Genetic and Environmental Determinants of GPRC6A Regulation of Energy Metabolism Using Genetically Engineered Mice and Systems Biology

代谢综合征（MetS）是一种以糖和脂肪代谢的多种异常为特征的复杂性状，涉及各种器官之间的生物网络不完全理解，并受遗传和环境（GXE）相互作用的影响。GPRC 6A是一种营养感应G蛋白偶联受体，参与能量代谢的独特调节。在基因工程小鼠模型（GEMM）中，GPRC 6A通过直接的组织特异性效应和协调器官间代谢功能的激素释放来调节葡萄糖和脂肪代谢，并预防高脂饮食（HFD）诱导的代谢并发症。GPRC 6A调控的细胞和全身代谢网络的复杂性、GEMM中的可变表型以及对GPRC 6A在人类中功能的有限理解是确定GPRC 6A在预防和治疗MetS及其并发症中的作用的关键障碍。我们的中心假设是GXE相互作用影响GPRC 6A调节能量稳态。目的1将使用GEMM和还原方法来检验GXE相互作用修饰GPRC 6A对肝脏和其他代谢活性器官中的葡萄糖和脂肪代谢的调节的假设。实验将使用野生型GPRC 6A-KGRKLP和GPRC 6A缺失小鼠、HFD和GPRC 6A激动剂来探索HFD和GPRC 6A功能的丧失和获得对小鼠能量代谢的影响。将在“人源化”Gprc6a_KGKY_敲入小鼠中测试最近进化的人GPRC6A_KGKY遗传多态性的功能意义。我们将描述肝细胞特异性Gprc 6a基因敲除小鼠（Gprc 6aliver-cko）的特征，以研究GPRC 6A在肝脏中的功能，作为控制葡萄糖和脂肪代谢的原型器官。在目标2中，我们将使用系统遗传学系统的突破性资源来测试遗传背景改变GPRC 6A和HFD的代谢效应的假设。我们将从用HFD和GPRC 6A激动剂骨钙素（OCN）处理的BXD重组近交系的肝脏中收集代谢表型和分子表达数据。然后，我们将应用系统生物学的方法来定义信号通路，代谢过程和基因网络涉及GPRC 6A调节肝脏脂肪和葡萄糖代谢。细胞、分子和小鼠遗传学方法将验证系统生物学预测的这些途径和网络.实验和计算系统生物学方法的预测能力，结合和整合不同水平的信息和GPRC 6A创建的复杂系统的科学知识，将提高GPRC 6A对MetS影响的临床前研究的严谨性和可重复性。我们的影响将是：1）建立GPRC 6AKGRKLP和GPRC 6AKGKY变体的器官特异性功能，并确定这些多态性是否改变MetS及其代谢并发症的易感性和治疗反应; 2）鉴定GPRC 6A调节的控制葡萄糖和脂肪代谢的基因网络，并确定影响GPRC 6A和HFD对MetS的作用的遗传修饰剂; GPRC 6A是一个独特的分子靶点，可用于了解MetS的发病机制和治疗。

Metabolic syndrome (MetS) is a complex trait characterized by multiple abnormalities in glucose and fat metabolism, involving incompletely understood biological networks between various organs, and influenced by genetic and environmental (GXE) interactions. GPRC6A is a nutrient sensing G-protein coupled receptor implicated in the unique regulation of energy metabolism. In genetically engineered mouse models (GEMMs), GPRC6A regulates glucose and fat metabolism and prevents high fat diet (HFD) induced metabolic complications through direct tissue-specific effects and the release of hormones that coordinate metabolic functions between organs. The complexity of the cellular and systemic metabolic networks regulated by GPRC6A, the variable phenotypes in GEMMs, and the limited understanding of GPRC6A functions in humans are critical barriers to defining the role of GPRC6A in preventing and treating MetS and its complications. Our central hypothesis is that GXE interactions influence GPRC6A regulation of energy homeostasis. Aim 1 will test the hypothesis that GXE inter- actions modify GPRC6A regulation of glucose and fat metabolism in the liver and other metabolically active organs using GEMMs and a reductionist approach. Experiments will use wild-type GPRC6A-KGRKLP and GPRC6A null mice, HFD, and GPRC6A agonists to explore the effects of HFD and loss- and gain-of GPRC6A function on energy metabolism in mice. The functional significance of the recently evolved human GPRC6A_KGKY genetic polymorphism will be tested in a “humanized” Gprc6a_KGKY_knockin mouse. We will characterize hepatocyte-specific Gprc6a knockout mice (Gprc6aliver-cko) to investigate GPRC6A’s function in liver, as a prototypic organ controlling glucose and fat metabolism. In Aim 2, we will use groundbreaking resources for systems genetics systems to test hypothesis that genetic backgrounds modify the metabolic effects of GPRC6A and HFD. We will collect metabolic phenotypes and molecular expression data from the livers of BXD recombinant inbred lines treated with a HFD and the GPRC6A agonist, osteocalcin (Ocn). Then we will apply systems biology approaches to define signaling pathways, metabolic processes and gene networks involving GPRC6A regulation of hepatic fat and glucose metabolism. Cell, molecular and mouse genetic approaches will validate these pathways and net- works predicted by systems biology. The predictive power of experimental and computational systems biology approaches to incorporate and integrate distinct levels of information and scientific knowledge of complex systems created by GPRC6A will improve the rigor and reproducibility of preclinical studies of GPRC6A effects on MetS. Our impact will be to: 1) establish the organ-specific functions of GPRC6AKGRKLP and GPRC6AKGKY variants and determine if these polymorphisms alter the susceptibility to and treatment responses of MetS and its metabolic complications; 2) identify the GPRC6A-regulated gene networks controlling glucose and fat metabolism and determine the genetic modifiers that influence the effects of GPRC6A and HFD on MetS; and 3) validate GPRC6A as a unique molecular target for understanding the pathogenesis and treatment of MetS.