Original ArticlesInsulin receptor expression in primary and cultured osteoclast-like cells
Introduction
Insulin is known to have generally anabolic actions in bone, and insulin deficiency, as exemplified by insulin-dependent diabetes mellitus, is associated with decreased bone mass in animal models and in diabetic patients.3 These effects are currently ascribed to deficiency of insulin action upon osteoblasts within bone. Osteoblasts are known to express insulin receptors,13, 18, 24 and insulin has been shown to modulate collagen synthesis,4 alkaline phosphatase activity5, 14 and parathyroid hormone responsiveness23 in studies using in vitro osteoblast cultures. Insulin sensitivity is decreased in osteoporotic bone using rat models, in which insulin has been shown to stimulate alkaline phosphatase activity, cell number, and bone mineralization.28
Bone is the product of opposing, but coordinated, dynamic processes of formation and resorption. Osteoblasts synthesize osteoid that subsequently mineralizes, while the resorption of bone is chiefly carried out by osteoclasts. It is tempting to speculate that the anabolic actions of insulin might result from coordinated regulation of bone formation and resorption. Surprisingly, few published data exist on the actions of insulin on bone resorption, and these data are principally derived from calvarial organ culture prelabelled with 45Ca. Insulin was found to have little effect on 45Ca release in this system.20 However, this model is relatively insensitive and difficult to interpret, because calcium release is the net result of multiple cellular and physical processes.
The development of culture systems, which permit the generation of authentic functional osteoclasts for study in vitro, has provided the opportunity to investigate the role of insulin on osteoclast differentiation and function. The present study describes the use of immunocytochemistry and insulin binding studies to demonstrate the expression of insulin receptors by both mononuclear and multinucleated osteoclast-like cells. Evidence has also been obtained for a functional role for insulin in inhibiting pit formation by osteoclast-like cells on dentine slices.
Section snippets
Cell culture
Mouse osteoclast-like multinucleated cells were prepared as previously reported.22 Primary osteoblastic cells were obtained from newborn mouse calvaria and nucleated marrow cells were obtained from the long bones of 7–9-week-old male C57BL/6J mice. The mice were killed by cervical dislocation and the femora, tibiae, and humeri aseptically removed and stripped of adherent tissue. The ends of the bones were removed and the marrow cavity flushed out using 1 mL settling medium [α-Modified Eagles
Insulin receptor immunocytochemistry
As shown previously,22 coculture of mouse marrow cells with primary osteoblasts in the presence of 1,25(OH)2 vitamin D resulted in the generation of osteoclast-like cells. The osteoclast-like nature of these cell was indicated by positive TRAP staining and CTR expression (data not shown), and the ability to resorb bone on dentine slices (see below). Figure 1A demonstrates the absence of staining in either osteoblasts or osteoclast-like cells in the absence of primary insulin receptor antibody
Discussion
This article provides, to our knowledge, the first evidence that osteoclasts possess insulin receptors. This finding has been demonstrated by immunocytochemistry and by ligand binding using 125I-labeled insulin. The true osteoclastic nature of multi- and mononucleated cells grown in cocultures was demonstrated by TRAP staining, bone resorption, and calcitonin binding. In the cocultures, osteoclast-like cell enrichment resulted in increased specific insulin binding, in parallel with increased
Acknowledgements
The authors would like to express gratitude to Professor T. J. Martin, Dr. S. D. Rogers, and Dr. K. W. Ng for their helpful advice and support. D.M.T. was the recipient of the John Hayden Fellowship.
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