Dinesh Gautam

Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development

Muscarinic acetylcholine receptors (mAChRs), M1–M5, regulate the activity of numerous fundamental central and peripheral functions. The lack of small-molecule ligands that can block or activate specific mAChR subtypes with high selectivity has remained a major obstacle in defining the roles of the individual receptor subtypes and in the development of novel muscarinic drugs. Recently, phenotypic analysis of mutant mouse strains deficient in each of the five mAChR subtypes has led to a wealth of new information regarding the physiological roles of the individual receptor subtypes. Importantly, these studies have identified specific mAChR-regulated pathways as potentially novel targets for the treatment of various important disorders including Alzheimers disease, schizophrenia, pain, obesity and diabetes.

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

Impaired functioning of pancreatic β cells is a key hallmark of type 2 diabetes. β cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific β cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that β cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific β cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in β 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 β cell Gq/11 or Gs G proteins. Here we report the findings that conditional and selective activation of β 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 β cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of β 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 β cell function in vivo.

Muscarinic acetylcholine receptors: novel opportunities for drug development

The muscarinic acetylcholine receptors are a subfamily of G protein-coupled receptors that regulate numerous fundamental functions of the central and peripheral nervous system. The past few years have witnessed unprecedented new insights into muscarinic receptor physiology, pharmacology and structure. These advances include the first structural views of muscarinic receptors in both inactive and active conformations, as well as a better understanding of the molecular underpinnings of muscarinic receptor regulation by allosteric modulators. These recent findings should facilitate the development of new muscarinic receptor subtype-selective ligands that could prove to be useful for the treatment of many severe pathophysiological conditions.

RGS4 is a negative regulator of insulin release from pancreatic beta-cells in vitro and in vivo.

Therapeutic strategies that augment insulin release from pancreatic β-cells are considered beneficial in the treatment of type 2 diabetes. We previously demonstrated that activation of β-cell M3 muscarinic receptors (M3Rs) greatly promotes glucose-stimulated insulin secretion (GSIS), suggesting that strategies aimed at enhancing signaling through β-cell M3Rs may become therapeutically useful. M3R activation leads to the stimulation of G proteins of the Gq family, which are under the inhibitory control of proteins known as regulators of G protein signaling (RGS proteins). At present, it remains unknown whether RGS proteins play a role in regulating insulin release. To address this issue, we initially demonstrated that MIN6 insulinoma cells express functional M3Rs and that RGS4 was by far the most abundant RGS protein expressed by these cells. Strikingly, siRNA-mediated knockdown of RGS4 expression in MIN6 cells greatly enhanced M3R-mediated augmentation of GSIS and calcium release. We obtained similar findings using pancreatic islets prepared from RGS4-deficient mice. Interestingly, RGS4 deficiency had little effect on insulin release caused by activation of other β-cell GPCRs. Finally, treatment of mutant mice selectively lacking RGS4 in pancreatic β-cells with a muscarinic agonist (bethanechol) led to significantly increased plasma insulin and reduced blood glucose levels, as compared to control littermates. Studies with β-cell-specific M3R knockout mice showed that these responses were mediated by β-cell M3Rs. These findings indicate that RGS4 is a potent negative regulator of M3R function in pancreatic β-cells, suggesting that RGS4 may represent a potential target to promote insulin release for therapeutic purposes.

Role of the M3 muscarinic acetylcholine receptor in β‐cell function and glucose homeostasis

The release of insufficient amounts of insulin in the presence of elevated blood glucose levels is one of the key features of type 2 diabetes. Various lines of evidence indicate that acetylcholine (ACh), the major neurotransmitter of the parasympathetic nervous system, can enhance glucose‐stimulated insulin secretion from pancreatic β‐cells. Studies with isolated islets prepared from whole body M3 muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose‐dependent insulin secretion is exclusively mediated by the M3 muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M3 receptors only in pancreatic β‐cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M3 receptors in pancreatic β‐cells showed a pronounced increase in glucose tolerance and insulin secretion and were resistant to diet‐induced glucose intolerance and hyperglycaemia. These findings indicate that β‐cell M3 muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through β‐cell M3 muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

Muscarinic acetylcholine receptor knockout mice show distinct synaptic plasticity impairments in the visual cortex

In the present report, we focused our attention on the role played by the muscarinic acetylcholine receptors (mAChRs) in different forms of long‐term synaptic plasticity. Specifically, we investigated long‐term potentiation (LTP) and long‐term depression (LTD) expression elicited by theta‐burst stimulation (TBS) and low‐frequency stimulation (LFS), respectively, in visual cortical slices obtained from different mAChR knockout (KO) mice. A normal LTP was evoked in M1/M3 double KO mice, while LTP was impaired in the M2/M4 double KO animals. On the other hand, LFS induced LTD in M2/M4 double KO mice, but failed to do so in M1/M3 KO mice. Interestingly, LFS produced LTP instead of LTD in M1/M3 KO mice. Analysis of mAChR single KO mice revealed that LTP was affected only by the simultaneous absence of both M2 and M4 receptors. A LFS‐dependent shift from LTD to LTP was also observed in slices from M1 KO mice, while LTD was simply abolished in slices from M3 KO mice. Using pharmacological tools, we showed that LTP in control mice was blocked by pertussis toxin, an inhibitor of Gi/o proteins, but not by raising intracellular cAMP levels. In addition, the inhibition of phospholipase C by U73122 induced the same shift from LTD to LTP after LFS observed in M1 single KO and M1/M3 double KO mice. Our results indicate that different mAChR subtypes regulate different forms of long‐term synaptic plasticity in the mouse visual cortex, activating specific G proteins and downstream intracellular mechanisms.

Acetylcholine modulates cortical synaptic transmission via different muscarinic receptors, as studied with receptor knockout mice

The central cholinergic system plays a crucial role in synaptic plasticity and spatial attention; however, the roles of the individual cholinergic receptors involved in these activities are not well understood at present. In the present study, we show that acetylcholine (ACh) can facilitate or depress synaptic transmission in occipital slices of mouse visual cortex. The precise nature of the ACh effects depends on the ACh concentration, and is input specific, as shown by stimulating different synaptic pathways. Pharmacological blockade of muscarinic receptor (mAChR) subtypes and the use of M1–M5 mAChR‐deficient mice showed that specific mAChR subtypes, together with the activity of the cholinesterases (ChEs), mediate facilitation or depression of synaptic transmission. The present data suggest that local ACh, acting through mAChRs, regulates the cortical dynamics making cortical circuits respond to specific stimuli.

Beneficial Metabolic Effects Caused by Persistent Activation of β-Cell M3 Muscarinic Acetylcholine Receptors in Transgenic Mice

Previous studies have shown that β-cell M(3) muscarinic acetylcholine receptors (M3Rs) play a key role in maintaining blood glucose homeostasis by enhancing glucose-dependent insulin release. In this study, we tested the hypothesis that long-term, persistent activation of β-cell M3Rs can improve glucose tolerance and ameliorate the metabolic deficits associated with the consumption of a high-fat diet. To achieve the selective and persistent activation of β-cell M3Rs in vivo, we generated transgenic mice that expressed the Q490L mutant M3R in their pancreatic β-cells (β-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 15 mm glucose and the absence of M3R ligands, isolated perifused islets prepared from β-M3-Q490L Tg mice released considerably more insulin than wild-type control islets. This effect could be completely blocked by incubation of the transgenic islets with atropine (10 μm), an inverse muscarinic agonist, indicating that the Q490L mutant M3R exhibited ligand-independent signaling (constitutive activity) in mouse β-cells. In vivo studies showed that β-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 β-cell M3Rs may represent a useful approach to boost insulin output in the long-term treatment of type 2 diabetes.

Metabolic Roles of the M3 Muscarinic Acetylcholine Receptor Studied with M3 Receptor Mutant Mice: A Review

The M3 muscarinic acetylcholine (ACh) receptor (M3 mAChR) is expressed in many central and peripheral tissues. It is a prototypic member of the superfamily of G protein-coupled receptors and preferentially activates G proteins of the Gq family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M3 mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M3 receptors in pancreatic β -cells indicated that β -cell M3 mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through β -cell M3 mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M3 mAChR knockout mice showed that the absence of M3 receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M3 receptor-deficient mice showed greatly ameliorated impairments in glucose homeostasis and insulin sensitivity, reduced food intake, and a significant elevation in basal and total energy expenditure, most likely due to increased central sympathetic outflow and increased rate of fatty acid oxidation. These findings are of potential interest for the development of novel therapeutic approaches for the treatment of obesity and associated metabolic disorders.

Hepatic Muscarinic Acetylcholine Receptors Are Not Critically Involved in Maintaining Glucose Homeostasis in Mice

OBJECTIVE An increase in the rate of hepatic glucose production is the major determinant of fasting hyperglycemia in type 2 diabetes. A better understanding of the signaling pathways and molecules that regulate hepatic glucose metabolism is therefore of great clinical importance. Recent studies suggest that an increase in vagal outflow to the liver leads to decreased hepatic glucose production and reduced blood glucose levels. Since acetylcholine (ACh) is the major neurotransmitter of the vagus nerve and exerts its parasympathetic actions via activation of muscarinic ACh receptors (mAChRs), we examined the potential metabolic relevance of hepatocyte mAChRs. RESEARCH DESIGN AND METHODS We initially demonstrated that the M3 mAChR is the only mAChR subtype expressed by mouse liver/hepatocytes. To assess the physiological role of this receptor subtype in regulating hepatic glucose fluxes and glucose homeostasis in vivo, we used gene targeting and transgenic techniques to generate mutant mice lacking or overexpressing M3 receptors in hepatocytes only. RESULTS Strikingly, detailed in vivo phenotyping studies failed to reveal any significant metabolic differences between the M3 receptor mutant mice and their control littermates, independent of whether the mice were fed regular or a high-fat diet. Moreover, the expression levels of genes for various key transcription factors, signaling molecules, and enzymes regulating hepatic glucose fluxes were not significantly altered in the M3 receptor mutant mice. CONCLUSIONS This rather surprising finding suggests that the pronounced metabolic effects mediated by activation of hepatic vagal nerves are mediated by noncholinergic signaling pathways.