Discovery of an Osteocalcin Sensing GPCR Regulating Beta-Cell Function

描述（由申请人提供）：新出现的证据支持这样的假设，即骨骼也是一个内分泌器官，通过释放成骨细胞衍生激素骨钙素（Ocn）来调节能量代谢。这一新的假设是有争议的，因为我们对Ocn在多器官中的生理作用以及Ocn给药引起的其他激素网络的复杂变化的认识仍有重要的空白有待填补。了解骨对能量代谢的综合调节的关键步骤将是识别和表征Ocn受体的功能。我们已经发现了OcnR的主要候选者GPRC6A，这是一种氨基酸传感GPCR，在细胞中高度表达，并在体外和体内被重组Ocn激活。GPRC6A的整体消融产生OcnR-/-小鼠，导致类似OcnR-/-小鼠的表型，包括葡萄糖耐受不良、循环胰岛素水平降低和胰岛素抵抗。从OcnR-/-小鼠分离的胰岛表现出葡萄糖刺激胰岛素分泌异常和胰岛发育不全，表明OcnR也调节胰岛素分泌和¿-细胞质量。因此，我们提出验证以下假设：[a]该GPCR是生物学相关的OcnR， [b]它定义了将骨代谢与胰岛素分泌和细胞增殖的代谢调节联系起来的分子机制。此外，OcnR还被l -精氨酸和睾酮激活，重组Ocn调节睾丸产生睾酮，以及肝脏、肌肉和脂肪的代谢功能，表明OcnR可能是连接多个内分泌网络的多配体受体。具体目的是：1)通过转基因小鼠模型和离体胰岛组织特异性功能丧失实验，检测OcnR在胰腺细胞中的特异性功能；2)证实OcnR介导重组Ocn对胰腺细胞的直接作用；3)确定羧化和欠羧化形式的Ocn与OcnR的结合动力学。总的来说，这些研究将定义OcnR的功能，以整合连接骨骼、Ocn和其他潜在配体与细胞功能的生理网络，并提供有助于简化骨骼内分泌功能与其他器官分泌的影响骨骼和能量代谢的激素之间复杂的相互依赖性的知识。

DESCRIPTION (provided by applicant): Emerging evidence supports the hypothesis that the skeleton is also an endocrine organ that regulates energy metabolism through the release of the osteoblast-derived hormone, osteocalcin (Ocn). This novel hypothesis is controversial, because important gaps remain to be filled in our knowledge of the physiological effects of Ocn in multiple organs and the complex alterations in other hormonal networks induced by Ocn administration. Key steps toward understanding the integrative regulation of energy metabolism by bone would be to identify and characterize the function of the receptor for Ocn. We have discovered a prime candidate for the OcnR, GPRC6A, an amino-acid sensing GPCR that is highly expressed in ¿-cells and is activated by recombinant Ocn in vitro and in vivo. Global ablation of GPRC6A to create OcnR-/- mice results in a phenotype that resembles Ocn-/- mice, including glucose intolerance, reductions in circulating insulin levels, and insulin resistance. Pancreatic islets isolated from OcnR-/- mice exhibit abnormalities of glucose-stimulated insulin secretion and pancreatic islet hypoplasia, suggesting that the OcnR also regulates insulin secretion and ¿-cell mass. Thus, we propose to test the hypotheses that [a] this GPCR is the biologically relevant OcnR and [b] it defines a molecular mechanism for linking bone metabolism with metabolic regulation of insulin secretion and ¿-cell proliferation. In addition, OcnR is also activated by L-arginine and testosterone, and recombinant Ocn regulates testosterone production by the testes, as well as metabolic functions of liver, muscle and fat, suggesting that OcnR may be a multi-liganded receptor connecting multiple endocrine networks. The Specific Aims are to: 1) Test the specific functions of OcnR in pancreatic ¿-cells by tissue-specific loss-of-function experiments in transgenic mouse models and in isolated pancreatic islets ex vivo; 2) Confirm that OcnR mediates the direct effects of recombinant Ocn in pancreatic ¿-cells ; and 3) Determine the binding kinetics of carboxylated and undercarboxylated forms of Ocn for OcnR. Collectively these studies will define the functions of OcnR to integrate physiological networks linking bone, Ocn and other potential ligands to ¿-cell functions and provide knowledge that will help to simplify the complex interdependency between the endocrine functions of bone and hormones secreted by other organs that affect bone and energy metabolism.