GPRC6A mediates responses to osteocalcin in β-cells in vitro and pancreas in vivo

Abstract

A bone-pancreas endocrine loop has been identified recently that involves insulin secreted from β-cells in the pancreas stimulating insulin receptors in osteoblasts, leading to osteoblastic differentiation and increased secretion of osteocalcin (Ocn), a bone-derived hormone that regulates insulin secretion in β-cells. The identity of the Ocn-sensing receptor in β-cells is a missing component of this endocrine loop. The abnormalities in glucose homeostasis in Gprc6a null mice suggests that this pertussis toxin-sensitive G protein-coupled receptor is a candidate for mediating the effects of Ocn on insulin secretion in the pancreas. In support of this possibility, we found that transfection of non-Gprc6a-expressing HEK-293 cells with a full-length Gprc6a cDNA imparted a dose-dependent response to Ocn (5 to 60 ng/mL), as measured by PKD1 and ERK phosphorylation. In addition, Gprc6a is highly expressed in mouse pancreatic tissue and in the mouse TC-6 pancreatic β-cell line. Ocn also stimulated ERK activity in TC-6 pancreatic β-cells. Finally, intraperitoneal injection of Ocn stimulated ERK activity in the pancreas and increased serum insulin levels in wild-type mice, but these responses were markedly attenuated in Gprc6a(-/-) mice. These findings suggest that GPRC6A is a candidate for mediating the response to Ocn in the bone-pancreas endocrine loop regulating insulin signaling.

Fig. 1

Evidence for GPRC6A sensing of Ocn in HEK-293 cells transfected with Gprc6a. (A) Dose-dependent effects of recombinant human Ocn, l-arginine, and calcium to stimulate GPRC6A. Oc, l-arginine, and calcium stimulated ERK activation in HEK-293 cells transfected with Gprc6a cDNA but had no effect in nontransfected HEK-293 cells. (B) Involvement of PLC and PKC in GPRC6A signaling. Ocn-stimulated activation of phospho-ERK in Gprc6a-expressing HEK-293 cells was inhibited by the PLC inhibitor U73122 (10 µM) and the PKC inhibitor Ro31-8220 (5 µM). Neither U73122 nor Ro31-8200 alone exhibited effects on phospho-ERK in unstimulated HEK-293 cells stably transfected with Gprc6a. Representative blots are shown, and the results were verified in at least three independent experiments.

Fig. 2

In vitro and in vivo effects of Ocn on TC-6 mouse β cells and pancreatic tissues in wild-type and Gprc6a null mice. (A) RT-PCR analysis showing that Gprc6a message is expressed in TC-6 cells and mouse pancreas but is not present in the pancreas from Gprc6a^−/− mice. (B) Effects of Ocn, l-arginine, and calcium on pancreatic β-cell signaling. TC-6 cells expressing endogenous Gprc6a exhibited a dose-dependent stimulation of ERK activation in response to the addition of recombinant human Ocn, l-arginine, and calcium to the culture medium. (C, D) Gprc6a deficiency attenuates the pancreatic response to systemic Ocn administration. (C) ERK phosphorylation. Recombinant human Ocn (1 or 3 µg/kg) or PBS vehicle was injected intraperitoneally into wild-type or Gprc6a^−/− male mice. Western blot analysis of ERK phosphorylation in pancreatic tissue assessed 20 minutes after injection of Ocn shows loss of Ocn-mediated ERK activation in the pancreas obtained from Gprc6a null mice but markedly stimulated ERK activity in the pancreas of wild-type mice. Representative blots are shown, and the results were verified in at least three independent experiments. (D) Serum insulin level. Recombinant human Ocn 1 µg/kg or PBS vehicle was injected intraperitoneally into wild-type (n = 5 each group) or Gprc6a^−/− male mice (n = 4 each group). The serum was collected 20 minutes after injection of Ocn. Serum insulin level was measured by insulin (mouse) ultrasensitive ELISA kit (ALPCO Immunoassays) according to the manufacturer’s instructions. (E) Pancreatic insulin expression. Recombinant human Ocn 1 µg/kg or PBS vehicle was injected intraperitoneally into wild-type (n = 4 each group) or Gprc6a^−/− male mice (n = 4 each group). Pancreas were collected 4 hours after injection of Ocn.