M3 muscarinic receptor (M3R)-based designer GPCRs Armbruster et al. (PNAS 104, 5163-8, 2007) first described a set of muscarinic receptor-based designer GPCRs, which they 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 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). In the following, I will briefly summarize two recent studies that demonstrate the usefulness of these new tools to explore the physiological relevance of Gq-dependent signaling pathways in the regulation of glucose homeostasis. Specifically, we generated transgenic mice that express the M3R-based Gq DREADD in pancreatic beta-cells or hepatocytes. For the sake of simplicity, I refer to this particular Gq DREADD as 'Rq'. SUMMARY In vivo metabolic consequences of activating a designer Gq-coupled receptor selectively expressed in mouse pancreatic beta-cells We previously reported that acute CNO treatment of transgenic mice selectively expressing the Rq designer receptor in pancreatic beta-cells (beta-Rq mice) resulted in striking metabolic changes in vivo, including increased insulin release, reduced blood glucose levels, and greatly improved glucose tolerance (Guettier et al., PNAS 106, 19197-202, 2009). Recently, we subjected beta-Rq mice to additional studies in order to explore the in vivo metabolic effects caused by chronic activation of a Gq-coupled GPCR expressed by pancreatic beta-cells. CNO treatment of beta-Rq mice led to pronounced increases in pancreatic insulin content and beta-cell mass by simulating beta-cell proliferation and transcriptional processes that result in increased insulin synthesis. In addition, qRT-PCR studies showed that the expression of Pdx1 and several other key beta-cell transcription factors was significantly increased in islets prepared from beta-Rq mice chronically treated with CNO. In agreement with this finding, the expression of many functionally critical beta-cell genes (e.g. Ins2, Pcks1, Pcks2, or Glut2) was also significantly elevated in islets prepared from this group of mice. Interesting, chronic activation of Rq signaling in pancreatic beta-cells also led to greatly elevated IRS2 transcript and protein levels, triggering an increase in the activity of Akt and ERK1/2. It is therefore likely that enhanced signaling through the IRS2-Akt-ERK1/2 cascade plays an important role in mediating the beneficial effects on beta-cell function of chronic activation of Rq. To gain insight into the molecular mechanisms responsible for the increase in IRS2 expression mediated by activation of a beta-cell Gq-coupled receptor, we carried out additional studies with an M3R-expressing insulinoma cell line and islets from beta-Rq and wild-type (WT) control mice. We found that activation of Gq signaling in pancreatic beta-cells stimulates the PLC-beta/PKC pathway (as expected), resulting in the activation of ERK1/2 which in turn stimulates the activity of the Irs2 promoter. Studies with IRS2-deficient mice showed that the Rq-mediated beneficial effects on beta-cell function were dependent on the presence of IRS2. To explore the potential clinical relevance of these findings, we generated a mouse model of diabetes by injecting mice, both beta-Rq and WT control mice, with a relatively low dose of streptozotocin (STZ) over several days, a protocol which causes a severe but not complete loss of beta-cell mass. In WT mice and non-CNO-treated beta-Rq mice, this treatment led to severe diabetes, including hyperglycemia, low plasma insulin, glucose intolerance, and impaired glucose-induced insulin release. Strikingly, chronic CNO treatment of the beta-Rq mice was able to prevent all STZ-induced metabolic deficits. We also observed that chronic CNO treatment of the STZ-exposed beta-Rq mice led to significant increases in pancreatic insulin content and beta-cell mass, accompanied by the enhanced expression of many genes critical for beta-cell function and growth, including Irs2 and various beta-cell transcription factors. These findings clearly indicate that chronic activation of a designer Gq-coupled receptor selectively expressed in pancreatic beta-cells can prevent STZ-induced diabetes, most likely by stimulating pathways that promote beta-cell function and proliferation. We obtained very similar results when we studied beta-Rq and WT control mice maintained on a high fat diet. These findings should stimulate the development of novel classes of antidiabetic drugs that exert their beneficial actions on beta-cell function by activating Gq-coupled receptors or downstream signaling pathways. In vivo metabolic consequences of activating a designer Gq-coupled receptor selectively expressed in mouse hepatocytes An increase in the rate of hepatic glucose production (HGP) is the major contributor to fasting hyperglycemia in T2D. Thus, a better understanding of the signaling pathways that regulate hepatic glucose fluxes is of great potential clinical relevance. For a number of reasons, the in vivo metabolic roles of hepatic Gq-coupled GPCRs remain poorly understood. To shed light on this issue, we generated a transgenic mouse line that expresses the Rq designer receptor selectively in hepatocytes (Hep-Rq mice). We demonstrated that acute treatment of Hep-Rq mice with CNO caused pronounced, dose-dependent increases in blood glucose levels, without any concomitant changes in plasma insulin levels. In addition, 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 increase in HGP. Injection of Hep-Rq mice with maximally effective doses of glucagon and CNO indicated that the rise in blood glucose levels induced by CNO was similar in magnitude to the corresponding glucagon response. In addition, isotope-labeling studies demonstrated that hepatocyte Rq receptors mediated increases in HGP by stimulating both gluconeogenesis and glycogenolysis. Taken together, these data raise the possibility that hepatocyte GPCRs that signal through Gq-type G proteins play important roles in regulating blood glucose levels. Interestingly, qRT-PCR studies showed that the expression levels of several Gq-coupled receptors were significantly increased in the liver of 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 elevated HGP. This finding raises the possibility that V1b vasopressin antagonists may prove useful to reduce HGP for the treatment of T2D.
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