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Beta-cell M3 muscarinic acetylcholine receptors as potential targets for novel antidiabetic drugs
International Immunopharmacology 81 (2020) 106267
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Beta-cell M3 muscarinic acetylcholine receptors as potential targets for novel antidiabetic drugs Lu Zhua,1, Mario Rossia, Nicolai M. Dolibab, Jürgen Wessa, a b
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Molecular Signaling Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
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Keywords: Acetylcholine Muscarinic receptor G protein-coupled receptor Allosteric modulator Beta-cell Insulin release Diabetes
A key feature of type 2 diabetes (T2D) is that beta-cells of the pancreatic islets fail to release sufficient amounts of insulin to overcome peripheral insulin resistance. Glucose-stimulated insulin secretion (GSIS) is regulated by the activity of numerous neurotransmitters, hormones and paracrine factors that act by stimulating specific G protein-coupled receptors expressed by pancreatic beta-cells. Studies with both mouse and human islets suggest that acetylcholine (ACh) acts on beta-cell M3 muscarinic receptors (M3Rs) to promote GSIS. In mouse islets, beta-cell M3Rs are thought to be activated by ACh released from parasympathetic nerve endings. Interestingly, studies with human pancreatic islets suggest that ACh is synthesized, stored and released by alpha-cells, which, in human pancreatic islets, are intermingled with beta-cells. Independent of the source of pancreatic islet ACh, recent studies indicate that beta-cell M3Rs represent a potential target for drugs capable of promoting insulin release for therapeutic purposes. In this review, we will provide an overview about signaling pathways and molecules that regulate the activity of beta-cell M3Rs. We will also discuss a novel pharmacological strategy to stimulate the activity of these receptors to reduce the metabolic impairments associated with T2D.
1. Introduction Type 2 diabetes (T2D) has emerged as a major threat to human health throughout the world, primary driven by the ongoing obesity epidemic [1–3]. At the cellular level, T2D is characterized by two major deficits. First, key metabolic tissues, including liver, fat, and skeletal muscle do not properly respond to insulin, a phenomenon referred to as peripheral insulin resistance [4,5]. Second, beta-cell function is impaired due to several factors including chronically increased blood glucose and lipid levels [4,5]. As a result, beta-cells cannot produce and release sufficient insulin to overcome peripheral insulin resistance. These factors lead to chronically elevated blood glucose levels which is the hallmark of T2D. Like other cell types, beta-cells express a large number of G proteincoupled receptors (GPCRs) on their cell surface [6–8]. Previous work has shown that activation of Gq- and Gs-coupled GPCRs promotes glucose-stimulated insulin secretion (GSIS) [6,9]. Several studies demonstrated that both mouse and human beta-cells express the M3 muscarinic acetylcholine (ACh) receptor subtype (M3R), which, following ACh binding, triggers the activation of G proteins of the Gq family
which in turn activate intracellular signaling pathways leading to enhanced GSIS [10–12] (Fig. 1). Interestingly, several years ago, Caicedo and colleagues reported that human pancreatic alpha-cells are able to synthesize, store and secrete ACh [13]. These authors demonstrated that ACh released by human alpha-cells can act as a paracrine signal on adjacent beta-cells to promote insulin secretion [13]. In contrast to human alpha-cells, mouse alpha-cells do not seem to contain ACh [13]. In this case, beta-cell M3Rs are predicted to be activated by ACh released from parasympathetic nerve endings [14]. However, in both species, ACh-induced activation of beta-cell M3Rs represents a strong stimulus to promote insulin secretion. To assess the physiological relevance of beta-cell M3Rs, we used Cre/loxP technology to create mutant mice lacking M3Rs only in pancreatic beta-cells. In parallel, we also generated transgenic mice that selectively overexpressed M3Rs in pancreatic beta-cells. We found that the beta-cell-specific M3R KO mice displayed impaired glucose tolerance and significantly reduced insulin release, the two key features of T2D [11]. On the other hand, transgenic mice overexpressing M3Rs in pancreatic beta-cells showed greatly improved glucose tolerance and
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Corresponding author at: Laboratory of Bioorganic Chemistry, Molecular Signaling Section, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 8A, Room B1A-05, 8 Center Drive, Bethesda, MD 20892, USA. E-mail address: [email protected] (J. Wess). 1 Current affiliation: Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China. https://doi.org/10.1016/j.intimp.2020.106267 Received 29 November 2019; Received in revised form 25 January 2020; Accepted 27 January 2020 1567-5769/ Published by Elsevier B.V.
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can modulate signaling through beta-cell M3Rs (Fig. 1A). This observation was consistent with previous reports indicating that SPL functions as an adaptor molecule to recruit members of the RGS protein family to ligand-activated GPCR/G protein signaling complexes [21]. For example, we found that M3R-mediated increases in insulin secretion were significantly augmented in pancreatic islets prepared from SPL−/− mice, as compared with islets obtained from control WT [19]. In agreement with these in vitro findings, bethanechol treatment of SPL−/− mice led to increased insulin secretion and more robust blood glucose-lowering effects, as compared with WT littermates [19]. Additional in vitro experiments indicated that SPL acts as an adaptor protein that is able to recruit RGS4 to the ligand-activated M3R/G protein complex, thus shortening the lifetime of M3R-activated Gαq/11 subunits [19]. Since beta-cell M3Rs play a key role in the maintenance of proper glucose homeostasis [11], approaches aimed at inhibiting beta-cell RGS4 or SPL may prove beneficial to enhance beta-cell M3R function for therapeutic purposes. Like most other GPCRs, activated M3Rs are phosphorylated by various kinases including GRKs and CK2 [22–26]. In most cases, the physiological relevance of these various phosphorylation events remains unclear. We recently demonstrated that CK2 phosphorylation of beta-cell M3Rs plays an important role in regulating the activity of these receptors [27]. In vitro studies demonstrated that decreased CK2mediated phosphorylation of beta-cell M3Rs led to an increase in M3Rstimulated insulin release. This phenomenon was observed with both mouse and human islets [27]. Treatment of obese mice with a pharmacological CK2 inhibitor (CX4945) reduced the metabolic deficits associated with obesity, including hyperglycemia and glucose intolerance. This effect was not observed with M3R-deficient mice, indicating that the beneficial metabolic effects of CX4945 required the presence of M3Rs [27]. These data clearly indicated that CK2 exerts an inhibitory effect on the function of beta-cell M3Rs and that pharmacological inhibition of beta-cell CK2 may prove useful to enhance M3R-mediated insulin release for therapeutic purposes (Fig. 1B).
Fig. 1. Pathways and molecules that regulate M3R signaling in pancreatic betacells. (A) Recent work suggest that RGS4 [18] and spinophilin (SPL) [19] act as potent negative regulators of M3R signaling in pancreatic β-cells. Most likely, SPL functions as an adaptor protein that is able to recruit RGS4 to the ligandactivated M3R/Gq protein complex, thus limiting the lifetime of M3R-activated G protein αq/11 subunits. (B) CK2-mediated phosphorylation of beta-cell M3Rs reduces the efficiency of M3R/Gq coupling, resulting in impaired insulin release [27]. The figure was modified based on a scheme depicted in one of our previous publications [45]. ACh, acetylcholine; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; ER, endoplasmic reticulum; K-ATP, ATP-sensitive K+ channel; VDCC, voltage-dependent Ca2+ channel.
3. Metabolic studies with a positive allosteric modulator of M3R function
increased insulin release [11]. In addition, these mice were protected against the metabolic deficits associated with the consumption of an obesogenic diet [11]. These results prompted us to speculate that agents able to enhance signaling through beta-cell M3Rs might become therapeutically useful as new antidiabetic drugs. Interestingly, in a recent study, Ito et al. [15] chronically administered bethanechol, a muscarinic agonist that acts on all five MR subtypes (M1R-M5R), to diabetic mice. The authors found that this treatment led to beneficial effects on glucose homeostasis and pancreatic beta-cell maintenance. However, bethanechol treatment was also associated with significant side effects, including increased salivation and lacrimation, in a large percentage of mice [15].
3.1. Background During the past decade, many academic and industrial labs have focused on developing subtype-specific muscarinic allosteric modulators for the treatment of various human diseases, including severe brain disorders such as Alzheimer’s disease and schizophrenia [28–31]. In Fig. 2, the green square represents the binding site for ACh, the socalled orthosteric binding site. The amino acid side chains that contribute to the ACh binding site are highly conserved among all five MR subtypes [30,32]. Interestingly, all MRs feature another ligand binding site, shown in purple in Fig. 2, which is located on the extracellular surface of the receptor protein. This so-called allosteric site is less well conserved than the orthosteric site, making it possible to develop subtype-selective allosteric modulators of MR function [33,34]. Muscarinic PAMs are positive allosteric modulators that enhance the affinity and/ or efficacy of ACh at MRs, while muscarinic NAMs are negative allosteric modulators that impair MR function [28–31] (Fig. 2). The potential clinical use of allosteric modulators of GPCR function is predicted to provide several benefits [28–31]. First, it is easier to develop allosteric rather than orthosteric ligands that are endowed with high receptor subtype-selectivity. Second, allosteric agents respect the spatio-temporal control of receptor activation. They exert their actions only during the release of the endogenous orthosteric agonist. Finally, based on their mechanism of action, allosteric modulators show a socalled ‘ceiling effect’, resulting in fewer side effects [28–31].
2. Identification of negative regulators of beta-cell M3R function During the past decade, we identified several factors (proteins) that regulate M3R function in pancreatic beta-cells. For example, we found that RGS4 acts as a potent negative regulator of M3R function in this cell type (Fig. 1A). RGS4 is a member of the B/R4 subfamily of RGS proteins, acting as a GTPase-activating protein (GAP) for Gq- and Gitype G protein α-subunits [16,17]. Interestingly, in vivo studies revealed that bethanechol treatment of mice selectively lacking RGS4 in pancreatic beta-cells showed enhanced insulin release and improved glucose homeostasis, as compared to control littermates [18]. Importantly, experiments with beta-cell-specific M3R KO mice demonstrated that these beneficial metabolic effects of bethanechol were dependent on the presence of beta-cell M3Rs [18]. In another study [19], we showed that spinophilin (SPL), a scaffolding protein known to regulate various functions of the CNS [20], 2
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Fig. 2. Agents that can modulate MR function by binding to an allosteric binding site. ACh and other orthosteric muscarinic agonists bind to a highly conserved receptor site located within the transmembrane core of the M1-M5 receptors. Allosteric modulators of MR function usually bind to a less well conversed receptor region located ‘above’ the orthosteric binding site. This scheme was modified based on a recently published figure [46]. PAM, positive allosteric modulator; NAM, negative allosteric modulator.
human islets, we perifused human islets with increasing concentrations of glucose, either in the absence or presence of VU0119498 (5 μM) [37]. When the glucose concentration in the medium was high (8 mM), we exposed islets to increasing concentrations of ACh. This treatment caused a significant increase in insulin secretion (Fig. 3B). Strikingly, this ACh-stimulated insulin release was greatly augmented in the presence of the VU0119498 (Fig. 3B), suggesting that this PAM also promotes signaling through M3Rs expressed by human beta-cells.
3.2. Identification of a PAM that enhances signaling via M3Rs and other Gq-coupled MRs Orthosteric agonists that selectively target the M3R subtype are not available at present. However, several years ago, Jeff Conn, Craig Lindsley, and their colleagues identified a compound, referred to as VU0119498, that acts as a PAM at M3Rs [35,36]. Moreover, this agent showed PAM activity at M1Rs and M5Rs, which, like the M3R, are also linked to Gq-type G proteins [35,36]. Since the latter two receptor subtypes are found mostly in the brain, we recently explored the possibility that VU0119498 has potential to act as an antidiabetic drug by promoting ACh-induced insulin secretion.
3.4. In vivo metabolic studies with VU0119498 We next carried out a set of experiments to explore whether VU0119498 was also able to stimulate insulin secretion in vivo [37]. For these studies, we initially used WT C57BL/6 mice. In agreement with the in vitro insulin release studies, we found that the PAM-treated WT mice showed a significant increase in GSIS. This effect was associated with a striking improvement in glucose tolerance, as compared with vehicle-injected mice. To confirm that these beneficial metabolic effects of VU0119498 were caused by binding of the PAM to beta-cell M3Rs, we carried out analogous studies with mice that lacked M3Rs selectively in their pancreatic beta-cells [37]. As expected, the PAM had no significant effect on GSIS and glucose tolerance in this mutant mouse strain, strongly suggesting that VU0119498 acts on beta-cell M3Rs to stimulate GSIS, which in turn promotes a more rapid clearance of glucose from the blood.
3.3. In vitro insulin secretion studies with VU0119498 Initially, we performed a series of in vitro insulin release studies using pancreatic islets prepared from wild-type (WT) and M3R KO mice [37]. These studies where carried out in the presence of a stimulatory concentration of glucose (16 mM). As expected, treatment of WT islets with ACh stimulated insulin secretion in a concentration-dependent fashion (Fig. 3A). This response was completely absent in islets prepared from mice lacking M3Rs (Fig. 3A), indicating that this ACh activity was mediated by M3Rs. Strikingly, ACh-induced insulin secretion was significantly enhanced in the presence of VU0119498 (20 μM) (Fig. 3A), suggesting that this agent acts as a PAM on beta-cell M3Rs. To explore whether VU0119498 displayed similar properties in
Fig. 3. Treatment of isolated mouse and human islets with VU0119498 (PAM) promotes ACh-stimulated insulin release in an M3R-dependent fashion. (A) Studies with isolated islets from WT mice and whole-body M3R KO mice. ACh-induced increases in insulin release were studied in the absence or presence of 20 μM PAM. The amount of insulin secreted into the medium during the 1 hr incubation period was normalized to the total insulin content of each well (islets plus medium). Data are given as means ± SEM of at least three independent experiments. **P < 0.01 (two-way ANOVA followed by Tukey's post hoc test). (B) Studies with perifused human islets. Perifused human islets were incubated in glucose solution (G4 = 4 mM; G8 = 8 mM) in the absence or presence of 5 μM PAM. During the 8G perifusion period, islets were exposed to increasing concentrations of ACh (0–1 μM). Each curve represents the mean ± SEM of three independent perfusion experiments (180 human islets per group and perifusion). **P < 0.001 (two-tailed Student’s t-test). Data taken from Zhu et al. [37]. 3
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detailed studies are needed to distinguish between these different possibilities. 3.6. Potential functional role of M5 receptors (M5Rs) in human beta-cells In a previous study, we did not detect M5R RNA in mouse pancreatic islets [10]. However, recent data suggest that human beta-cells may not only express M3Rs but also M5Rs [43], suggesting that both of these receptors represent potential therapeutic targets. Interestingly, a recent RNAseq study indicates that M3R transcript levels are unchanged in human beta-cells in T2D [44]. On the other hand, M5R RNA levels appear to be increased in beta-cells of T2D patients [44]. Fig. 4. Treatment of WT mice with VU0119498 (PAM) has little or no effect on pupil diameter. WT mice were injected i.p. with either vehicle or increasing doses of VU0119498 (0.1, 0.5, and 2 mg/kg). For control purposes, carbachol, an orthosteric muscarinic agonist, was applied topically to one eye (1–2 μl of a 100 μM solution). Pupil constriction tests were performed using video analysis software. Data are given as means ± SEM (n = 9 or 10 male mice per group). **P < 0.01 vs. vehicle (two-way ANOVA followed by Tukey's post hoc test). Data taken from Zhu et al. [37].
4. Conclusion The studies summarized above strongly suggest that selective M3R PAMs may prove beneficial to enhance insulin release for therapeutic purposes. Since many GPCRs feature allosteric binding sites, our findings are likely to have a more general impact on the development of GPCR PAMs as novel antidiabetic agents.
To generate a mouse model of T2D, we maintained male WT mice (strain: C57BL/6 mice) on a high-fat diet for at least 8 weeks [38]. Chronic consumption of this diet led to severe obesity, associated with hyperglycemia and glucose intolerance, two of the key features of T2D [37]. Strikingly, VU0119498 treatment of the obese WT mice caused significant improvements in glucose tolerance and GSIS, indicative of the therapeutic potential of M3R PAMs [37].
Declaration of Competing Interest None of the authors has any conflicts of interest regarding this submission. Acknowledgements This research was funded by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH. We thank all of our colleagues and collaborators for their invaluable contributions to the studies summarized in this review.
3.5. Side effect profile of VU0119498 M3Rs are not only expressed by pancreatic beta-cells but are also present in other peripheral tissues such as smooth muscle and exocrine glands [39,40]. For this reason, we also examined whether VU0119498 caused side effects by enhancing M3R signaling in these tissues [37]. For example, it is well known that M3Rs play a key role in mediating ACh-induced salivary secretion [41]. We found that treatment of WT mice with increasing doses of VU0119498 had absolutely no effect on salivary secretion [37]. On the other hand, administration of pilocarpine (1 mg/kg i.p.), an orthosteric muscarinic agonist, triggered copious salivation in all experimental animals [37]. M3Rs expressed by smooth muscle tissues are known to stimulate smooth muscle contraction [39,40]. For this reason, we also examined whether treatment of WT mice with VU0119498 affected smooth muscle contractility [37]. Since activation of smooth muscle M3Rs of the eye causes the pupil to constrict [42], we carried out a series of pupil constriction tests [37]. Specifically, we injected WT mice i.p. with either vehicle or increasing doses of VU0119498 and then monitored pupil size over a 60 min time period using video analysis software. In general, PAM treatment had little or no effect on pupil diameter (Fig. 4). Only the highest PAM dose used (2 mg/kg i.p.) caused a significant reduction in pupil size at the 45-time point. Importantly, lower doses of VU0119498 (e.g. 0.5 mg/kg i.p.) that caused pronounced beneficial metabolic effects had no significant effect on pupil diameter (Fig. 4). On the other hand, carbachol, an orthosteric muscarinic agonist, caused maximum pupillary constriction in all animals used (Fig. 4). At this point, we can only speculate why VU0119498 had little or no effect on salivary secretion and pupil diameter at doses that strongly simulated insulin secretion in vivo. It is possible that actual ACh concentrations differ in the various M3R-expressing tissues, leading to quantitatively different PAM effects. Other possibilities are that M3Rresponse coupling is particularly efficacious in beta-cells or that VU0119498 accumulates at higher concentrations in pancreatic islets as compared to other tissues. Different M3R densities in different tissues may also affect tissue responsiveness to PAM administration. More
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Beta-cell M3 muscarinic acetylcholine receptors as potential targets for novel antidiabetic drugs Lu Zhua,1, Mario Rossia, Nicolai M. Dolibab, Jürgen Wessa, a b
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Molecular Signaling Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Acetylcholine Muscarinic receptor G protein-coupled receptor Allosteric modulator Beta-cell Insulin release Diabetes
A key feature of type 2 diabetes (T2D) is that beta-cells of the pancreatic islets fail to release sufficient amounts of insulin to overcome peripheral insulin resistance. Glucose-stimulated insulin secretion (GSIS) is regulated by the activity of numerous neurotransmitters, hormones and paracrine factors that act by stimulating specific G protein-coupled receptors expressed by pancreatic beta-cells. Studies with both mouse and human islets suggest that acetylcholine (ACh) acts on beta-cell M3 muscarinic receptors (M3Rs) to promote GSIS. In mouse islets, beta-cell M3Rs are thought to be activated by ACh released from parasympathetic nerve endings. Interestingly, studies with human pancreatic islets suggest that ACh is synthesized, stored and released by alpha-cells, which, in human pancreatic islets, are intermingled with beta-cells. Independent of the source of pancreatic islet ACh, recent studies indicate that beta-cell M3Rs represent a potential target for drugs capable of promoting insulin release for therapeutic purposes. In this review, we will provide an overview about signaling pathways and molecules that regulate the activity of beta-cell M3Rs. We will also discuss a novel pharmacological strategy to stimulate the activity of these receptors to reduce the metabolic impairments associated with T2D.
1. Introduction Type 2 diabetes (T2D) has emerged as a major threat to human health throughout the world, primary driven by the ongoing obesity epidemic [1–3]. At the cellular level, T2D is characterized by two major deficits. First, key metabolic tissues, including liver, fat, and skeletal muscle do not properly respond to insulin, a phenomenon referred to as peripheral insulin resistance [4,5]. Second, beta-cell function is impaired due to several factors including chronically increased blood glucose and lipid levels [4,5]. As a result, beta-cells cannot produce and release sufficient insulin to overcome peripheral insulin resistance. These factors lead to chronically elevated blood glucose levels which is the hallmark of T2D. Like other cell types, beta-cells express a large number of G proteincoupled receptors (GPCRs) on their cell surface [6–8]. Previous work has shown that activation of Gq- and Gs-coupled GPCRs promotes glucose-stimulated insulin secretion (GSIS) [6,9]. Several studies demonstrated that both mouse and human beta-cells express the M3 muscarinic acetylcholine (ACh) receptor subtype (M3R), which, following ACh binding, triggers the activation of G proteins of the Gq family
which in turn activate intracellular signaling pathways leading to enhanced GSIS [10–12] (Fig. 1). Interestingly, several years ago, Caicedo and colleagues reported that human pancreatic alpha-cells are able to synthesize, store and secrete ACh [13]. These authors demonstrated that ACh released by human alpha-cells can act as a paracrine signal on adjacent beta-cells to promote insulin secretion [13]. In contrast to human alpha-cells, mouse alpha-cells do not seem to contain ACh [13]. In this case, beta-cell M3Rs are predicted to be activated by ACh released from parasympathetic nerve endings [14]. However, in both species, ACh-induced activation of beta-cell M3Rs represents a strong stimulus to promote insulin secretion. To assess the physiological relevance of beta-cell M3Rs, we used Cre/loxP technology to create mutant mice lacking M3Rs only in pancreatic beta-cells. In parallel, we also generated transgenic mice that selectively overexpressed M3Rs in pancreatic beta-cells. We found that the beta-cell-specific M3R KO mice displayed impaired glucose tolerance and significantly reduced insulin release, the two key features of T2D [11]. On the other hand, transgenic mice overexpressing M3Rs in pancreatic beta-cells showed greatly improved glucose tolerance and
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Corresponding author at: Laboratory of Bioorganic Chemistry, Molecular Signaling Section, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 8A, Room B1A-05, 8 Center Drive, Bethesda, MD 20892, USA. E-mail address: [email protected] (J. Wess). 1 Current affiliation: Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China. https://doi.org/10.1016/j.intimp.2020.106267 Received 29 November 2019; Received in revised form 25 January 2020; Accepted 27 January 2020 1567-5769/ Published by Elsevier B.V.
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can modulate signaling through beta-cell M3Rs (Fig. 1A). This observation was consistent with previous reports indicating that SPL functions as an adaptor molecule to recruit members of the RGS protein family to ligand-activated GPCR/G protein signaling complexes [21]. For example, we found that M3R-mediated increases in insulin secretion were significantly augmented in pancreatic islets prepared from SPL−/− mice, as compared with islets obtained from control WT [19]. In agreement with these in vitro findings, bethanechol treatment of SPL−/− mice led to increased insulin secretion and more robust blood glucose-lowering effects, as compared with WT littermates [19]. Additional in vitro experiments indicated that SPL acts as an adaptor protein that is able to recruit RGS4 to the ligand-activated M3R/G protein complex, thus shortening the lifetime of M3R-activated Gαq/11 subunits [19]. Since beta-cell M3Rs play a key role in the maintenance of proper glucose homeostasis [11], approaches aimed at inhibiting beta-cell RGS4 or SPL may prove beneficial to enhance beta-cell M3R function for therapeutic purposes. Like most other GPCRs, activated M3Rs are phosphorylated by various kinases including GRKs and CK2 [22–26]. In most cases, the physiological relevance of these various phosphorylation events remains unclear. We recently demonstrated that CK2 phosphorylation of beta-cell M3Rs plays an important role in regulating the activity of these receptors [27]. In vitro studies demonstrated that decreased CK2mediated phosphorylation of beta-cell M3Rs led to an increase in M3Rstimulated insulin release. This phenomenon was observed with both mouse and human islets [27]. Treatment of obese mice with a pharmacological CK2 inhibitor (CX4945) reduced the metabolic deficits associated with obesity, including hyperglycemia and glucose intolerance. This effect was not observed with M3R-deficient mice, indicating that the beneficial metabolic effects of CX4945 required the presence of M3Rs [27]. These data clearly indicated that CK2 exerts an inhibitory effect on the function of beta-cell M3Rs and that pharmacological inhibition of beta-cell CK2 may prove useful to enhance M3R-mediated insulin release for therapeutic purposes (Fig. 1B).
Fig. 1. Pathways and molecules that regulate M3R signaling in pancreatic betacells. (A) Recent work suggest that RGS4 [18] and spinophilin (SPL) [19] act as potent negative regulators of M3R signaling in pancreatic β-cells. Most likely, SPL functions as an adaptor protein that is able to recruit RGS4 to the ligandactivated M3R/Gq protein complex, thus limiting the lifetime of M3R-activated G protein αq/11 subunits. (B) CK2-mediated phosphorylation of beta-cell M3Rs reduces the efficiency of M3R/Gq coupling, resulting in impaired insulin release [27]. The figure was modified based on a scheme depicted in one of our previous publications [45]. ACh, acetylcholine; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; ER, endoplasmic reticulum; K-ATP, ATP-sensitive K+ channel; VDCC, voltage-dependent Ca2+ channel.
3. Metabolic studies with a positive allosteric modulator of M3R function
increased insulin release [11]. In addition, these mice were protected against the metabolic deficits associated with the consumption of an obesogenic diet [11]. These results prompted us to speculate that agents able to enhance signaling through beta-cell M3Rs might become therapeutically useful as new antidiabetic drugs. Interestingly, in a recent study, Ito et al. [15] chronically administered bethanechol, a muscarinic agonist that acts on all five MR subtypes (M1R-M5R), to diabetic mice. The authors found that this treatment led to beneficial effects on glucose homeostasis and pancreatic beta-cell maintenance. However, bethanechol treatment was also associated with significant side effects, including increased salivation and lacrimation, in a large percentage of mice [15].
3.1. Background During the past decade, many academic and industrial labs have focused on developing subtype-specific muscarinic allosteric modulators for the treatment of various human diseases, including severe brain disorders such as Alzheimer’s disease and schizophrenia [28–31]. In Fig. 2, the green square represents the binding site for ACh, the socalled orthosteric binding site. The amino acid side chains that contribute to the ACh binding site are highly conserved among all five MR subtypes [30,32]. Interestingly, all MRs feature another ligand binding site, shown in purple in Fig. 2, which is located on the extracellular surface of the receptor protein. This so-called allosteric site is less well conserved than the orthosteric site, making it possible to develop subtype-selective allosteric modulators of MR function [33,34]. Muscarinic PAMs are positive allosteric modulators that enhance the affinity and/ or efficacy of ACh at MRs, while muscarinic NAMs are negative allosteric modulators that impair MR function [28–31] (Fig. 2). The potential clinical use of allosteric modulators of GPCR function is predicted to provide several benefits [28–31]. First, it is easier to develop allosteric rather than orthosteric ligands that are endowed with high receptor subtype-selectivity. Second, allosteric agents respect the spatio-temporal control of receptor activation. They exert their actions only during the release of the endogenous orthosteric agonist. Finally, based on their mechanism of action, allosteric modulators show a socalled ‘ceiling effect’, resulting in fewer side effects [28–31].
2. Identification of negative regulators of beta-cell M3R function During the past decade, we identified several factors (proteins) that regulate M3R function in pancreatic beta-cells. For example, we found that RGS4 acts as a potent negative regulator of M3R function in this cell type (Fig. 1A). RGS4 is a member of the B/R4 subfamily of RGS proteins, acting as a GTPase-activating protein (GAP) for Gq- and Gitype G protein α-subunits [16,17]. Interestingly, in vivo studies revealed that bethanechol treatment of mice selectively lacking RGS4 in pancreatic beta-cells showed enhanced insulin release and improved glucose homeostasis, as compared to control littermates [18]. Importantly, experiments with beta-cell-specific M3R KO mice demonstrated that these beneficial metabolic effects of bethanechol were dependent on the presence of beta-cell M3Rs [18]. In another study [19], we showed that spinophilin (SPL), a scaffolding protein known to regulate various functions of the CNS [20], 2
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Fig. 2. Agents that can modulate MR function by binding to an allosteric binding site. ACh and other orthosteric muscarinic agonists bind to a highly conserved receptor site located within the transmembrane core of the M1-M5 receptors. Allosteric modulators of MR function usually bind to a less well conversed receptor region located ‘above’ the orthosteric binding site. This scheme was modified based on a recently published figure [46]. PAM, positive allosteric modulator; NAM, negative allosteric modulator.
human islets, we perifused human islets with increasing concentrations of glucose, either in the absence or presence of VU0119498 (5 μM) [37]. When the glucose concentration in the medium was high (8 mM), we exposed islets to increasing concentrations of ACh. This treatment caused a significant increase in insulin secretion (Fig. 3B). Strikingly, this ACh-stimulated insulin release was greatly augmented in the presence of the VU0119498 (Fig. 3B), suggesting that this PAM also promotes signaling through M3Rs expressed by human beta-cells.
3.2. Identification of a PAM that enhances signaling via M3Rs and other Gq-coupled MRs Orthosteric agonists that selectively target the M3R subtype are not available at present. However, several years ago, Jeff Conn, Craig Lindsley, and their colleagues identified a compound, referred to as VU0119498, that acts as a PAM at M3Rs [35,36]. Moreover, this agent showed PAM activity at M1Rs and M5Rs, which, like the M3R, are also linked to Gq-type G proteins [35,36]. Since the latter two receptor subtypes are found mostly in the brain, we recently explored the possibility that VU0119498 has potential to act as an antidiabetic drug by promoting ACh-induced insulin secretion.
3.4. In vivo metabolic studies with VU0119498 We next carried out a set of experiments to explore whether VU0119498 was also able to stimulate insulin secretion in vivo [37]. For these studies, we initially used WT C57BL/6 mice. In agreement with the in vitro insulin release studies, we found that the PAM-treated WT mice showed a significant increase in GSIS. This effect was associated with a striking improvement in glucose tolerance, as compared with vehicle-injected mice. To confirm that these beneficial metabolic effects of VU0119498 were caused by binding of the PAM to beta-cell M3Rs, we carried out analogous studies with mice that lacked M3Rs selectively in their pancreatic beta-cells [37]. As expected, the PAM had no significant effect on GSIS and glucose tolerance in this mutant mouse strain, strongly suggesting that VU0119498 acts on beta-cell M3Rs to stimulate GSIS, which in turn promotes a more rapid clearance of glucose from the blood.
3.3. In vitro insulin secretion studies with VU0119498 Initially, we performed a series of in vitro insulin release studies using pancreatic islets prepared from wild-type (WT) and M3R KO mice [37]. These studies where carried out in the presence of a stimulatory concentration of glucose (16 mM). As expected, treatment of WT islets with ACh stimulated insulin secretion in a concentration-dependent fashion (Fig. 3A). This response was completely absent in islets prepared from mice lacking M3Rs (Fig. 3A), indicating that this ACh activity was mediated by M3Rs. Strikingly, ACh-induced insulin secretion was significantly enhanced in the presence of VU0119498 (20 μM) (Fig. 3A), suggesting that this agent acts as a PAM on beta-cell M3Rs. To explore whether VU0119498 displayed similar properties in
Fig. 3. Treatment of isolated mouse and human islets with VU0119498 (PAM) promotes ACh-stimulated insulin release in an M3R-dependent fashion. (A) Studies with isolated islets from WT mice and whole-body M3R KO mice. ACh-induced increases in insulin release were studied in the absence or presence of 20 μM PAM. The amount of insulin secreted into the medium during the 1 hr incubation period was normalized to the total insulin content of each well (islets plus medium). Data are given as means ± SEM of at least three independent experiments. **P < 0.01 (two-way ANOVA followed by Tukey's post hoc test). (B) Studies with perifused human islets. Perifused human islets were incubated in glucose solution (G4 = 4 mM; G8 = 8 mM) in the absence or presence of 5 μM PAM. During the 8G perifusion period, islets were exposed to increasing concentrations of ACh (0–1 μM). Each curve represents the mean ± SEM of three independent perfusion experiments (180 human islets per group and perifusion). **P < 0.001 (two-tailed Student’s t-test). Data taken from Zhu et al. [37]. 3
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detailed studies are needed to distinguish between these different possibilities. 3.6. Potential functional role of M5 receptors (M5Rs) in human beta-cells In a previous study, we did not detect M5R RNA in mouse pancreatic islets [10]. However, recent data suggest that human beta-cells may not only express M3Rs but also M5Rs [43], suggesting that both of these receptors represent potential therapeutic targets. Interestingly, a recent RNAseq study indicates that M3R transcript levels are unchanged in human beta-cells in T2D [44]. On the other hand, M5R RNA levels appear to be increased in beta-cells of T2D patients [44]. Fig. 4. Treatment of WT mice with VU0119498 (PAM) has little or no effect on pupil diameter. WT mice were injected i.p. with either vehicle or increasing doses of VU0119498 (0.1, 0.5, and 2 mg/kg). For control purposes, carbachol, an orthosteric muscarinic agonist, was applied topically to one eye (1–2 μl of a 100 μM solution). Pupil constriction tests were performed using video analysis software. Data are given as means ± SEM (n = 9 or 10 male mice per group). **P < 0.01 vs. vehicle (two-way ANOVA followed by Tukey's post hoc test). Data taken from Zhu et al. [37].
4. Conclusion The studies summarized above strongly suggest that selective M3R PAMs may prove beneficial to enhance insulin release for therapeutic purposes. Since many GPCRs feature allosteric binding sites, our findings are likely to have a more general impact on the development of GPCR PAMs as novel antidiabetic agents.
To generate a mouse model of T2D, we maintained male WT mice (strain: C57BL/6 mice) on a high-fat diet for at least 8 weeks [38]. Chronic consumption of this diet led to severe obesity, associated with hyperglycemia and glucose intolerance, two of the key features of T2D [37]. Strikingly, VU0119498 treatment of the obese WT mice caused significant improvements in glucose tolerance and GSIS, indicative of the therapeutic potential of M3R PAMs [37].
Declaration of Competing Interest None of the authors has any conflicts of interest regarding this submission. Acknowledgements This research was funded by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH. We thank all of our colleagues and collaborators for their invaluable contributions to the studies summarized in this review.
3.5. Side effect profile of VU0119498 M3Rs are not only expressed by pancreatic beta-cells but are also present in other peripheral tissues such as smooth muscle and exocrine glands [39,40]. For this reason, we also examined whether VU0119498 caused side effects by enhancing M3R signaling in these tissues [37]. For example, it is well known that M3Rs play a key role in mediating ACh-induced salivary secretion [41]. We found that treatment of WT mice with increasing doses of VU0119498 had absolutely no effect on salivary secretion [37]. On the other hand, administration of pilocarpine (1 mg/kg i.p.), an orthosteric muscarinic agonist, triggered copious salivation in all experimental animals [37]. M3Rs expressed by smooth muscle tissues are known to stimulate smooth muscle contraction [39,40]. For this reason, we also examined whether treatment of WT mice with VU0119498 affected smooth muscle contractility [37]. Since activation of smooth muscle M3Rs of the eye causes the pupil to constrict [42], we carried out a series of pupil constriction tests [37]. Specifically, we injected WT mice i.p. with either vehicle or increasing doses of VU0119498 and then monitored pupil size over a 60 min time period using video analysis software. In general, PAM treatment had little or no effect on pupil diameter (Fig. 4). Only the highest PAM dose used (2 mg/kg i.p.) caused a significant reduction in pupil size at the 45-time point. Importantly, lower doses of VU0119498 (e.g. 0.5 mg/kg i.p.) that caused pronounced beneficial metabolic effects had no significant effect on pupil diameter (Fig. 4). On the other hand, carbachol, an orthosteric muscarinic agonist, caused maximum pupillary constriction in all animals used (Fig. 4). At this point, we can only speculate why VU0119498 had little or no effect on salivary secretion and pupil diameter at doses that strongly simulated insulin secretion in vivo. It is possible that actual ACh concentrations differ in the various M3R-expressing tissues, leading to quantitatively different PAM effects. Other possibilities are that M3Rresponse coupling is particularly efficacious in beta-cells or that VU0119498 accumulates at higher concentrations in pancreatic islets as compared to other tissues. Different M3R densities in different tissues may also affect tissue responsiveness to PAM administration. More
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