and body weight gain in female
C57BL/6J ob/ob mice may not be mediated by peptide acti-
Schneider Children’s Medical Center of Israel
Institute of Endocrinology and Diabetes
National Center for Childhood Diabetes
14 Kaplan Street
Petah Tiqva 49202, Israel
vation of the long isoform of the leptin receptor. Diabetes Received in original form June 6, 2001; in revised form November
48:2204–2209. 30, 2001; accepted January 7, 2002.