Glucose transport in osteoblast-enriched bone explants: Characterization and insulin regulation

Insulin has potent effects on osteoblast function both in vivo and in vitro. In various insulin-sensitive tissues, stimulation of glucose transport and metabolism are hallmarks of insulin action, and have been postulated to play a role in insulin regulation of cellular function. However, insulin effects on glucose metabolism in osteoblast-like cells have not been demonstrated. Therefore we examined the in vitro effects of insulin on hexose uptake in an osteoblast-enriched rat bone explant preparation. Uniform 5-mm-diameter punch sections were obtained from the cartilage-free frontal portions of the calvaria of 3-day-old rats, and the periosteum was removed. The resulting sections contained a highly enriched population of osteoblast-like cells as determined by histologic criteria, elimination of calcitonin-stimulatable cAMP generation, and enhancement of PTH-stimulatable cAMP generation per microgram of DNA. Sections were incubated for 24 hr at 37°C in BGJb medium and then transferred to modified glucose-free Krebs-Ringer bicarbonate buffer for 2-deoxy-D-glucose (2-DG) uptake studies. 3H-2-DG uptake was linear with time over 60 min, temperature sensitive, and inhibited by 5 mA/phloridzin. Kinetic analysis of 2-DG uptake at 25°C demonstrated a saturable transport mechanism with a Km of 2.2 mM, similar to that observed for 2-DG transport in other tissues. Studies of competitive inhibition by other sugars demonstrated a transport specificity for 2-DG that was comparable to that previously observed in fat and muscle cells. 2-DG uptake was rapidly stimulated by insulin (0.5 nM-1 μM), with maximal insulin effect observed at 50 nM. Concanavalin A (10–50 μg/ml) produced a similar stimulation of 2-DG transport. Both basal and insulin-stimulated uptake were inhibited reversibly by cytochalasin B (50 μM). Insulin stimulation of 2-DG uptake was the result of increased transport Vmax, with Km remaining constant. These studies provide the first demonstration that the cell population of osteoblast-enriched bone explants possesses an insulin-stimulatable glucose transport system with characteristics similar to those found in other insuln-sensitive tissues.