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Institute for Endocrinology and Diabetes, National Center for Childhood Diabetes, Schneider Children’s Medical Center of Israel and Felsenstein Medical Research Center (G.G.-Y., T.B.-A., B.S., O.P., O.M., R.E., M.P.), Petah Tikva 49202, Israel; Sackler School of Medicine, Tel Aviv University (G.G.-Y., T.B.-A., B.S., O.P., O.M., R.E., M.P.), Tel Aviv 69978, Israel; Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine (G.M.), Technion, Haifa 31096, Israel; and Laboratory of Molecular Endocrinology, Ben Gurion University of the Negev (Y.S.), Beer Sheva 84105, Israel
Address all correspondence and requests for reprints to: Moshe Phillip, M.D., Institute for Endocrinology and Diabetes, National Center for Childhood Diabetes, Schneider Children’s Medical Center of Israel, 14 Kaplan Street, Petah-Tikva 49202, Israel. E-mail: mosheph{at}post.tau.ac.il.
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Abstract |
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Introduction |
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Leptin, a hormone secreted from the adipocytes, was originally described as a circulating hormone involved in feeding behavior and energy homeostasis (1). Later, it was found to be a pleiotropic hormone involved in the regulation of a wide range of physiological processes (2, 3, 4, 5, 6, 7, 8), including bone density (9, 10). The effect of leptin on bone remains controversial (9, 10, 11, 12, 13, 14). Only a few reports on its effect on chondrocytes and cartilage have been published.
Low caloric intake due to malnutrition or malabsorption, particularly in critical periods of growth and development, is often the underlying cause of longitudinal growth failure and short stature. At the other extreme, hyperphagia and obesity during infancy and childhood might be associated with accelerated growth (15). In our recent series of ex vivo experiments using the well-known mandibular condyle model of endochondral ossification, we found that leptin stimulates both the proliferation and differentiation of growth plate chondrocytes (16).
In the present work we describe a set of in vivo experiments designed to determine whether leptin stimulates longitudinal growth also in vivo even in the presence of caloric restriction.
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Materials and Methods |
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Anti-IGF-I receptor (anti-IGF-IR
) was obtained from Santa Cruz Biotechnology, Inc. (catalog no. sc-712, Santa Cruz, CA), antiproliferating cell nuclear antigen (anti-PCNA) was purchased from Zymed Laboratories, Inc. (catalog no. 08-0110), antitype II collagen were obtained from Chemicon International (MAB8887, Temecula, CA), and antitype X collagen was purchased from NeoMarkers (MS-852-B0 (Labvision Corp., Fremont, CA).
Animals
Prepubertal male ICR mice (25 d old), purchased from Harlan (Jerusalem, Israel), were housed in the animal care facility of Felsenstein Medical Research Center. The animals were maintained on standard rodent chow, given water ad libitum, and housed individually under circadian lighting conditions (12-h light, 12-h dark cycle; lights off at 1800 h). All animals had access to a large tube within their cage for shelter. The animals were observed daily, and all remained bright, alert, and active, with no evidence of any disorder, throughout the study. The Tel Aviv University animal care committee approved all procedures.
Experiment 1
The animals were divided into three groups of six mice each. Two groups were fed ad libitum and received ip injections of either vehicle only (1 mg/ml BSA in PBS) or leptin in vehicle (8 µg/g body weight·d ovine leptin) (17). These concentrations were previously shown to have a central effect on mouse appetite and body weight (18) as well as on puberty (19). To dissociate leptin’s effect on growth from its effect on food consumption, the third group was pair-fed with the leptin group and given injections with carrier only (1 mg/ml BSA in PBS). To accurately measure the amount of food consumed by the leptin-treated group, the sawdust at the bottom of the cage was replaced by paper covered with a fine metal mesh. The same amount of food was then given to the pair-fed group the following day. All injections were administered at 1700 h on the basis of earlier findings that leptin affects food consumption primarily during the dark phase (19). The animals and their chow were weighed at the same time.
Experiment 2
The animals were divided into three groups. The first group was fed ad libitum and received injections of vehicle alone. The other two groups were fed 60% of the food intake of the first group and received injections of either leptin or vehicle only.
Animals in both experiments were killed on d 12, and changes in body weight and tibia length were determined. Trunk blood was collected, the tibias were removed, and morphological and functional changes were assayed by specific staining or immunohistochemistry.
Biochemical studies
Serum levels of GH and IGF-I were measured with a double antibody RIA (20). Acidic extraction was used to detach IGF-I from its binding proteins; therefore, the results represent the total IGF-I level.
Morphological studies
For morphological analysis, the tibias were processed for paraffin embedding. Paraffin sections (5 µm) were deparaffinized in xylene, hydrated in graduated ethanols, and pretreated with 3% acetic acid for 3 min. These were then stained with 1% Alcian Blue at pH 2.5 for 30 min, thoroughly rinsed with tap water, and counterstained with hematoxylin-eosin. We analyzed the proximal growth plate, which grows faster and for a longer time then the distal growth plate (21). The size of the EGP was measured by drawing a straight line from the apical border of the reserve zone cells to the lower border of the mineralized cartilage. The findings presented represent the average of 11 measurements in 2 sections from each animal. Two individuals blinded to the source of the slide made each measurement. Statistical analyses showed no significant differences in measurements between the 2. Morphometric analyses were performed with an Olympus DP-10 digital camera with appropriate morphometric software (Olympus DP-soft, Olympus Optical Co., New Hyde Park, NY). The proliferating zone was defined as the layer between the apical border and the end of the proliferative columns. The hypertrophic zone was defined as the remaining area. These two zones were divided to give the ratio of proliferation to hypertrophy.
5-Bromo-2'-deoxyuridine (BrdU) incorporation into DNA
To follow the proliferation of chondroprogenitor cells in the skeletal growth centers, newly synthesized DNA was labeled with BrdU by injection of the mice with 50 µg/g body weight BrdU 3 h before the mice were killed. Deparaffinized sections served for the detection of BrdU-labeled cells, using the BrdU staining kit (catalog no. 93-3943, Zymed Laboratories, Inc., San Francisco, CA) in accordance with the manufacturer’s instructions.
Immunohistochemistry
Deparaffinized sections were incubated for 25 min in 3% H2O2 in methanol to inactivate endogenous peroxidases, blocked with 10% nonimmune serum compatible with the second antibody, and incubated with a specific antibody. Positive binding was visualized with the appropriate biotinylated second antibody and streptavidin-peroxidase conjugated with aminoetyl carbazole as a substrate (Histostatin-SP kit, Zymed Laboratories, Inc.). Counterstaining was performed with hematoxylin. To detect type II and X collagen, an additional step of protein digestion was necessary. For type II, the slides were incubated with 1 mg/ml pepsin in Tris-HCl, pH 2.0, for 10 min at 37 C. For type X, 5 min of microwave boiling in retrieval buffer (2 mM citric acid and 8 mM sodium citrate, pH 6.0) was required before pepsin treatment. Negative controls were incubated with a nonimmune serum of the same species in which the first antibody was raised.
Preparation of probes for in situ hybridization
The probe for IGF-I receptor contained a 417-bp EcoRI/BamHI fragment encoding exon 3, cloned into pBluescript SK+. A specific sense probe was produced from the same plasmid to serve as a negative control.
Antisense and sense RNA probes for in situ hybridization were produced by transcription using the (T7/T3) digoxigenin (Dig) RNA labeling kit (Roche, Mannheim, Germany) in accordance with the manufacturer’s instructions.
In situ hybridization
Deparaffinized sections loaded on precleaned poly-L-lysine-coated slides were treated with proteinase K (12.5 mg/ml in Tris-EDTA buffer) for 15 min at 37 C, acetylated in 0.1 M Tris-HCl (pH 8.0) in 0.5% acetic anhydride, and postfixed for 5 min in 4% paraformaldehyde (in 1 M PBS, pH 7.4). Prehybridization was performed by 10-min incubation in 2x SSC (20x SSC = 0.3 M sodium citrate and 3 M sodium chloride), followed by 30 min in a hybridization buffer (50% formamide, 0.5 mg/ml salmon sperm DNA, 4x SSC, and 1x Denhardt’s solution). Hybridization was performed overnight (18 h) at 42 C in maximal humidity with a 5 ng/µl Dig-labeled probe. At the end of the incubation period, slides were rinsed in SSC at increasing stringency conditions. Hybrids were detected using anti-Dig antibodies conjugated with biotin and a secondary antibody conjugate with streptavidin-peroxidase, as described for immunohistochemistry.
RNA extraction and cDNA preparation
Total RNA was prepared from the EGP tissue by homogenization in a guanidinium isothiocyanate solution essentially as previously described (22). Homogenization was followed by 20-min incubation with 0.72 g/liter proteinase K in 19 mM Tris-HCl (pH 7.0), extraction with phenol/chloroform/isoamylalcohol, and precipitation with isopropanol. To avoid possible contamination of genomic DNA, the samples were treated with RQ1 ribonuclease-free deoxyribonuclease (Promega, Madison, WI) before cDNA synthesis. cDNA was synthesized with the EZ-First Strand cDNA kit (Biological Industries, Beit Hemeek, Israel) and random primers according to the manufacturer’s instructions. The product was then employed as a template for PCR amplification under standard conditions. The primers used for the amplification were as follows: primers for collagen type II: forward, TTA GAA AGG GGA GCA CAG TCC; reverse, TAC ACT GCC ATG AAG CAT GG; and primers for collagen type X: forward, CAG AGG AAG CCA GGA AAG C; reverse, GGT GTC CAG GAC TTC CAT AGC (23). To specifically identify the long form of the leptin receptor (Ob-Rb) we used primers derived from exon 18 of the leptin receptor (24): forward primer, GGT CTC AGA GCA CCC AGG TA; and reverse primer, TGG ATA AAC CCT TGC TCT TCA. The reaction consisted of an initial denaturation step at 94 C for 4 min, followed by 40 cycles of denaturation at 94 C (45 sec); annealing at 46.4 C (collagen II), 57 C (collagen X), or 60 C (OB-Rb; 45 sec); and extension at 72 C (1 min). A final extension step of 10 min at 72 C terminated the reaction. The products were analyzed on 2% agarose gel to confirm the success and specificity of the reaction.
Statistical analysis
The data were analyzed using BMDP (25). Groups were analyzed using ANOVA with Bonferonni’s correction for multiple comparisons. Longitudinal data were compared using ANOVA with repeated measures. P 
0.05 was considered significant.
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Results |
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, lane 1 in B and C, respectively). A clear Ob-Rb-specific PCR product was observed in the EGP lane (Fig. 1A, lane 1), indicating that the EGP tissue expresses the long form of leptin receptor. We next compared the longitudinal growth of the leptin-treated and pair-fed animals. The experiment yielded several important results.
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). The percent weight gain in the control mice was 26.4% compared with only 16.0% in the leptin-treated and pair-fed mice. By the end of the experiment, the average weights of the leptin-treated group (27.63 g) and the pair-fed group (27.8 g) were indistinguishable (P = 0.873) and significantly lower than that of the fed ad libitum control group (31.63 g; P < 0.001).
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) and significantly longer than that in the weight-matched pair-fed group.
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Leptin did not distort the architecture of the EGP. Bone growth is associated with endochondral ossification, which involves the proliferation of chondrocytes, followed by their maturation into hypertrophic chondrocytes that secrete extracellular matrix. At this stage, cells cease to divide and undergo programmed cell death. This is accompanied by vascular invasion, mineralization of the extracellular matrix, and replacement of the cartilage scaffold with bone tissue. Figure 3 demonstrates the general histological architecture of the proximal EGP in the control pair-fed and leptin-treated mice. The characteristic arrangement of the EGP was unchanged after leptin treatment.
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), thus indicating a greater proliferation rate.
Leptin stimulated differentiation activity in the chondrocytes of the tibia growth plate. The effect of leptin on the differentiation of the growth plate was studied by using monoclonal antibodies directed against two specific markers of differentiation: type II collagen, an early and abundant marker of chondrocytes, and type X collagen, a nonfibrillar, network-forming collagen, characteristic of mature chondrocytes (28). Immunohistochemical analysis clearly showed that leptin induced differentiation of the chondrocytes at both the young and mature chondrocytic phases (Fig. 4, B and D compared with A and C).
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, the levels of IGF-IR protein (A and B; determined by immunohistochemistry with an antibody directed against IGF-IR) and mRNA (C and D; detected by an IGF-IR mRNA probe in in situ hybridization) were elevated in the mature, prehypertrophic cells. No IGF-IR mRNA was detected in young chondrocytes in either the presence or absence of leptin. The effects on the protein and mRNA levels colocalized to the same cellular zone.
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shows that the average weight of both groups was similar and significantly lower than that of the control group fed ad libitum (P < 0.001; see Table 2).
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). This effect was achieved despite the reduced body weight compared with the ad libitum control group and the similar weight gain compared with the other semistarved group. These results indicate that caloric deprivation by itself reduced tibial length by 5%. Peripheral leptin administration corrected this growth deficiency by 3%, bringing the tibia to almost normal size.
Starvation reduced the growth of the EGP. Measurement of EGP width on deparaffinized slides exposed to hematoxylin-eosin staining showed an average reduction of 26% in the semistarved mice (Table 2, weight control group) with no disorganization. No significant difference was noted in the length of the EGP between the two semistarved groups.
Unlike the marked effect shown in the pair-fed experiment, there were no significant differences in either proliferation (measured both by BrdU incorporation or PCNA immunohistochemistry) or differentiation (measured by following the expression of collagen II and collagen X) after 12 d in the leptin-treated mice compared with controls.
Serum GH levels were measured in all mice. Using ANOVA, we failed to demonstrate any significant difference in serum GH levels between the groups (data not shown).
Leptin further reduced circulating IGF-I levels. Semistarved mice showed a 70% decrease in circulating free IGF-I compared with animals fed ad libitum. In the semistarved, leptin-treated mice, serum levels of IGF-I fell even further, reaching a level only 10% of that in control mice fed ad libitum. The reduction in IGF-I was statistically significant (P < 0.001; Fig. 7).
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Discussion |
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To the best of our knowledge, this study demonstrates for the first time the presence of the long form of the leptin receptor mRNA in the mice EGP tissue, thus indicating that leptin may indeed have a direct effect on the chondrocytes of the EGP.
The present report documents a significant stimulatory effect of leptin on longitudinal growth in a mouse model, even in the presence of low caloric intake. The results clearly support our previous ex vivo findings; namely, that leptin is a skeletal growth factor and can induce longitudinal growth. Moreover, in our first experiment, although leptin administration led to reduced food consumption and reduced body weight, the treated animals had longer tibia. Normal skeletal growth requires coupling of proliferation and differentiation processes, and the relative rate of each affects the extent to which the growth plate elements continue to grow. We found that leptin stimulation of the EGP was balanced, positively affecting both proliferation and differentiation, so that the ratio between the proliferating and hypertrophic chondrocytes remained constant.
The second experiment tested the potency of leptin as a growth factor in the presence of a more severe caloric restriction. To minimize stress, we used a semistarvation protocol, reducing the animal’s caloric intake by only 40% for 12 d. As anticipated, the caloric deprivation had a significant effect on growth. The average body weight of the semistarved leptin group was similar to that of the semistarved controls. These results suggest that when food is available, leptin affects the animals’ appetite and hence their body weight. However, when food is scarce, leptin cannot suppress the need for food or reduce food consumption any further.
In both experiments, the length of the tibia increased significantly in the leptin-treated animals compared with the pair-fed controls in the first experiment and the semistarved weight-matched mice in the second. In the first experiment elongation of the EGP was significantly greater in the leptin-treated mice; in the second, we could not detect significant differences in the width of the EGP. It is possible that this difference was due to the difference in the amount of food consumed. The length of the EGP in the first experiment was reduced by only 4% after caloric restriction (which was leptin induced), whereas in the second experiment (semistarvation model) it was reduced by 26% compared with that in the group of mice fed ad libitum. Therefore, this may be the reason for the reduced histological effect of leptin on proliferation and differentiation after prolonged exposure to a significant caloric restriction.
In the calorie-restricted experiment (experiment 2), serum IGF-I levels dropped in association with the caloric deprivation. Leptin injections failed to increase serum IGF-I levels. The mechanism by which leptin was able to stimulate tibial length is not clear and is difficult to understand in light of the different effects leptin exerts in the serum and locally within the EGP. In starvation experiments performed in rodents, it was shown that the serum GH level dropped in response to a reduction of food consumption (33, 34, 35, 36, 37, 38, 39). Leptin administration to fasted rats restored their blunted GH secretion (33, 34, 39). Our study performed in semistarved mice showed a similar trend in serum GH level; however, the results did not reach statistical significance. The reduction in serum IGF-I levels after semistarvation was more pronounced and statistically significant and did not increase in response to leptin injections, similar to the results observed in fasted mice (40, 41).
We have shown previously, using an isolated organ model of the mandibular condyle, that leptin significantly stimulates both proliferation and hypertrophy of the chondrocytes. In the organ culture, leptin increases IGF-IR at both the mRNA and protein levels despite the absence of GH (16). Moreover, immunoblocking of IGF-I with anti-IGF-I antibodies partly abolished the effect of leptin on proliferation and differentiation. In the present in vivo system, leptin also stimulated mRNA expression and protein abundance of IGF-IR in mature prehypertrophic and hypertrophic chondrocytes in the EGP. We do not yet know what is the dominant mediator of leptin’s effect on tibial growth in the in vivo model. Is it due to a small increment in serum GH levels (which we failed to detect), which might directly stimulate the EGP? Does GH stimulate small changes in local IGF-I gene expression, which interact with the elevated IGF-IR? Does leptin stimulate other growth mechanisms entirely independent of the GH-IGF-I axis? More studies are needed to clarify the precise mechanism mediating the presently documented effect leptin has on tibial growth and its ability to overcome the growth deceleration of caloric restriction.
In conclusion, leptin injections in mice can overcome calorie-deprived longitudinal growth arrest. Leptin can at least partially bypass the caloric restriction effect and might therefore link nutritional status with bone growth.
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Acknowledgments |
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Footnotes |
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Received July 21, 2003.
Accepted for publication September 25, 2003.
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) compared with leptin-treated (
) and pair-fed animals (
). Starting from initial weights that were essentially identical, control mice increased in weight by 26.4%, whereas leptin-treated and pair-fed mice gained only 16.0%. Day 1 is the first day of the experiment.
