Osteocalcin enhances glucose uptake by mediating AMP-activated protein kinase (AMPK) in human skeletal muscle cell

Abstract

Osteocalcin (OC) is an osteoblast-derived bone matrix protein which also presents in blood circulation in the form of carboxylated OC (cOC) or uncarboxylated OC (ucOC) according to its γ-carboxylation status of glutamyl residues at positions 17, 21, and 24. Evidences from mice showed that ucOC is a hormone exerting metabolic effects on glucose metabolism by enhancing insulin sensitivity and promoting insulin secretion. GPRC6A is suggested as a receptor relaying metabolic effects of OC by triggering Gq/11 signaling pathway with IP3. However, GPRC6A is atypical that it is also coupled to Gαs/i protein and was reported to increase cAMP and ERK phosphorylation in other studies.In this study, the effects of both human native OC (nOC; a mixture of cOC and ucOC) and recombinant OC (rOC, all ucOC) on 2-NBDG uptake and the roles of GPRC6A were investigated in human primary muscle cells.Both nOC and rOC significantly increased 2-NBDG uptake in human primary skeletal muscle cells; this increase was mediated by AMPK. AMPK inhibition blunted OC-mediated 2-NBDG uptake. Intracellular IP3 production and subsequent Ca2+ mobilization increased by nOC and rOC; these responses were attenuated by inhibition of PLC. These suggest that OC activates PLC to trigger phosphatidylinositol signal pathway and GPRC6A is coupled to Gq/11– protein in human skeletal muscle. Increased cytosolic free Ca2+ acted as a secondary messenger to regulate CaMMKα, a direct upstream kinase of AMPK. Decreasing free cytosolic free Ca2+ by BAPTA-AM, U-73122, and 2-APB attenuated rOC-mediated 2-NBDG uptake and AMPKαThr172 phoshorylation; direct inhibition of CaMKKα also attenuated 2-NBDG uptake and AMPKαThr172 phoshorylation. GPRC6A inactivation attenuated rOC-mediated 2-NBDG uptake, AMPKαThr172 phoshorylation, IP3 production, and intracellular Ca2+ mobilization, supporting that GPRC6A is an OC receptor

relaying its physiological responses by triggering phosphatidylinositol signal pathway in human skeletal muscle.This study was first to show that both nOC and rOC increased glucose uptake by mediating AMPK in human primary skeletal muscle cells. Two different forms of OC had no functional differences in triggering phosphatidylinositol signal pathway by activating its physiological receptor, GPRC6A. Further investigations for identification of tissue- or species specific roles of GPRC6A are suggested for thorough understanding of the effects of OC-GPRC6A interaction.