Role of G protein-coupled receptors in regulating glucose and energy homeostasis

使用设计者GPCR研究关键代谢途径的GPCR调控 Armbruster等人（PNAS 104，5163-8，2007）首先描述了一组基于毒蕈碱受体的设计者GPCR，其现在通常被称为DREADD（“仅由设计者药物激活的设计者受体”）。这些设计受体不能结合内源性毒蕈碱受体激动剂，乙酰胆碱，由于两个单点突变引入跨膜受体核心。重要的是，DREADD可以被一种叫做氯氮平-N-氧化物（CNO）的化合物有效地激活，这种化合物在其他方面是惰性的。开发的第一个DREADD代表分别选择性激活Gq或Gi家族的G蛋白的GPCR。 我们最近产生了额外的DREADD赋予不同的耦合特性。例如，我们设计了能够选择性激活Gs的M3毒蕈碱受体（M3 R）/β 1-肾上腺素能受体杂合物DREADD（Guettier等人，PNAS 106，19197-202，2009）。最近，我们还产生了基于M3 R的DREADD，其不能与G蛋白偶联，但保留了抑制蛋白依赖性信号传导（Nakajima和Wess，Mol Pharmacol 82，575-82，2012）。 我们目前正在代谢相关细胞类型中表达具有不同偶联特性的DREADD。这些细胞类型包括脂肪细胞、胰腺β细胞、骨骼肌细胞和肝细胞。我们还使用“柔性开关”技术在下丘脑的不同神经元亚群中选择性地表达各种DREADD。初步结果表明，CNO处理这些突变小鼠品系中的一些对葡萄糖和能量稳态具有显著影响（K。Nakajima，M.罗西湖Zhu，D.骨，Z。Cui等人;未公布的结果）。 以下段落总结了最近的一项研究，其中我们使用DREADD技术来探索Gq信号在调节肝脏葡萄糖产生中的作用。 肝细胞中Gq信号的激活促进肝葡萄糖产生 肝葡萄糖生成（HGP）升高是T2 D患者空腹高血糖症的主要原因。因此，更好地了解调节肝脏葡萄糖通量的信号通路具有很大的潜在临床意义。目前，肝脏Gq偶联GPCR的体内代谢作用仍然知之甚少。为了解决这个问题，我们产生了一种转基因小鼠系，其在肝细胞中选择性地表达基于M3 R的Gq DREADD（Hep-Rq小鼠）。为了简单起见，我们将这个Gq DREADD简称为“Rq”。 用CNO对Hep-Rq小鼠进行急性治疗导致血糖水平显著的剂量依赖性增加。此外，在丙酮酸激发试验中，hoc注射的Hep-Rq小鼠显示葡萄糖耐量受损和血糖水平显著升高，这与Rq介导的HGP刺激一致。Rq介导的HGP增加的幅度与用胰高血糖素处理Hep-Rq小鼠后观察到的相似，胰高血糖素通过GS依赖性机制促进HGP。同位素标记研究表明，Gq信号通过刺激糖原合成和糖原分解导致HGP增强。这些发现表明，Gq连接的GPCR在调节肝脏葡萄糖通量中起着关键作用。 我们还表明，几个Gq偶联受体的表达水平显着增加瘦素缺乏小鼠（ob/ob小鼠）的肝脏中，与瘦同窝出生。在分析的所有GPCR基因中，V1 b加压素受体表现出受体转录水平最强的增加。引人注目的是，体内研究表明，用选择性V1 b受体拮抗剂SSR 149415治疗ob/ob小鼠，能够显著降低瘦素缺陷小鼠显示的HGP增加。我们目前正在进一步探索V1 b加压素拮抗剂可能被证明有助于改善与肥胖和T2 D相关的代谢缺陷的可能性。

Use of designer GPCRs to study GPCR regulation of key metabolic pathways Armbruster et al. (PNAS 104, 5163-8, 2007) first described a set of muscarinic receptor-based designer GPCRs which are now generally referred to as DREADDs ('designer receptors exclusively activated by designer drugs'). These designer receptors are unable to bind the endogenous muscarinic receptor agonist, acetylcholine, due to two single point mutations introduced into the transmembrane receptor core. Importantly, DREADDs can be efficiently activated by a compound called clozapine-N-oxide (CNO), an agent that is otherwise pharmacologically inert. The first DREADDs that were developed represent GPCRs that selectively activate G proteins of the Gq or Gi family, respectively. We recently generated additional DREADDs endowed with different coupling properties. For example, we designed an M3 muscarinic receptor (M3R)/beta1-adrenergic receptor hybrid DREADD that is able to selectively activate Gs (Guettier et al., PNAS 106, 19197-202, 2009). More recently, we also generated an M3R-based DREADD that is unable to couple to G proteins but retains arrestin-dependent signaling (Nakajima and Wess, Mol Pharmacol 82, 575-82, 2012). We are currently in the process of expressing DREADDs with different coupling properties in metabolically relevant cell types. These cell types include adipocytes, pancreatic beta-cells, skeletal muscle cells, and hepatocytes. We are also using "flex switch" technology to selectively express various DREADDs in distinct neuronal subpopulations of the hypothalamus. Preliminary results indicate that CNO treatment of some of these mutant mouse strains has pronounced effect on glucose and energy homeostasis (K. Nakajima, M. Rossi, L. Zhu, D. Bone, Z. Cui, et al.; unpublished results). The following paragraph summarizes a recent study in which we used DREADD technology to explore the role of Gq signaling in regulating hepatic glucose production. Activation of Gq signaling in hepatocytes promotes hepatic glucose production Elevated hepatic glucose production (HGP) is the major contributor to fasting hyperglycemia in T2D. For this reason, a better understanding of the signaling pathways that regulate hepatic glucose fluxes is of great potential clinical relevance. At present, the in vivo metabolic roles of hepatic Gq-coupled GPCRs remain poorly understood. To address this issue, we generated a transgenic mouse line that expresses the M3R-based Gq DREADD in selectively in hepatocytes (Hep-Rq mice). For the sake of simplicity, we refer to this Gq DREADD simply as 'Rq'. Acute treatment of Hep-Rq mice with CNO caused pronounced, dose-dependent increases in blood glucose levels. Moreover, CNO-injected Hep-Rq mice showed impaired glucose tolerance and significantly increased blood glucose levels in a pyruvate challenge test, consistent with an Rq-mediated stimulation of HGP. The Rq-mediated increase in HGP was similar in magnitude to that observed after treatment of Hep-Rq mice with glucagon which promotes HGP via Gs-dependent mechanisms. Isotope-labeling studies indicated that Gq signaling leads to enhanced HGP by stimulating both gluconeogenesis and glycogenolysis. These findings suggest that Gq-linked GPCRs play a critical role in regulating hepatic glucose fluxes. We also demonstrated that the expression levels of several Gq-coupled receptors were significantly increased in the liver of leptin-deficient mice (ob/ob mice), as compared to lean littermates. Among all GPCR genes analyzed, the V1b vasopressin receptor showed the most robust increase in receptor transcript levels. Strikingly, in vivo studies demonstrated that treatment of ob/ob mice with SSR149415, a selective V1b receptor antagonist, was able to significantly reduce the increase in HGP displayed by leptin-deficient mice. We are currently further exploring the possibility that V1b vasopressin antagonists might prove useful to ameliorate the metabolic deficits associated with obesity and T2D.