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.

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