Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

To evaluate the relationship between osteoblast differentiation and bioener getics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorb ate treatment, the rate of glucose consumption was 320 nmol . h(-1) . 10(6) cells(-1), respiration was 40 nmol . h(-1) . 10(6) cells(-1), and the rati o of lactate production to glucose consumption was similar to 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascor bate treatment for 14 days led to a fourfold increase in respiration, a thr eefold increase in ATP production, and a fivefold increase in ATP content c ompared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mat ure cells possessed abundant amounts of high-transmembrane-potential mitoch ondria, which were concentrated near the culture medium-facing surface. Acu te treatment of mature osteoblasts with metabolic inhibitors showed that th e rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabol ism and mitochondrial activity, which are likely to play key roles in osteo blast function.