Chemogenetic control of liver metabolism

肝脏葡萄糖产生(HGP)是糖原储存被分解为葡萄糖(糖原分解)或葡萄糖从非碳水化合物碳底物中产生(糖异生)的协调生化过程的结果。这些过程受肝细胞表面表达的许多G蛋白偶联受体(GPCRs)的调节。顾名思义，GPCRs与位于质膜上的G蛋白相互作用。当一个配体与GPCR结合时，它会引起构象变化，从而触发GPCR与附近的G蛋白之间的相互作用。然而，由于缺乏适当的工具来选择性地强烈刺激G蛋白，介导GPCR激活对HPG影响的下游信号事件仍然被误解。在这里，我建议利用化学遗传学的力量来选择性地操纵肝细胞中的GPCR信号，作为一种定义导致HGP的复杂生化反应的手段。化学遗传学(化学遗传学)是一种用来对受体进行基因工程的方法，这些受体与以前未被识别的小分子化学致动器相互作用。这种方法已经被用来创造修改的GPCRs，称为由设计师药物独有激活的设计师受体(DREADD)。DREADDS最初是为了控制神经元活动而开发的，现在正在成为在任何细胞类型或器官中选择性地对GPCR信号进行药物控制的关键工具。最近，我报道了一种新的化学发生方法的产生，该方法允许在脂肪细胞中急性刺激GsPCR和GiPCR信号。这一创新的模型揭示了脂肪组织中重要的、以前未被认识到的GPCR信号的复杂相互作用，并证明了化学遗传技术在更好地了解器官功能方面的有效性。我建议将最先进的代谢流技术与化学遗传学模型相结合，以更好地了解HGP的调节机制。特别是，我建议使用化学遗传学来剖析HGP的两条重要的代谢途径，即体外(原代肝细胞)、体外(灌流肝脏)和体内的糖原分解和糖异生。基于我以前在肝脏代谢和化学发生技术方面的工作和专业知识，这个项目旨在：1)确定肝脏GqPCR化学发生刺激刺激HGP的性别依赖机制；2)在小鼠肝脏灌流模型中评估GqPCR信号如何独立于外周激素的变化影响代谢通量；3)确定导致原代肝细胞HGP的分子级联反应。这项发现拨款将定义复杂的、此前未知的调节HGP的机制。了解这一生物学将为更好地了解不同系统如何直接调节肝脏代谢提供一个框架。这个项目还将为我的学生和该领域提供强大和受欢迎的科学和技术技能集，以了解涉及外周新陈代谢的其他调节因素。

Hepatic glucose production (HGP) is the result of coordinated biochemical processes by which glycogen storage are catabolized to generate glucose (glycogenolysis), or glucose is produced from non-carbohydrate carbon substrates (gluconeogenesis). These processes are regulated by many G protein coupled receptors (GPCRs) expressed at the surface of the hepatocytes. As their names imply, GPCRs interact with G proteins located in the plasma membrane. When a ligand binds to the GPCR, it causes a conformational change that triggers the interaction between the GPCR and a nearby G protein. However, the downstream signaling events mediating the effects of GPCR activation on HPG are still misunderstood because of the lack of appropriate tools to selectively stimulate G proteins acutely. Here, I propose to harness the power of chemogenetics to selectively manipulate GPCR signaling in hepatocytes, as a mean of defining the complex biochemical reactions leading to HGP. Chemical genetics (chemogenetics) is an approach use to genetically engineer receptors that interact with previously unrecognized small molecule chemical actuators. This approach has been used to create modified GPCRs, called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Originally developed to control neuronal activity, DREADDs are emerging as key tools for selective pharmacological control of GPCR signaling in any cell type or organ. Recently, I reported the generation of a novel chemogenetic approach allowing acute stimulation of GsPCR and GiPCR signaling in adipocytes. This innovative model revealed important, previously unappreciated complex interactions of GPCR signaling in adipose tissue and demonstrated the usefulness of chemogenetic technology to better understand organ functions. I propose to combine state-of-the-art metabolic flux techniques with chemogenetics models to better understand the mechanisms mediating HGP. In particular, I propose to use chemogenetics to dissect two important metabolic pathways for HGP, namely glycogenolysis and gluconeogenesis in vitro (primary hepatocytes), ex vivo (perfused livers) and in vivo. Building on my previous work and expertise on liver metabolism and chemogenetic technology, this program aims to: 1) determine the sex-dependent mechanisms by which chemogenetic stimulation of liver GqPCR stimulate HGP; 2) evaluate how GqPCR signaling affects metabolic flux independent of changes in peripheral hormones in a perfused mouse liver model; 3) define the molecular cascade leading to HGP in primary hepatocytes. This Discovery Grant will define complex previously unknown mechanisms mediating HGP. Understanding this biology will provide a framework to better understand how different systems directly regulates liver metabolism. This program will also provide a powerful and sought after scientific and technical skill set for my students and the field to understand other regulatory factors involved in peripheral metabolism.