Alokesh Duttaroy

Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States

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Publications (16)112.39 Total impact

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    D Gautam · S-J Han · A Duttaroy · D Mears · F F Hamdan · J H Li · Y Cui · J Jeon · J Wess
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    ABSTRACT: 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.
    Diabetes Obesity and Metabolism 12/2007; 9 Suppl 2(s2):158-69. DOI:10.1111/j.1463-1326.2007.00781.x · 5.46 Impact Factor
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    ABSTRACT: 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.
    Journal of Molecular Neuroscience 02/2006; 30(1-2):157-60. DOI:10.1385/JMN:30:1:157 · 2.76 Impact Factor
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    ABSTRACT: Pancreatic muscarinic acetylcholine receptors play an important role in stimulating insulin and glucagon secretion from islet cells. To study the potential role of the M(3) muscarinic receptor subtype in cholinergic stimulation of insulin release, we initially examined the effect of the muscarinic agonist, oxotremorine-M (Oxo-M), on insulin secretion from isolated pancreatic islets prepared from wild-type (WT) and M(3) receptor-deficient mice (M3(+/-) and M3(-/-) mice). At a stimulatory glucose level (16.7 mmol/l), Oxo-M strongly potentiated insulin output from islets of WT mice. Strikingly, this effect was completely abolished in islets from M3(-/-) mice and significantly reduced in islets from M3(+/-) mice. Additional in vitro studies showed that Oxo-M-mediated glucagon release was also virtually abolished in islets from M3(-/-) mice. Consistent with the in vitro data, in vivo studies showed that M3(-/-) mice displayed reduced serum insulin and plasma glucagon levels and a significantly blunted increase in serum insulin after an oral glucose load. Despite the observed impairments in insulin release, M3(-/-) mice showed significantly reduced blood glucose levels and even improved glucose tolerance, probably due to the reduction in plasma glucagon levels and the fact that M3(-/-) mice are hypophagic and lean. These findings provide important new insights into the metabolic roles of the M(3) muscarinic receptor subtype.
    Diabetes 08/2004; 53(7):1714-20. DOI:10.2337/diabetes.53.7.1714 · 8.47 Impact Factor
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    ABSTRACT: Until recently, little was known about the possible physiological functions of the M(5) muscarinic acetylcholine receptor subtype, the last member of the muscarinic receptor family (M(1)-M(5)) to be cloned. To learn more about the potential physiological roles of this receptor subtype, we generated and analyzed M(5) receptor-deficient mice (M5 -/- mice). Strikingly, acetylcholine, a potent dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5 -/- mice, suggesting that endothelial M(5) receptors mediate this activity in wild-type mice. This effect was specific for cerebral blood vessels, since acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5 -/- mice. In addition, in vitro neurotransmitter release experiments indicated that M(5) receptors located on dopaminergic nerve terminals play a role in facilitating muscarinic agonist-induced dopamine release in the striatum, consistent with the observation that the dopaminergic neurons innervating the striatum almost exclusively express the M(5) receptor subtype. We also found that the rewarding effects of morphine, the prototypical opiate analgesic, were substantially reduced in M5 -/- mice, as measured in the conditioned place preference paradigm. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5 -/- mice. It is likely that these behavioral deficits are caused by the lack of mesolimbic M(5) receptors, activation of which is known to stimulate dopamine release in the nucleus accumbens. These results convincingly demonstrate that the M(5) muscarinic receptor is involved in modulating several important pharmacological and behavioral functions. These findings may lead to novel therapeutic strategies for the treatment of drug addiction and certain cerebrovascular disorders.
    Life Sciences 01/2004; 74(2-3):345-53. DOI:10.1016/j.lfs.2003.09.022 · 2.30 Impact Factor
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    ABSTRACT: 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. KeywordsAcetylcholine–Analgesia–Gene targeting–Knockout mice–Morphine–Muscarinic agonists–Muscarinic receptors–Oxotremorine–Parasympathetic nervous system–Pilocarpine
    12/2003: pages 65-93;
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    ABSTRACT: A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M 1 -M 5 ) 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 used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M 1 -M 5 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 M 1 -M 5 mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.
    Receptors and Channels 07/2003; 9(4):279-290. DOI:10.1080/10606820308262
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    ABSTRACT: To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.
    Life Sciences 04/2003; 72(18-19):2047-54. DOI:10.1016/S0024-3205(03)00082-1 · 2.30 Impact Factor
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    ABSTRACT: A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M(1)-M(5)) 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 used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M(1)-M(5) 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 M(1)-M(5) mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.
    Receptors and Channels 02/2003; 9(4):279-90.
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    ABSTRACT: Centrally active muscarinic agonists display pronounced analgesic effects. Identification of the specific muscarinic acetylcholine receptor (mAChR) subtype(s) mediating this activity is of considerable therapeutic interest. To examine the roles of the M(2) and M(4) receptor subtypes, the two G(i)/G(o)-coupled mAChRs, in mediating agonist-dependent antinociception, we generated a mutant mouse line deficient in both M(2) and M(4) mAChRs [M(2)/M(4) double-knockout (KO) mice]. In wild-type mice, systemic, intrathecal, or intracerebroventricular administration of centrally active muscarinic agonists resulted in robust analgesic effects, indicating that muscarinic analgesia can be mediated by both spinal and supraspinal mechanisms. Strikingly, muscarinic agonist-induced antinociception was totally abolished in M(2)/M(4) double-KO mice, independent of the route of application. The nonselective muscarinic agonist oxotremorine showed reduced analgesic potency in M(2) receptor single-KO mice, but retained full analgesic activity in M(4) receptor single-KO mice. In contrast, two novel muscarinic agonists chemically derived from epibatidine, CMI-936 and CMI-1145, displayed reduced analgesic activity in both M(2) and M(4) receptor single-KO mice, independent of the route of application. Radioligand binding studies indicated that the two CMI compounds, in contrast to oxotremorine, showed >6-fold higher affinity for M(4) than for M(2) receptors, providing a molecular basis for the observed differences in agonist activity profiles. These data provide unambiguous evidence that muscarinic analgesia is exclusively mediated by a combination of M(2) and M(4) mAChRs at both spinal and supraspinal sites. These findings should be of considerable relevance for the development of receptor subtype-selective muscarinic agonists as novel analgesic drugs.
    Molecular Pharmacology 11/2002; 62(5):1084-93. DOI:10.1124/mol.62.5.1084 · 4.12 Impact Factor
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    ABSTRACT: Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1-M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M5 receptor-deficient mice (M5-/- mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M5-/- mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5-/- mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M5 receptors. The finding that M5 receptor activity modulates both morphine reward and withdrawal processes suggests that M5 receptors may represent a novel target for the treatment of opiate addiction.
    Proceedings of the National Academy of Sciences 09/2002; 99(17):11452-7. DOI:10.1073/pnas.162371899 · 9.81 Impact Factor
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    ABSTRACT: Oscillatory network activity at gamma frequencies is assumed to be of major importance in cortical information processing. Whereas the synaptic mechanisms of gamma oscillations have been studied in detail, the ionic currents involved at the cellular level remain to be elucidated. Here we show that in vitro gamma oscillations induced by muscarine require activation of M1 receptors on hippocampal CA3 pyramidal neurons and are absent in M1 receptor-deficient mice. M1 receptor activation depolarizes pyramidal neurons by increasing the mixed Na(+)/K(+) current I(h) and the Ca(2+)-dependent nonspecific cation current I(cat), but not by modulation of I(M). Our data provide important insight into the molecular basis of gamma oscillations by unequivocally establishing a novel role for muscarinic modulation of I(h) and I(cat) in rhythmic network activity.
    Neuron 03/2002; 33(4):615-24. DOI:10.1016/S0896-6273(02)00587-1 · 15.98 Impact Factor
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    ABSTRACT: This chapter discusses the phenotypical analysis and clinical implications of muscarinic acetylcholine (ACh) receptor knockout mice. Molecular cloning studies have revealed the existence of five molecularly distinct muscarinic acetylcholine (ACh) receptor (mAChR) subtypes (M1–M5). At a molecular level, the M1, M3, and M5 receptors preferentially couple to G proteins of the Gq/G11 family, whereas the M2 and M4 receptors are primarily linked to G proteins of the Gi/Go class. Studies with mAChR agonists and antagonists have shown that mAChRs are involved in the control of numerous fundamental physiological processes. Central mAChRs are known to regulate a large number of vegetative, sensory, and motor functions. Moreover, central muscarinic mechanisms play important roles in arousal, attention, rapid eye movement (REM) sleep, emotional responses, stress modulation, and higher cognitive processes such as memory and learning. The chapter reviews the major phenotypes displayed by mutant mice lacking M2, M3, or M4 mAChRs. The M2 and M4 mAChR genes were inactivated via homologous recombination in mouse embryonic stem (ES) cells. Homozygous M2-/- (M2R-/-) and M4-/- receptor (M4R-/-) mutant mice were obtained with the expected Mendelian frequency, indicating that there was no increase in embryonic or postnatal mortality. Moreover, wild-type (WT) and M2 and M4 receptor mutant mice did not differ in overall health, were fertile, and bred normally.
    Pharmacochemistry Library 01/2002; 32:97-113. DOI:10.1016/S0165-7208(02)80012-5
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    ABSTRACT: 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 Alzheimer's disease and focal cerebral ischemia, cerebrovascular M5 receptors may represent an attractive therapeutic target.
    Proceedings of the National Academy of Sciences 11/2001; 981(24):14096-14101. DOI:10.1073/pnas.251542998 · 9.81 Impact Factor
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    T Miyakawa · M Yamada · A Duttaroy · J Wess
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    ABSTRACT: Members of the muscarinic acetylcholine receptor family are thought to play key roles in the regulation of a large number of important functions of the CNS. However, the precise roles of the individual muscarinic receptor subtypes in modulating these processes are not well understood at present, primarily because of the lack of ligands with sufficient receptor subtype selectivity. To investigate the behavioral significance of the M(1) muscarinic receptor (M(1)R), which is abundantly expressed in the forebrain, we subjected M(1) receptor-deficient mice (M(1)R(-/-) mice) to a battery of behavioral tests. M(1)R(-/-) mice showed no significant impairments in neurological reflexes, motor coordination, pain sensitivity, and prepulse inhibition. Strikingly, however, M(1)R(-/-) mice consistently exhibited a pronounced increase in locomotor activity in various tests, including open field, elevated plus maze, and light/dark transition tests. Moreover, M(1)R(-/-) mice showed reduced immobilization in the Porsolt forced swim test and reduced levels of freezing after inescapable footshocks, suggesting that M(1)R(-/-) mice are hyperactive under stressful conditions as well. An increased number of social contacts was observed in a social interaction test. Surprisingly, M(1)R(-/-) mice displayed no significant cognitive impairments in the Morris water maze and in contextual fear conditioning. M(1)R(-/-) mice showed slight performance deficits in auditory-cued fear conditioning and in an eight-arm radial maze, most likely because of the hyperactivity phenotype displayed by the M(1)R(-/-) mice. Our results indicate that M(1) muscarinic receptors play an important role in the regulation of locomotor activity but appear to be less critical for cognitive processes, as generally assumed.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 08/2001; 21(14):5239-50. · 6.75 Impact Factor
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    ABSTRACT: Muscarinic acetylcholine receptors (M1–M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.
    Life Sciences 05/2001; 68(22-23):2457-2466. DOI:10.1016/S0024-3205(01)01039-6 · 2.30 Impact Factor
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    ABSTRACT: Members of the muscarinic acetylcholine receptor family (M1-M5) have central roles in the regulation of many fundamental physiological functions. Identifying the specific receptor subtype(s) that mediate the diverse muscarinic actions of acetylcholine is of considerable therapeutic interest, but has proved difficult primarily because of a lack of subtype-selective ligands. Here we show that mice deficient in the M3 muscarinic receptor (M3R -/- mice) display a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low levels of serum leptin and insulin. Paradoxically, hypothalamic messenger RNA levels of melanin-concentrating hormone (MCH), which are normally upregulated in fasted animals leading to an increase in food intake, are significantly reduced in M3R -/- mice. Intra-cerebroventricular injection studies show that an agouti-related peptide analogue lacked orexigenic (appetite-stimulating) activity in M3R -/- mice. However, M3R -/- mice remained responsive to the orexigenic effects of MCH. Our data indicate that there may be a cholinergic pathway that involves M3-receptor-mediated facilitation of food intake at a site downstream of the hypothalamic leptin/melanocortin system and upstream of the MCH system.
    Nature 03/2001; 410(6825):207-212. DOI:10.1038/35065604 · 42.35 Impact Factor