Yinghong Cui

Cholinergic dilation of cerebral blood vessels is abolished in M 5 muscarinic acetylcholine receptor knockout mice

The M5 muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M1-M5) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M5 receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M5 receptor-deficient mice (M5R−/− mice). M5R−/− mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M5 receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M5 receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M5 receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R−/− mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5R−/− mice. Our findings provide direct evidence that M5 muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimers disease and focal cerebral ischemia, cerebrovascular M5 receptors may represent an attractive therapeutic target.

A Subpopulation of Neuronal M4 Muscarinic Acetylcholine Receptors Plays a Critical Role in Modulating Dopamine-Dependent Behaviors

Acetylcholine (ACh) regulates many key functions of the CNS by activating cell surface receptors referred to as muscarinic ACh receptors (M1–M5 mAChRs). Like other mAChR subtypes, the M4 mAChR is widely expressed in different regions of the forebrain. Interestingly, M4 mAChRs are coexpressed with D1 dopamine receptors in a specific subset of striatal projection neurons. To investigate the physiological relevance of this M4 mAChR subpopulation in modulating dopamine-dependent behaviors, we used Cre/loxP technology to generate mutant mice that lack M4 mAChRs only in D1 dopamine receptor-expressing cells. The newly generated mutant mice displayed several striking behavioral phenotypes, including enhanced hyperlocomotor activity and increased behavioral sensitization following treatment with psychostimulants. These behavioral changes were accompanied by a lack of muscarinic inhibition of D1 dopamine receptor-mediated cAMP stimulation in the striatum and an increase in dopamine efflux in the nucleus accumbens. These novel findings demonstrate that a distinct subpopulation of neuronal M4 mAChRs plays a critical role in modulating several important dopamine-dependent behaviors. Since enhanced central dopaminergic neurotransmission is a hallmark of several severe disorders of the CNS, including schizophrenia and drug addiction, our findings have substantial clinical relevance.

Muscarinic Acetylcholine Receptor Knockout Mice

Muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous system. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M1-M5) has proven a difficult task, primarily due to the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues and organs express multiple mAChRs. To circumvent these difficulties, we and others have used gene targeting strategies to generate mutant mouse lines containing inactivating mutations of the M1-M5 mAChR genes. The different mAChR mutant mice and the corresponding wild-type control animals were subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. The M1-M5 mAChR mutant mice (MXR-/- mice) were all viable and reproduced normally. However, each mutant mouse line displayed distinct phenotypical changes. For example, M1R-/- mice showed a pronounced increase in locomotor activity, probably due to the increase in dopamine release in the striatum. In addition, pilocarpine-induced epileptic seizures were absent in M1R-/- mice. Pharmacological analysis of M2R-/- mice indicated that the M2 subtype plays a key role in mediating three of the most striking central muscarinic effects: tremor, hypothermia, and analgesia. As expected, muscarinic agonist-mediated bradycardia was abolished in M2R-/- mice. M3R-/- mice displayed a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low serum leptin and insulin levels. Additional studies showed that the M3 receptor subtype also plays a key role in mediating smooth muscle contraction and glandular secretion. Behavioral analysis of M4R-/- mice suggested that M4 receptors mediate inhibition of D1 dopamine receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons. Studies with M5R-/- mice indicated that vascular M5 receptors mediate cholinergic relaxation of cerebral arteries and arterioles. Behavioral and neurochemical studies showed that M5 receptor activity modulates both morphine reward and withdrawal processes, probably through activation of M5 receptors located on midbrain dopaminergic neurons. These results offer promising new perspectives for the rational development of novel muscarinic drugs.

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 (M) Receptors in Coronary Circulation: Gene-Targeted Mice Define the Role of M2 and M3 Receptors in Response to Acetylcholine

Objective—Determining the role of specific muscarinic (M) receptor subtypes mediating responses to acetylcholine (ACh) has been limited by the specificity of pharmacological agents. Deletion of the gene for M5 receptors abolished response to ACh in cerebral blood vessels but did not affect dilation of coronary arteries. The goal of this study was to determine the M receptors mediating responses to ACh in coronary circulation using mice deficient in M2 or M3 receptors (M2−/−, M3−/−, respectively). Methods and Results—Coronary arteries from respective wild-type, M2−/−, or M3−/− mice were isolated, cannulated, and pressurized. Diameter was measured with video microscopy. After preconstriction with U46619, ACh produced dose-dependent dilation of coronary arteries that was similar in wild-type and M2−/− mice. In contrast, dilation of coronary arteries from M3−/− mice to ACh was reduced by ≈80% compared with wild type. The residual response to ACh was atropine insensitive. Relaxation of coronary arteries to other stimuli was similar in M2−/− and M3−/− mice. Similar results were obtained in aorta rings. Conclusion—These findings provide the first direct evidence that relaxation to ACh in coronary circulation is mediated predominantly by activation of M3 receptors.

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.

Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake

Agouti-related peptide (AgRP) neurons of the hypothalamus play a key role in regulating food intake and body weight, by releasing three different orexigenic molecules: AgRP; GABA; and neuropeptide Y. AgRP neurons express various G protein-coupled receptors (GPCRs) with different coupling properties, including Gs-linked GPCRs. At present, the potential role of Gs-coupled GPCRs in regulating the activity of AgRP neurons remains unknown. Here we show that the activation of Gs-coupled receptors expressed by AgRP neurons leads to a robust and sustained increase in food intake. We also provide detailed mechanistic data linking the stimulation of this class of receptors to the observed feeding phenotype. Moreover, we show that this pathway is clearly distinct from other GPCR signalling cascades that are operative in AgRP neurons. Our data suggest that drugs able to inhibit this signalling pathway may become useful for the treatment of obesity.

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.

A Novel Experimental Strategy to Assess the Metabolic Effects of Selective Activation of a Gq-Coupled Receptor in Hepatocytes In Vivo

Increased hepatic glucose production is a key pathophysiological feature of type 2 diabetes. Like all other cell types, hepatocytes express many G protein-coupled receptors (GPCRs) that are linked to different functional classes of heterotrimeric G proteins. The important physiological functions mediated by G(s)-coupled hepatic glucagon receptors are well-documented. In contrast, little is known about the in vivo physiological roles of hepatocyte GPCRs that are linked to G proteins of the G(q) family. To address this issue, we established a transgenic mouse line (Hep-Rq mice) that expressed a G(q)-linked designer receptor (Rq) in a hepatocyte-selective fashion. Importantly, Rq could no longer bind endogenous ligands but could be selectively activated by a synthetic drug, clozapine-N-oxide. Clozapine-N-oxide treatment of Hep-Rq mice enabled us to determine the metabolic consequences caused by selective activation of a G(q)-coupled GPCR in hepatocytes in vivo. We found that acute Rq activation in vivo led to pronounced increases in blood glucose levels, resulting from increased rates of glycogen breakdown and gluconeogenesis. We also demonstrated that the expression of the V(1b) vasopressin receptor, a G(q)-coupled receptor expressed by hepatocytes, was drastically increased in livers of ob/ob mice, a mouse model of diabetes. Strikingly, treatment of ob/ob mice with a selective V(1b) receptor antagonist led to reduced glucose excursions in a pyruvate challenge test. Taken together, these findings underscore the importance of G(q)-coupled receptors in regulating hepatic glucose fluxes and suggest novel receptor targets for the treatment of type 2 diabetes.