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

在胰腺β细胞中选择性表达M3毒蕈碱受体组成型活性版本的转基因小鼠的研究 我们以前已经证明，β-细胞M3毒蕈碱乙酰胆碱受体（M3R）通过增强葡萄糖依赖性胰岛素释放在维持血糖稳态中起关键作用。为了检验β细胞M3R的长期、持续活化可以改善葡萄糖耐量并改善与高脂饮食消耗相关的代谢缺陷的假设，我们产生了在胰腺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具有组成型活性。在几个体外和体内代谢试验中检查转基因小鼠的代谢表型。在存在高浓度葡萄糖且不存在M3R配体的情况下，从β-M3-Q490L Tg小鼠制备的分离的灌流胰岛比野生型对照胰岛释放相当多的胰岛素。这种作用可以通过将转基因胰岛与阿托品（一种反向毒蕈碱激动剂）孵育而完全阻断，表明Q490L突变体M3R在小鼠β细胞中表现出配体非依赖性信号传导（组成性活性）。体内研究表明，β-M3-Q490L Tg小鼠显示出极大改善的葡萄糖耐量和增加的血清胰岛素水平以及对饮食诱导的葡萄糖耐受不良和高血糖症的抗性。这些结果表明，β-细胞M3R的慢性激活可能代表了在T2D的长期治疗中增加胰岛素输出的有用方法。 与胰腺β细胞中Gq型G蛋白的选择性激活相关的有益代谢效应 胰腺β细胞功能受损是T2D的标志之一。胰腺β细胞表达多种GPCR，其与不同功能类别的异源三聚体G蛋白（包括Gs和Gq）连接。刺激胰腺β细胞中Gs信号传导的药物，如GLP 1受体激动剂，最近已被批准用于治疗T2D的临床用途。相比之下，对Gq信号在调节β细胞功能中的作用知之甚少。为了检查Gq在体内对β细胞功能的调节，我们产生了仅在β细胞中表达Gq偶联设计受体的转基因小鼠（β-Rq小鼠）。这种设计受体不再结合其内源性激动剂（乙酰胆碱），但可以有效地激活外源性给药的配体，氯氮平-N-氧化物（CNO），否则是一种惰性化合物。CNO注射实验表明，β细胞Gq信号传导的急性激活导致胰岛素释放增强，并大大改善葡萄糖耐量。此外，长期CNO给药研究表明，β细胞Gq信号传导的长期激活与血清胰岛素升高和血糖水平降低、胰腺胰岛素含量增加、β细胞质量增加和β细胞增殖速率增强相关。β细胞Gq的慢性刺激还导致对β细胞功能和β细胞群维持重要的几种基因的表达增强。引人注目的是，链脲霉素诱导的糖尿病在长期用CNO治疗的β-Rq小鼠中得到了极大的改善。这些结果表明，旨在增强胰腺β细胞中的Gq信号传导的药物可以成为临床上有用的抗糖尿病药物。 β细胞M3R的刺激通过蛋白激酶D1的磷酸化/抑制蛋白依赖性激活促进胰岛素释放 （与莱斯特大学安德鲁·托宾博士小组合作） 为了解决M3R磷酸化在β细胞M3R活性中的作用，用表达M3R磷酸化缺陷突变体形式的敲入小鼠品系进行研究。该突变体M3R表现出生理表达模式，并且能够高效地激活Gq型G蛋白。然而，突变体M3R在其进行内化和招募抑制蛋白的能力受损。突变M3R基因敲入小鼠显示葡萄糖耐量和胰岛素分泌受损，表明胰腺β细胞表达的M3R通过受体磷酸化/抑制蛋白依赖性信号传导调节葡萄糖稳态。另外的研究表明，抑制蛋白向磷酸化M3R的募集导致蛋白激酶D1的活化，这进而触发胰岛素从胰腺β细胞的释放。这些发现支持了M3R介导的抑制蛋白募集促进胰岛素从胰腺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.