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

利用设计的GPCRs研究GPCRs对关键代谢途径的调控 ArmBruster等人。(PNAS 104,5163-8,2007)首先描述了一组基于M受体的设计者GPCRs，现在通常被称为DREADD(‘由设计者药物独占激活的设计者受体’)。由于跨膜受体核心中引入了两个单点突变，这些设计受体无法结合内源性M受体激动剂乙酰胆碱。重要的是，DREADDS可以被一种名为氯氮平-N-氧化物(CNO)的化合物有效地激活，这种药物在其他方面是惰性的。开发的第一批DREADD分别代表选择性激活GQ或GI家族G蛋白的GPCRs。 我们随后产生了另外的被赋予不同耦合性质的DREADD，包括Gs DREADD和功能混杂的DREADD(Guettier等人，PNAS106,19197-2022009年)。最近，我们产生了一个基于M3R的DREADD，它与G蛋白解偶联，但保留了arrestin依赖的信号(Nakajima和Wess，Mol Pharmacol 82,575-82,2012)。类似地，我们最近设计了一种基于M3R的DREADD，它显示了相反的偶联曲线：缺乏arrestin招募，但有效地偶联到GQ型G蛋白(Hu等人，J Biol Chem 291,7809-20,2016)(有关最近的综述，请参阅：Wess J.使用设计者G蛋白偶联受体来剖析代谢途径。趋势内分泌代谢。2016年9月；27(9)：600-3)。 我们目前正在各种代谢相关的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、胰岛β细胞、骨骼肌细胞、肝细胞和下丘脑的某些神经元亚群。初步结果表明，对其中一些突变小鼠品系的CNO处理对血糖和能量稳态有显著影响(未发表结果)。 下面总结的数据是在一项合作研究中获得的。 一种小鼠模型，在该模型中，Gs DREADD表达可以以特定细胞类型的、时间控制的方式诱导 许多G蛋白偶联受体(GPCRs)的激活导致Gs的刺激，Gs是一种促进cAMP形成的异源三聚体蛋白。为了促进体内特定细胞群体中cAMP信号的研究，我们使用DREADD技术创建了基于rosa26的敲入小鼠，用于Gs DREADD的条件表达。在表达Cre重组酶后，该DREADD可被选择性DREADD激动剂CNO激活。在体内将Cre重组酶病毒导入小鼠肝细胞后，Gs DREADD在肝细胞中选择性表达。在这些动物中，长期的DREADD活性会导致cAMP信号增强和糖原分解，并伴有高血糖。因此，这些新的突变小鼠代表了一个很好的工具来研究Gs信号的生理效应，无论是急性的还是慢性的，在任何组织或细胞类型中，转基因或病毒Cre驱动因素都是可用的。 (阿赫梅多夫D，门多萨-罗德里格斯MG，Rajendran K，Rossi M，Wess J，Berdeaux R. GS-DREADD敲入小鼠，用于组织特异性的、暂时刺激循环AMP信号。Mol Cell Biol.2017年4月14日；37(9)。PII：e00584-16) β-arrestin-2对胰岛β细胞的正常功能是必不可少的 我们最近发现，在胰腺β细胞中选择性缺乏β-arrestin-2(Barr2)的小鼠(beta-barr2-KO小鼠)表现出几种显著的代谢缺陷，包括葡萄糖刺激的胰岛素分泌和Ca~(2+)进入β细胞的严重障碍，以及当-barr2-KO小鼠保持高脂饮食时明显的糖耐量下降。我们证明了Barr2是正确激活CaMKII所必需的，并且这一途径的中断可以完全解释在β-barr2-KO小鼠中观察到的代谢缺陷。此外，下调barr2的表达几乎消除了葡萄糖诱导的人β细胞中胰岛素的释放。这些结果可能导致旨在调节β细胞中barr2功能的新药的开发，以达到治疗目的。 (朱L，Almaa J，Dadi PK，Hong H，Sakamoto W，Rossi M，Lee RJ，Viera NC，Lu H，Cui Y，McMillin SM，Perry NA，Gurevich VV，Lee A，Kuo B，Leapman RD，Matschinsky FM，Doliba NM，Urs NM，Caron MG，Jacobson DA，Caicedo A，Wess J.Beta-arrestin-2是生理和病理生理条件下胰腺β细胞功能的重要调节因子。纳特社区。2017年2月1日；8：14295。) 肝脏β-arrestin-2是维持正常血糖水平所必需的 2型糖尿病的一个重要特征是肝脏葡萄糖产量(HGP)增加。这种代谢缺陷严重依赖于通过肝高血糖素受体(GCGRs)增强的信号。在体内对GCGR活性的调节在很大程度上仍未被探索。我们发现，成年小鼠肝细胞中的β-arrestin-2(Barr2)选择性失活会导致肝脏GCGR信号的显著增加，导致显著的葡萄糖稳态失调。有趣的是，在肝细胞中选择性缺乏β-arrestin-1的小鼠并没有表现出任何葡萄糖稳态的变化。重要的是，在肝细胞中过度表达barr2的小鼠显示出大大减少了肝脏GCGR信号，并对高脂饮食引起的代谢缺陷具有保护作用。这些发现支持了一种新的概念，即旨在增强肝脏barr2活性的策略可能被证明对抑制HGP治疗2型糖尿病有用。 [朱L，罗西·M，崔勇，Lee RJ，Sakamoto W，Perry NA，Urs NM，Caron MG，Gurevich VV，Godlewski G，Kuos G，Chen M，Chen W，Wess J.肝脏β-arrestin 2对维持正常血糖是必不可少的。j Clin投资公司。2017年6月26日。PII：92913。DOI：10.1172/JCI92913。印刷前的ePub)

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 subsequently generated additional DREADDs endowed with different coupling properties, including a Gs DREADD and a functionally promiscuous DREADD (Guettier et al., PNAS 106, 19197-202, 2009). More recently, we generated an M3R-based DREADD that is uncoupled from G proteins but retains arrestin-dependent signaling (Nakajima and Wess, Mol Pharmacol 82, 575-82, 2012). Analogously, we recently designed an M3R-based DREADD that shows the opposite coupling profile: lack of arrestin recruitment but efficient coupling to Gq-type G proteins (Hu et al., J Biol Chem 291, 7809-20, 2016) (for a recent review see: Wess J. Use of Designer G protein-coupled receptors to dissect metabolic pathways. Trends Endocrinol Metab. 2016 Sep;27(9):600-3). We are currently in the process of expressing DREADDs with different coupling properties in various metabolically relevant cell types. These cell types include adipocytes, pancreatic beta-cells, skeletal muscle cells, hepatocytes, and certain 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 (unpublished results). The data summarized below were obtained in a collaborative study. A mouse model in which Gs DREADD expression can be induced in a cell type-specific, temporally controlled fashion Activation of many G protein-coupled receptors (GPCRs) leads to the stimulation of Gs, a heterotrimeric protein that promotes the formation of cAMP. To facilitate studies of cAMP signaling in specific cell populations in vivo, we used DREADD technology to create ROSA26-based knock-in mice for the conditional expression of a Gs DREADD. After expression of Cre recombinase, this DREADD can be activated by the administration CNO, a selective DREADD agonist. After viral delivery of Cre recombinase to mouse hepatocytes in vivo, the Gs DREADD is selectively expressed in hepatocytes. In these animals, long-term DREADD activity leads to enhanced cAMP signaling and glycogen breakdown, accompanied by hyperglycemia. Thus, these new mutant mice represent an excellent tool to study the physiological effects of Gs signaling, acutely or chronically, in any tissue or cell type for which transgenic or viral Cre drivers are available. (Akhmedov D, Mendoza-Rodriguez MG, Rajendran K, Rossi M, Wess J, Berdeaux R. Gs-DREADD knock-in mice for tissue-specific, temporal stimulation of cyclic AMP signaling. Mol Cell Biol. 2017 Apr 14;37(9). pii: e00584-16) Beta-Arrestin-2 is essential for the proper function of pancreatic beta-cells We recently found that mice selectively lacking beta-arrestin-2 (barr2) in pancreatic beta-cells (beta-barr2-KO mice) showed several striking metabolic deficits, including greatly impaired glucose-stimulated insulin secretion and Ca2+ entry into beta-cells, and a pronounced reduction of glucose tolerance when -barr2-KO mice were maintained on a high-fat diet. We demonstrated that barr2 is required for the proper activation of CAMKII and that disruption of this pathway can fully account for the metabolic deficits observed with the beta-barr2-KO mice. Moreover, knockdown of barr2 expression virtually abolished glucose-induced insulin release in human beta-cells. These results may lead to the development of novel drugs aimed at modulating barr2 function in beta-cells for therapeutic purposes. (Zhu L, Almaa J, Dadi PK, Hong H, Sakamoto W, Rossi M, Lee RJ, Vierra NC, Lu H, Cui Y, McMillin SM, Perry NA, Gurevich VV, Lee A, Kuo B, Leapman RD, Matschinsky FM, Doliba NM, Urs NM, Caron MG, Jacobson DA, Caicedo A, Wess J. Beta-arrestin-2 is an essential regulator of pancreatic beta-cell function under physiological and pathophysiological conditions. Nat Commun. 2017 Feb 1;8:14295.) Hepatic beta-arrestin-2 by is required for maintaining normal blood glucose levels A key feature of type 2 diabetes is an increase in hepatic glucose production (HGP). This metabolic deficit critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). The regulation of GCGR activity in vivo remains largely unexplored. We showed that selective inactivation of beta-arrestin-2 (barr2) in hepatocytes of adult mice leads to greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. Interestingly, mice selectively lacking beta-arrestin-1 in hepatocytes do not show any changes in glucose homeostasis. Importantly, mice that over-express barr2 in hepatocytes display greatly reduced hepatic GCGR signaling and are protected against the metabolic deficits caused by the consumption of a high-fat diet. These findings support the novel concept that strategies aimed at enhancing hepatic barr2 activity could prove useful to suppress HGP for the treatment of type 2 diabetes. (Zhu L, Rossi M, Cui Y, Lee RJ, Sakamoto W, Perry NA, Urs NM, Caron MG, Gurevich VV, Godlewski G, Kunos G, Chen M, Chen W, Wess J. Hepatic beta-arrestin 2 is essential for maintaining euglycemia. J Clin Invest. 2017 Jun 26. pii: 92913. doi: 10.1172/JCI92913. Epub ahead of print)