Leptin is a major hormonal product of the adipocyte which regulates appetite and reproductive function through its hypothalamic receptors. It has now become clear that leptin receptors are much more widely distributed than just the hypothalamus, and the skeleton has emerged as an important site of action of leptin. The signalling form of the leptin receptor has been found in several cell types including human osteoblasts, rat osteoblasts and human chondrocytes. In vitro we have shown leptin to an anabolic factor, stimulating osteoblast proliferation and inhibiting osteoclastogenesis. Leptin increases bone mass and reduces bone fragility when administered peripherally but has an indirect inhibitory effect on bone mass via the hypothalamus when administered directly into the central nervous system. Data from animal models where there is an absence of either leptin production (ob/ob) or its receptor (db/db) have been contradictory. In this study we compared the bone phenotype of leptin receptor-deficient (db/db) and wild-type (WT) mice. Micro-CT analysis was done on proximal tibiae using a Skyscan 1172 scanner. Db/db mice had significantly reduced trabecular bone volume, trabecular thickness and trabecular number and a higher degree of trabecular separation. Cortical bone was also significantly lower in db/db animals in volume, cross-sectional thickness and perimeter. These results demonstrate that in the absence of leptin signalling there is reduced bone mass indicating that leptin indeed acts in vivo as a bone anabolic factor, mimicking the in vitro results.
Adiponectin, a hormone secreted by adipocytes, regulates energy homeostasis and glucose and lipid metabolism. Plasma levels of adiponectin are negatively correlated with body fat mass. Adiponectin inhibits the formation and activity of osteoclasts and increases the proliferation and differentiation of osteoblasts in vitro. The aim of our study was to determine the bone phenotype of adiponectin knockout mice. Male adiponectin-deficient (Ad-KO) and wild-type (WT) C57BL/6J mice were sacrificed at 8, 14 and 22 weeks of age. Body weights did not differ between Ad-KO and WT mice. We scanned the left proximal tibia using micro-CT at 5μm resolution and analysed bone microarchitecture by 3D analysis. We found significant increases in trabecular bone volume (BV/TV) (15.9±1.63 vs. 12.2±0.72%, p=0.02) and trabecular number (3.20±0.18mm-1 vs. 2.32±0.12mm-1, p=0.0009) in 14-week old Ad-KO mice compared to controls. Similar differences between WT and Ad-KO were present in 8 and 22-week old animals but these did not reach statistical significance. Trabecular thickness was significantly greater (0.053±0.001mm vs. 0.048±0.002mm, p=0.04) in 22-week old Ad-KO mice compared to WT. Ad-KO mice have increased number and volume of trabeculae at 14 weeks of age indicating that the net effect of adiponectin on bone accrual in vivo is inhibitory. These effects are age-dependent. Our data concur with the observations from epidemiological studies in humans that adiponectin negatively correlates with both fat mass and bone mass. Therefore, adiponectin may be a contributor to the link between fat and bone mass.
Paget’s disease of bone is a common disorder characterised by focal areas of increased bone resorption coupled to increased and disorganised bone formation. Pagetic osteoclasts have been studied extensively, however, due to the integral cross-talk between osteoclasts and osteoblasts, we propose that pagetic osteoblasts may also play a key role in the pathogenesis of Paget’s disease. Any phenotypic changes in the diseased osteoblasts are likely to result from alterations in the expression levels of specific genes. To determine any differences in expression between pagetic and non-pagetic osteoblasts and their precursors the gene expression profiles of RANK, RANKL, OPG, VEGF, IL-1beta, IL-6, MIP-1, TNF and M-CSF were investigated in primary cultures of human osteoblasts and in the osteoblast precursor population of bone marrow stromal cells. We present preliminary data of this study. Trabecular bone explants were finely chopped, washed free of marrow and cellular debris then either snap frozen in liquid nitrogen or placed in flasks to culture outgrowth osteoblast-like cells. Mononuclear stromal cells from bone marrow were isolated and grown in culture flasks. RNA and conditioned media were collected from cultured osteoblasts and stromal cells at confluency. The innovative method of Real-Time PCR, the most accurate technique available at present to quantitatively measure gene expression, was used for the comparison of gene expression levels in our samples. 18S ribosomal RNA was used as an endogenous control to normalise the expression in the various samples. RANK, MIP-1 and TNF were only detected in stromal cells whereas RANKL, OPG, VEGF, IL-1beta, IL-6 and M-CSF were detected in both osteoblasts and stromal cells. OPG displayed higher expression in osteoblasts while IL-1beta showed higher expression in stromal cells. To date we have not seen any significant differences in gene expression between pagetic and non-pagetic subjects when comparing a small number of samples. A larger cohort is currently being investigated. We are also comparing levels of secreted proteins in the conditioned media from pagetic and non-pagetic cell cultures. This may lead to further candidate genes involved in the pathology of the pagetic lesion.
