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

在胰岛β细胞中选择性表达M3 M受体的转基因小鼠的研究 我们以前已经证明，β细胞M3 M受体(M3Rs)通过促进葡萄糖依赖的胰岛素释放，在维持血糖动态平衡方面发挥关键作用。为了验证长期、持续激活β细胞M3R可以改善葡萄糖耐量和改善与高脂饮食相关的代谢缺陷的假设，我们培育了在胰腺β细胞中选择性表达Q490L突变M3R的转基因小鼠(β-M3-Q490L TG小鼠)。已知Q490L点突变使M3R具有结构性活性。对转基因小鼠的代谢表型进行了体内和体外代谢试验。在高浓度葡萄糖存在和缺乏M3R配体的情况下，从β-M3-Q490L转基因小鼠制备的灌流胰岛比野生型对照胰岛释放相当多的胰岛素。这种作用可以通过转基因胰岛与阿托品孵育而完全被阻断，阿托品是一种反向毒碱激动剂，表明Q490L突变体M3R在小鼠β细胞中表现出配体无关的信号(构成活性)。体内研究表明，β-M3-Q490L转基因小鼠表现出极大的糖耐量改善和血清胰岛素水平的提高，以及对饮食诱导的葡萄糖耐量和高血糖的抵抗。这些结果表明，在T2D的长期治疗中，β细胞M3Rs的慢性激活可能是一种提高胰岛素输出的有用方法。 选择性激活Gq-型G蛋白对胰岛β细胞的有益代谢作用 胰岛β细胞功能受损是T2D的特征之一。胰岛β细胞表达大量的GPCRs，它们与不同功能类别的异源三聚体G蛋白有关，包括Gs和Gq。刺激胰岛β细胞Gs信号的药物，如GLP1受体激动剂，最近已被批准用于临床治疗T2D。相比之下，人们对GQ信号在调节β细胞功能中的作用知之甚少。为了在体内检测GQ对β细胞功能的调节，我们培育了仅在β细胞中表达GQ偶联设计者受体的转基因小鼠(β-RQ小鼠)。这种设计受体不再与其内源性激动剂(乙酰胆碱)结合，但可以被外源性配基氯氮平-N-氧化物(CNO)有效地激活，这是一种药理上的惰性化合物。CNO注射实验表明，急性激活β细胞GQ信号导致胰岛素释放增加，并极大地改善了糖耐量。此外，慢性给予CNO的研究表明，长期激活β细胞GQ信号与血清胰岛素水平升高和血糖水平下降、胰腺胰岛素含量增加、β细胞质量增加和β细胞增殖率增加有关。对β细胞Gq的慢性刺激也导致了几个对β细胞功能和维持β细胞质量重要的基因的表达增强。值得注意的是，在长期服用CNO的β-RQ小鼠中，链脲佐菌素诱导的糖尿病得到了极大的改善。这些结果表明，旨在增强胰岛β细胞GQ信号的药物可能成为临床上有用的抗糖尿病药物。 刺激β细胞M3Rs通过蛋白激酶D1的磷酸化/arrestin依赖的激活促进胰岛素释放 (与莱斯特大学安德鲁·托宾博士团队合作) 为了研究M3R磷酸化在β细胞M3R活性中的作用，用表达M3R磷酸化缺陷突变版本的敲入小鼠品系进行了研究。该突变体M3R具有生理表达模式，能够高效激活Gq型G蛋白。然而，突变的M3R在经历内化和招募拦阻因子的能力方面受到了损害。突变的M3R基因敲入小鼠表现出糖耐量和胰岛素分泌受损，表明胰岛β细胞表达的M3R通过受体磷酸化/arrestin依赖的信号调节葡萄糖稳态。更多的研究表明，阻滞素在磷酸化的M3Rs上的募集导致蛋白激酶D1的激活，进而触发胰岛β细胞释放胰岛素。这些发现支持M3R介导的抑制素募集促进胰岛素从胰岛β细胞释放的新概念。

Studies with transgenic mice that express a constitutively active version of the M3 Muscarinic receptor selectively in pancreatic beta-cells We have demonstrated previously that beta-cell M3 muscarinic acetylcholine receptors (M3Rs) play a key role in maintaining blood glucose homeostasis by enhancing glucose-dependent insulin release. To test the hypothesis that long-term, persistent activation of beta-cell M3Rs can improve glucose tolerance and ameliorate the metabolic deficits associated with the consumption of a high-fat diet, we generated transgenic mice that expressed the Q490L mutant M3R selectively in pancreatic beta-cells (beta-M3-Q490L Tg mice). The Q490L point mutation is known to render the M3R constitutively active. The metabolic phenotypes of the transgenic mice were examined in several in vitro and in vivo metabolic tests. In the presence of a high concentration of glucose and the absence of M3R ligands, isolated perifused islets prepared from beta-M3-Q490L Tg mice released considerable more insulin than wild-type control islets. This effect could be completely blocked by incubation of the transgenic islets with atropine, an inverse muscarinic agonist, indicating that the Q490L mutant M3R exhibited ligand-independent signaling (constitutive activity) in mouse beta-cells. In vivo studies showed that beta-M3-Q490L Tg mice displayed greatly improved glucose tolerance and increased serum insulin levels as well as resistance to diet-induced glucose intolerance and hyperglycemia. These results suggest that chronic activation of beta-cell M3Rs may represent a useful approach to boost insulin output in the long-term treatment of T2D. Beneficial metabolic effect associated with the selective activation of Gq-type G proteins in pancreatic beta-cells Impaired function of pancreatic beta-cells is one of the hallmarks of T2D. Pancreatic beta-cells express a multitude of GPCRs which are linked to different functional classes of heterotrimeric G proteins, including Gs and Gq. Drugs that stimulate Gs signaling in pancreatic beta-cells, such as GLP1 receptor agonists, have recently been approved for clinical use for the treatment of T2D. In contrast, much less is known about the role of Gq signaling in regulating beta-cell function. To examine the regulation of beta cell function by Gq in vivo, we generated transgenic mice that express a Gq-coupled designer receptor in beta-cells only (beta-Rq mice). This designer receptor does no longer bind its endogenous agonist (acetylcholine), but can be efficiently activated by an exogenously administered ligand, clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. CNO injection experiments showed that acute activation of beta-cell Gq signaling led to enhanced insulin release and greatly improved glucose tolerance. Moreover, chronic CNO administration studies demonstrated that prolonged activation of beta-cell Gq signaling was associated with elevated serum insulin and decreased blood glucose levels, increased pancreatic insulin content, increased beta-cell mass, and enhanced rate of beta-cell proliferation. Chronic stimulation of beta-cell Gq also led to enhanced expression of several genes important for eta-cell function and maintenance of beta-cell mass. Strikingly, streptozotocin-induced diabetes was greatly ameliorated in beta-Rq mice treated chronically with CNO. These results suggest that agents aimed at enhancing Gq signaling in pancreatic beta-cells could become clinically useful as antidiabetic drugs. Stimulation of beta-cell M3Rs promotes insulin release via phosphorylation/arrestin-dependent activation of protein kinase D1 (collaboration with the group of Dr. Andrew Tobin, University of Leicester) To address the role of M3R phosphorylation in the activity of beta-cell M3Rs, studies were carried out with a knock-in mouse strain expressing a phosphorylation-deficient mutant version of the M3R. This mutant M3R showed a physiological pattern of expression and was able to activate Gq-type G proteins with high efficacy. However, the mutant M3R was impaired in its ability to undergo internalization and to recruit arrestins. The mutant M3R knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M3Rs expressed by pancreatic beta-cells regulate glucose homeostasis via receptor phosphorylation/arrestin-dependent signaling. Additional studies suggested that recruitment of arrestins to phosphorylated M3Rs leads to the activation of protein kinase D1, which in turn triggers the release of insulin from pancreatic beta-cells. These findings support the novel concept that M3R-mediated recruitment of arrestins facilitates the release of insulin from pancreatic beta-cells.