Inhibition of CHRM3 Alleviates Necrosis Via the MAPK-p38/miR-31-5p/RIP3 Axis in L-Arginine-Induced Severe Acute Pancreatitis.

OBJECTIVES: Pancreatic acinar necrosis is a typical feature in the early phase of severe acute pancreatitis (SAP). Muscarinic acetylcholine receptor M3 (CHRM3) has been reported to play important roles in promoting insulin secretion and tumor cell proliferation, but its effect on necrosis remains unknown. This study revealed the important role of CHRM3 in regulating L-arginine-induced SAP and the molecular mechanisms. METHODS: To verify the function of CHRM3, pancreatic tissues and primary acinar cells of CRISPR/Cas9-mediated Chrm3 knockout mice were used in CHRM3 knockdown experiments, and to ascertain the CHRM3 overexpression, PLV-EGFP-Chrm3 plasmids were transfected in acinar cells in vitro. RESULTS: In L-arginine-induced SAP, CHRM3 is activated and regulates SAP through the mitogen-activated protein kinase/p38 pathway. Moreover, the expression of miR-31-5p decreased in the SAP model both in vitro and in vivo. Mir-31-5p effects the necrosis of acinar cells in SAP by upregulating the target gene RIP3, and miR-31-5p is a downstream miRNA of CHRM3. CONCLUSIONS: Necrosis in L-arginine-induced SAP is promoted by CHRM3 through the mitogen-activated protein kinase-p38/miR-31-5p/RIP3 axis.

Type 3 muscarinic receptors contribute to intestinal mucosal homeostasis and clearance of Nippostrongylus brasiliensis through induction of TH2 cytokines.

Despite increased appreciation for the role of nicotinic receptors in the modulation of and response to inflammation, the contribution of muscarinic receptors to mucosal homeostasis, clearance of enteric pathogens, and modulation of immune cell function remains relatively undefined. Uninfected and Nippostrongylus brasiliensis-infected wild-type and type 3 muscarinic receptor (M3R)-deficient (Chrm3(-/-)) mice were studied to determine the contribution of M3R to mucosal homeostasis as well as host defense against the TH2-eliciting enteric nematode N. brasiliensis Intestinal permeability and expression of TH1/TH17 cytokines were increased in uninfected Chrm3(-/-) small intestine. Notably, in Chrm3(-/-) mice infected with N. brasiliensis, small intestinal upregulation of TH2 cytokines was attenuated and nematode clearance was delayed. In Chrm3(-/-) mice, TH2-dependent changes in small intestinal function including smooth muscle hypercontractility, increased epithelial permeability, decreased epithelial secretion and absorption, and goblet cell expansion were absent despite N. brasiliensis infection. These findings identify an important role for M3R in host defense and clearance of N. brasiliensis, and support the expanding role of cholinergic muscarinic receptors in maintaining mucosal homeostasis.

Effect of M3 muscarinic acetylcholine receptor deficiency on collagen antibody-induced arthritis.

BACKGROUND: There is increasing evidence that the non-neuronal cholinergic system might be of importance for the pathology of rheumatoid arthritis. The role of M3 muscarinic acetylcholine receptor (M3R) in this regard has, however, not been investigated to date. Thus, in the present study we analyzed if M3R deficiency might have a protective effect on experimentally induced arthritis. METHODS: Collagen antibody-induced arthritis (CAIA) was evoked in M3R-deficient (M3R(-/-)) mice and wild-type (WT) littermates. Severity of arthritis was assessed by scoring of paw swelling. The joints of arthritic and nonarthritic animals were analyzed for histopathological changes regarding synovial tissue, cartilage degradation and bone destruction. Further, gene expression analysis of respective markers was performed. Systemic and local inflammatory response was determined by flow cytometry and immunohistochemistry for leukocytes as well as mRNA and protein measurements for pro-inflammatory cytokines and chemokines. RESULTS: In arthritic M3R(-/-) mice the number of leukocytes, specifically neutrophils, was enhanced even though clinical arthritis score was not significantly different between WT and M3R(-/-) mice with CAIA. In M3R(-/-) mice, levels of neutrophil chemoattractant chemokine C-X-C-motif ligand 2 (CXCL2) as well as the pro-inflammatory cytokine interleukin-6 were already strongly increased in mice with low arthritis score, whereas WT mice only showed prominent expression of these markers when reaching high arthritis scores. Furthermore, arthritic M3R(-/-) mice displayed a stronger degradation of collagen II in the articular cartilage and, most strikingly, histopathological evaluation revealed more severe bone destruction in arthritic mice with M3R deficiency compared to WT littermates. Moreover, in M3R(-/-) mice, gene expression of markers for bone degradation (matrix metalloproteinase 13, cathepsin K and receptor activator of nuclear factor-κB ligand) was already increased in mice with low arthritis score. CONCLUSIONS: Taken together, the present study shows that while M3R(-/-) mice were not protected from CAIA, they had a tendency toward a higher inflammatory response after arthritis induction than WT mice. Further, arthritis-induced joint destruction was significantly stronger in mice with M3R deficiency, indicating that stimulation of M3R might have protective effects on arthritis.

Muscarinic M3 receptors contribute to allergen-induced airway remodeling in mice.

Asthma is a chronic obstructive airway disease, characterized by inflammation and remodeling. Acetylcholine contributes to symptoms by inducing bronchoconstriction via the muscarinic M3 receptor. Recent evidence suggests that bronchoconstriction can regulate airway remodeling, and therefore implies a role for the muscarinic M3 receptor. The objective of this work was to study the contribution of the muscarinic M3 receptor to allergen-induced remodeling using muscarinic M3 receptor subtype-deficient (M3R(-/-)) mice. Wild-type (WT), M1R(-/-), and M2R(-/-) mice were used as controls. C57Bl/6 mice were sensitized and challenged with ovalbumin (twice weekly for 4 wk). Control animals were challenged with saline. Allergen exposure induced goblet cell metaplasia, airway smooth muscle thickening (1.7-fold), pulmonary vascular smooth muscle remodeling (1.5-fold), and deposition of collagen I (1.7-fold) and fibronectin (1.6-fold) in the airway wall of WT mice. These effects were absent or markedly lower in M3R(-/-) mice (30-100%), whereas M1R(-/-) and M2R(-/-) mice responded similarly to WT mice. In addition, airway smooth muscle and pulmonary vascular smooth muscle mass were 35-40% lower in saline-challenged M3R(-/-) mice compared with WT mice. Interestingly, allergen-induced airway inflammation, assessed as infiltrated eosinophils and T helper type 2 cytokine expression, was similar or even enhanced in M3R(-/-) mice. Our data indicate that acetylcholine contributes to allergen-induced remodeling and smooth muscle mass via the muscarinic M3 receptor, and not via M1 or M2 receptors. No stimulatory role for muscarinic M3 receptors in allergic inflammation was observed, suggesting that the role of acetylcholine in remodeling is independent of the allergic inflammatory response, and may involve bronchoconstriction.

Distinct roles of M1 and M3 muscarinic acetylcholine receptors controlling oscillatory and non-oscillatory [Ca2+]i increase.

We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3-3 µM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03-0.3 µM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca(2+)-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation.

Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome.

Urinary bladder malformations associated with bladder outlet obstruction are a frequent cause of progressive renal failure in children. We here describe a muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) homozygous frameshift mutation in familial congenital bladder malformation associated with a prune-belly-like syndrome, defining an isolated gene defect underlying this sometimes devastating disease. CHRM3 encodes the M3 muscarinic acetylcholine receptor, which we show is present in developing renal epithelia and bladder muscle. These observations may imply that M3 has a role beyond its known contribution to detrusor contractions. This Mendelian disease caused by a muscarinic acetylcholine receptor mutation strikingly phenocopies Chrm3 null mutant mice.

A novel clinical syndrome revealing a deficiency of the muscarinic M3 receptor.

OBJECTIVES: No clinical disorders have been caused by dysfunction of any of the 5 subtypes (M1-M5) of muscarinic receptors. We present a patient with a novel clinical syndrome that we suggest results from a deficiency of the muscarinic M3 receptor. METHODS: We conducted a comprehensive workup of autonomic function. The patient's disorder was compared to the phenotypic features of male M3 knockout mice. M3 protein quantity was assessed by Western blot and radioligand binding in peripheral blood lymphocytes. Tests for autoantibodies and genetic abnormalities were performed. RESULTS: The disease pattern was characterized by disturbances in micturition, pupil constriction, body weight, and sudomotor function, with normal accommodation, gastrointestinal motility, salivation, and lacrimation, similar to features of male M3 knockout mice. M3 protein quantity was reduced. Genetic tests were unrevealing, but unspecific antinuclear antibodies were present. CONCLUSIONS: The presented clinical syndrome suggests a deficiency of the muscarinic M3 receptor. These results and future evaluation of patients with autonomic deficits may provide insights into the site and functional role of the muscarinic M3 receptor in humans.

Muscarinic receptor subtypes mediating Ca2+ sensitization of intestinal smooth muscle contraction: studies with receptor knockout mice.

In the present study, we have characterized muscarinic receptor subtypes that mediate carbachol-induced Ca2+ sensitization of contraction in intestinal smooth muscle, using mutant mice lacking M(2) or M(3) muscarinic receptors or both receptor subtypes. In alpha-toxin-permeabilized muscle strips from wild-type (WT) mice, isometric tension responses to Ca2+ applied cumulatively (pCa 7.0-5.0) were increased when the muscarinic agonist carbachol (100 microM) was added to the medium, as judged from shifts of pCa-tension curves in both 50% effective concentration (EC(50)) and maximum response (E(max)) of pCa-tension curve. In preparations from M(2)-knockout (KO) mice, pCa-tension curves were also shifted by carbachol (100 microM), and the extents of the EC(50) and E(max) changes resembled those observed in preparations from WT mice. In preparations from M(3)-KO or M(2)/M(3)-double KO mice, however, no significant changes in pCa-tension curves were obtained after carbachol application. The G(q/11)-type G-protein inhibitor YM-254890 (1 microM) completely blocked the Ca2+ sensitization of contraction induced by carbachol in M(2)-KO or WT preparations. The results strongly support the idea that the muscarinic activation of Ca2+ sensitization in intestinal smooth muscles is mediated by the M(3) muscarinic receptor coupled to G(q/11)-type G-proteins, without any significant involvement of the other muscarinic receptor subtypes including M(2).

Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways.

Cholinergic bronchoconstriction is mediated by M(2) and M(3) muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-ß-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R(-/-) and M3R(-/-) mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.

M3 muscarinic receptors mediate positive inotropic responses in mouse atria: a study with muscarinic receptor knockout mice.

The potential functional roles of M(3) muscarinic receptors in mouse atria were examined by pharmacological and molecular biological techniques, using wild-type mice, muscarinic M(2) or M(3) receptor single knockout (M(2)KO, M(3)KO), and M(2) and M(3) muscarinic receptor double knockout mice (M(2)/M(3)KO). Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that the M(2) receptor mRNA was expressed predominantly in mouse atria but that the M(1), M(3), M(4), and M(5) receptor subtypes were also expressed at low levels. Carbachol (10 nM-30 microM) decreased the spontaneous beating frequency of right atria isolated from wild-type mice. Studies with subtype-preferring antagonists and atria from M(2)KO mice confirmed that this activity is mediated by the M(2) receptor subtype. In left atria from wild-type mice, carbachol decreased the amplitude of electrical field stimulation-evoked contractions (negative inotropic action), but this inhibition was transient and was followed by a gradual increase in contraction amplitude (positive inotropic response). In atria from M(3)KO mice, the transient negative inotropic action of carbachol changed to a sustained negative inotropic action. In contrast, in atria from M(2)KO mice, carbachol showed only positive inotropic activity. In atria from M(2)/M(3) double KO mice, carbachol was devoid of any inotropic activity. These observations, complemented by functional studies with subtype-preferring antagonists, convincingly demonstrate that atrial M(3) muscarinic receptors mediate positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M(2) receptor activation on atrial contractility.

N-methyl-D-aspartate receptors mediate the phosphorylation and desensitization of muscarinic receptors in cerebellar granule neurons.

Changes in synaptic strength mediated by ionotropic glutamate N-methyl-D-asparate (NMDA) receptors is generally considered to be the molecular mechanism underlying memory and learning. NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs). In this study we investigate the ability of NMDA receptors to regulate the signaling of GPCRs by focusing on the G(q/11)-coupled M(3)-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-muscarinic receptors through a mechanism dependent on NMDA-mediated calcium influx and the activity of calcium-calmodulin-dependent protein kinase II. Our study reveals a complex pattern of regulation where GPCRs (M(3)-muscarinic) and NMDA receptors can feedback on each other in a process that is likely to influence the threshold value of signaling networks involved in synaptic plasticity.

Metabolic roles of the M3 muscarinic acetylcholine receptor studied with M3 receptor mutant mice: a review.

The M(3) muscarinic acetylcholine (ACh) receptor (M(3) 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 G(q) family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M(3) mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M(3) receptors in pancreatic beta -cells indicated that beta -cell M(3) mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through beta -cell M(3) mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M(3) mAChR knockout mice showed that the absence of M(3) receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M(3) 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.

Role of the M3 muscarinic acetylcholine receptor in beta-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 beta-cells. Studies with isolated islets prepared from whole body M(3) muscarinic ACh receptor knockout mice showed that cholinergic amplification of glucose-dependent insulin secretion is exclusively mediated by the M(3) muscarinic receptor subtype. To investigate the physiological relevance of this muscarinic pathway, we used Cre/loxP technology to generate mutant mice that lack M(3) receptors only in pancreatic beta-cells. These mutant mice displayed impaired glucose tolerance and significantly reduced insulin secretion. In contrast, transgenic mice overexpressing M(3) receptors in pancreatic beta-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 beta-cell M(3) muscarinic receptors are essential for maintaining proper insulin secretion and glucose homeostasis. Moreover, our data suggest that enhancing signalling through beta-cell M(3) muscarinic receptors may represent a new avenue in the treatment of glucose intolerance and type 2 diabetes.

Three distinct muscarinic signalling pathways for cationic channel activation in mouse gut smooth muscle cells.

Using mutant mice genetically lacking certain subtypes of muscarinic receptor, we have studied muscarinic signal pathways mediating cationic channel activation in intestinal smooth muscle cells. In cells from M2 subtype-knockout (M2-KO) or M3-KO mice, carbachol (100 microM) evoked a muscarinic cationic current (mI(Cat)) as small as approximately 10% of mI(Cat) in wild-type (WT) cells. No appreciable current was evoked in M2/M3 double-KO cells. All mutant type cells preserved normal G-protein-cationic channel coupling. The M3-KO and WT mI(Cat) each showed a U-shaped current-voltage (I-V) relationship, whereas the M2-KO mI(Cat) displayed a linear I-V relationship. Channel analysis in outside-out patches recognized 70-pS and 120-pS channels as the major muscarinic cationic channels. Active patches of M2-KO cells exhibited both 70-pS and 120-pS channel activity usually together, either of which consisted of brief openings (the respective mean open times O(tau) = 0.55 and 0.23 ms). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (O(tau) = 0.55, 3.1 and 17.4 ms). In WT patches, besides the M2-KO and M3-KO types, another type of channel activity was also observed that consisted of 70-pS channel openings with four open states (O(tau) = 0.62, 2.7, 16.9 and 121.1 ms), and patch current of this channel activity showed a U-shaped I-V curve similar to the WT mI(Cat). The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M2/M3 pathway targeting 70-pS channels, serves as the major contributor to mI(Cat) generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2-Go protein and the M3-PLC systems predicted to control cationic channels.

Endocrine precursor cells from mouse islets are not generated by epithelial-to-mesenchymal transition of mature beta cells.

We previously presented evidence that proliferative human islet precursor cells may be derived in vitro from adult islets by epithelial-to-mesenchymal transition (EMT) and show here that similar fibroblast-like cells can be derived from mouse islets. These mouse cell populations exhibited changes in gene expression consistent with EMT. Both C-peptide and insulin mRNAs were undetectable in expanded cultures of mouse islet-derived precursor cells (mIPCs). After expansion, mIPCs could be induced to migrate into clusters and differentiate into hormone-expressing islet-like aggregates. Although early morphological changes suggesting EMT were observed by time-lapse microscopy when green fluorescent protein-labeled beta cells were placed in culture, the expanded precursor cell population was not fluorescent. Using two mouse models in which beta cells were permanently made either to express alkaline phosphatase or to have a deleted M(3) muscarinic receptor, we provide evidence that mIPCs in long term culture are not derived from beta cells.

M2 and M3 muscarinic receptors are involved in enteric nerve-mediated contraction of the mouse ileum: Findings obtained with muscarinic-receptor knockout mouse.

The involvement of muscarinic receptors in neurogenic responses of the ileum was studied in wild-type and muscarinic-receptor (M-receptor) knockout (KO) mice. Electrical field stimulation to the wild-type mouse ileum induced a biphasic response, a phasic and sustained contraction that was abolished by tetrodotoxin. The sustained contraction was prolonged for an extended period after the termination of electrical field stimulation. The phasic contraction was completely inhibited by atropine. In contrast, the sustained contraction was enhanced by atropine. Ileal strips prepared from M2-receptor KO mice exhibited a phasic contraction similar to that seen in wild-type mice and a sustained contraction that was larger than that in wild-type mice. In M3-receptor KO mice, the phasic contraction was smaller than that observed in wild-type mice. Acetylcholine exogenously administrated induced concentration-dependent contractions in strips isolated from wild-type, M2- and M3-receptor KO mice. However, contractions in M3-receptor KO mice shifted to the right. The sustained contraction was inhibited by capsaicin and neurokinin NK2 receptor antagonist, suggesting that it is mediated by substance P (SP). SP-induced contraction of M2-receptor KO mice did not differ from that of wild-type mice. SP immunoreactivity was located in enteric neurons, colocalized with M2 receptor immunoreactivity. These results suggest that atropine-sensitive phasic contraction is mainly mediated via the M3 receptor, and SP-mediated sustained contraction is negatively regulated by the M2 receptor at a presynaptic level.

M1-M3 muscarinic acetylcholine receptor-deficient mice: novel phenotypes.

The five muscarinic acetylcholine receptors (M1-M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R-/- mice, M2R-/- mice, M3R-/- mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion.

Beneficial metabolic effects of M3 muscarinic acetylcholine receptor deficiency.

Most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity. The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation. Here, we tested the hypothesis that the absence of M3 receptors in mice might protect against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. In all cases, the lack of M3 receptors greatly ameliorated impairments in glucose homeostasis and insulin sensitivity but had less robust effects on overall adiposity. Under all experimental conditions tested, M3 receptor-deficient mice showed a significant elevation in basal and total energy expenditure, most likely due to enhanced central sympathetic outflow and increased rate of fatty-acid oxidation. These findings suggest that the M3 receptor may represent a potential pharmacologic target for the treatment of obesity and associated metabolic disorders.

A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.

One of the hallmarks of type 2 diabetes is that pancreatic beta cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic beta cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M3 muscarinic acetylcholine receptor subtype in pancreatic beta cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.