Background: Bone and glucose metabolism are closely associated with each other. It has been shown that undercarboxylated osteocalcin regulates glucose homeostasis, and that the activity of osteocalcin requires its production by osteoblasts and decarboxylation by osteoclasts. Because osteocytes play important roles in bone remodeling by producing RANKL and sclerostin, osteocytes may be involved in glucose homeostasis. However, it is unknown whether osteocytes regulate the expression of RANKL, osteoprotegerin, and sclerostin as well as osteocalcin activity by sensing extracellular glucose levels.

Methods and results: We used MLO-Y4-A2 a murine long bone-derived osteocytic cell line, and confirmed that glucose transporter (GLUT) 1 was expressed in MLO-Y4-A2 cells, but not GLUT2, 3, or 4. Furthermore, treatment with phloretin, a GLUT inhibitor, (10-100 mM) significantly inhibited glucose uptake. Real-time PCR and western blot analysis showed that phloretin significantly and dose-dependently decreased the expression of RANKL and osteocalcin, whereas osteoprotegerin and sclerostin were not affected. Because phloretin activated AMP-activated protein kinase (AMPK), which is an intracellular energy sensor, we investigated whether AMPK activation would modulate the RANKL and osteocalcin expression. Coincubation of Ara-A, an AMPK inhibitor, with phloretin, significantly canceled the phloretin-induced decrease in osteocalcin expression, but not RANKL. In contrast, phloretin suppressed phosphorylation of ERK1/2, JNK, and p38 MAPK, and treatments with a JNK inhibitor SP600125, a p38 inhibitor SB203580, or a MEK inhibitor PD98059, significantly decreased the expression of RANKL and osteocalcin.

Conclusion: This is the first study to show that glucose uptake by GLUT1 is required for RANKL and osteocalcin expression in osteocytes, and that inhibition of glucose uptake decreases their expression through AMPK and MAPK pathways. These findings suggest that lowering glucose uptake into osteocytes may contribute to maintain blood glucose levels by decreasing osteocalcin expression and RANKL-induced bone resorption, resulting in suppressing the activation of osteocalcin.