A chemical-genetic approach to study G protein regulation of β cell function in vivo

Abstract

Impaired functioning of pancreatic ␤ cells is a key hallmark of type 2 diabetes. ␤ cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific ␤ cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that ␤ cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemicalgenetic approach that allows for the conditional and selective activation of specific ␤ cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in ␤ cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (Gq/11 versus Gs). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either ␤ cell Gq/11 or Gs G proteins. Here we report the findings that conditional and selective activation of ␤ cell Gq/11 signaling in vivo leads to striking increases in both first-and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated ␤ cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of ␤ cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of ␤ cell function in vivo.beta cells ͉ G protein-coupled receptors ͉ transgenic mice ͉ type 2 diabetes T ype 2 diabetes has emerged as one of the major threats to human health in the 21st century (1). Impaired function of pancreatic ␤ cells is one of the key hallmarks of type 2 diabetes, and therapies targeted at improving ␤ cell function are predicted to offer considerable therapeutic benefit (2).␤ Cell function is modulated by the actions of different classes of heterotrimeric G proteins which are the immediate downstream targets of a multitude of G protein-coupled receptors (GPCRs). Like most other cell types, pancreatic ␤ cells are predicted to express many different GPCRs (3-5). Several lines of evidence suggest that activation of G s -coupled receptors expressed by pancreatic ␤ cells, including the glucagon-like peptide (GLP-1) receptor, improves ␤ cell function and can increase in ␤ cell mass via cAMP-dependent mechanisms (5-7). Pancreatic ␤ cells also express several G q/11 -coupled receptors, including the M 3 muscarinic acetylcholine (ACh) receptor (M3R) and GPR40, which can promote insulin release in an agonist-dependent fashion [for recent reviews, see (5,8)].Studies with GLP-1 receptor agonists have yielded detailed information about the beneficial effects of G s signaling on ␤ cell function and whole body glucose homeostasis (note that the GLP-1 receptor is enriched in pancreatic ␤ cells) (5-7). In contrast, much...

Medial habenula cholinergic signaling regulates cocaine‐associated relapse‐like behavior

“…While it has been shown that CNO is not significantly back metabolized to clozapine in mice, it is possible that the limited clozapine produced is sufficient to induce DREADD activity reported in Gomez et al, while being subthreshold for off-target effects (Guettier et al 2009). Moreover, while it has been demonstrated that systemic clozapine is able to induce changes in neural activity, these changes occur in response to significantly higher doses (35 mg/kg) than what could be back metabolized from our doses of CNO (3 mg/kg) (Werme et al 2000).…”

Chemogenetic and Optogenetic Activation of Gαs Signaling in the Basolateral Amygdala Induces Acute and Social Anxiety-Like States

“…We first utilized chemogenetics to selectively control generic Gαs signaling in excitatory neurons of the BLA through the use of the Gαs DREADD rM3Ds (designer receptors exclusively activated by designer drugs) (Armbruster et al, 2007;Farrell et al, 2013;Guettier et al, 2009). Here we show that activation of generic Gαs signaling via within the BLA is sufficient to induce acute and social anxiety-like behavioral states.…”

Selective Manipulation of Neural Circuits

“…Like optogenetics, designer receptor exclusively activated by designer drug (DREADDs) can either activate or silence neurons. The first DREADD system mutated the muscarinic acetylcholine receptor so that it is only activated by clozapine-N-oxide, an inactive metabolite of the central nervous system drug clozapine [146,148,149]. The muscarinic DREADD receptor is coupled to Gq, which is an excitatory channel that depolarizes neurons and enhances their excitability through phospholipase C [146,150].…”

“…The muscarinic DREADD receptor is coupled to Gq, which is an excitatory channel that depolarizes neurons and enhances their excitability through phospholipase C [146,150]. A chimeric muscarinic-adrenergic receptor DREADD known as GsD also activates neurons but via cyclic adenosine monophosphate-mediated signaling [149,151]. In contrast, the human M4 muscarinic DREADD receptor is coupled to Gi and leads to neuronal silencing through opening inwardly rectifying potassium channels [146].…”