טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBen-Eliezer Miri
SubjectLeptin as a Skeletal Growth Factor
DepartmentDepartment of Biotechnology
Supervisors Professor Raymond Coleman
Professor Moshe Philip
Dr. Gila Maor


Abstract

Childhood obesity is frequently associated with an increase in height velocity and acceleration of epiphyseal growth plate maturation, despite low levels of serum growth hormone (GH). Obesity is also associated with higher circulating levels of leptin, a16 kD protein, which is secreted from adipocytes. In the present 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 μg/ml and 1.0 μg/ml stimulated the width of the chondroprogenitor zone and the overall condylar length, whereas higher concentrations had an inhibitory effect. We also found that leptin can induce both proliferation and differentiation activities in the mandibular condyle, in a dose-dependent manner. The influence on proliferation was confirmed by our findings of an increase in BrdU (Bromo-deoxy-Uridine) incorporation into DNA and an increase in PCNA (Proliferating Cell Nuclear Antigen) levels. These factors represents two different indicators of cell replication: BrdU is a labeled nucleotide that incorporates into the DNA during cell division, while PCNA is a DNA-polymerase cofactor, that is expressed during the S phase of the cell cycle. Using Alcian blue staining of the cartilaginous matrix, as well as immunohistochemistry and in situ hybridization techniques, we were able to prove that leptin also caused an elevation in the lavels of collagen type II, chondroitin-6-Sulfate and proteoglycans. The synthesis rate of these factors by the chondrocytes reflects the extent of differentiation processes in the mandibular condyle. Next we studied leptin's mode of action in stimulating growth, and were able to identify its specific receptor (OB-R) in the condyle. Using a Western blot analysis, we demonstrated that the mandibular condyle contains a long, and probably functioning, isoform of the OB-R. Leptin also increased the abundance of IGF-I and its receptors’ levels and transcription rate within the chondrocytes and the progenitor cell population. IGF-I activity seems to be pivotal for the leptin-induced increased skeletal growth, since immunoinhibition of IGF-I abolished the inductive effect of leptin.
Our results indicate that leptin acts as a skeletal growth factor, and that its influence on bone may be mediated by increasing production of and sensitivity to local IGF-I. We believe that high circulating levels of leptin might contribute to normal height velocity in obese children.