GPR55: From orphan to metabolic regulator?

JPT-06701; No of Pages 8 Pharmacology & Therapeutics xxx (2014) xxx–xxx

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GPR55: From orphan to metabolic regulator? Bo Liu, Shuang Song, Peter M. Jones, Shanta J. Persaud ⁎ Diabetes Research Group, Division of Diabetes & Nutritional Sciences, King's College London, London SE1 1UL, UK

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Keywords: GPR55 Cannabinoids Lysophosphatidylinositol Energy homeostasis Obesity Diabetes

a b s t r a c t GPR55 belongs to the class A family of G-protein coupled receptor (GPCRs) and its activity is regulated by a range of synthetic and endogenous cannabinoids, and by lipid-derived ligands. Cannabinoids are known to be important in controlling appetite and metabolic balance, and it is now emerging that GPR55 may have a role to play in energy homeostasis through the regulation of food intake, fuel storage in adipocytes, gut motility and insulin secretion. This review summarises our current knowledge of expression and function of GPR55 in tissues involved in metabolic regulation, the signalling cascades through which GPR55 is reported to act to exert its effects, and it comments on the difficulties in reaching firm conclusions when using GPR55 ligands of poor specificity. Understanding the role of GPR55 in energy homeostasis may provide a novel target for therapeutic intervention in obesity and type 2 diabetes mellitus. © 2014 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . 2. GPR55 expression and signalling . . . . . . 3. Action of GPR55 in metabolically active tissues 4. Conclusions and perspectives . . . . . . . Conflict of interest statement . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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1. Introduction 1.1. Dysregulated energy homeostasis in obesity and diabetes Energy homeostasis is a metabolic process through which fuel levels are tightly regulated by the central nervous system and peripheral tissues, providing a balance between energy input from food intake and the energy requirements of cells for metabolism, growth and repair. An imbalance in this system, in which dietary fuel provision exceeds requirements, leads to metabolic disorders such as type 2 diabetes mellitus (T2DM) and obesity, which are now two of the most serious healthcare issues worldwide. Obesity has been classified as a disease by the American Medical Association and its incidence has been

⁎ Corresponding author at: 2.9N Hodgkin Building, King's College London, Guy's Campus, London SE1 1UL, UK. Tel.: +44 20 7848 6275. E-mail address: [email protected] (S.J. Persaud).

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increasing globally (Finucane et al., 2011) as has that of diabetes, with current predictions that there will be 592 million people with diabetes by 2035 (IDF, 2013). T2DM is characterised by chronic hyperglycaemia as a result of insulin resistance of peripheral tissues, combined with insufficient insulin secretion from islet β-cells to compensate for the reduced insulin sensitivity. Obesity is closely linked to the development of T2DM and the hypertrophic adipocytes secrete peptides that have been implicated in the onset of insulin resistance (Kahn et al., 2006; Hajer et al., 2008). Chronically elevated levels of some saturated fatty acids are also known to be involved in metabolic dysregulation, through decreased production of the insulin sensitising fat-derived hormone adiponectin, reduced insulin-stimulated glucose uptake and increased β-cell death (Kennedy et al., 2009; Kusminski et al., 2009; Ziemke & Mantzoros, 2010). In recent years it has become apparent that adipocytes also secrete phospholipid-derived messengers known as endocannabinoids, which show elevated levels in obesity (Bluher et al., 2006) and may contribute to insulin resistance (Li et al., 2011). Classically, the endocanabinoid system (ECS; see Glossary) consists of cannabinoid receptors 1 and 2 (CB1 and CB2) and the endogenous ligands 2-

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Please cite this article as: Liu, B., et al., GPR55: From orphan to metabolic regulator? Pharmacology & Therapeutics (2014), http://dx.doi.org/ 10.1016/j.pharmthera.2014.06.007

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arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA). The enzymes responsible for the synthesis and degradation of the endocannabinoids also form part of the ECS. Thus, 2-AG is synthesised from diacylglycerol by diacylglycerol lipases (DAG-lipases) and degraded to arachidonic acid and glycerol by monoacylglycerol lipase (MGL), while AEA is synthesised from N-arachidonoyl-phosphatidylethanolamine by N-acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) and degraded to arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH; Li et al., 2011). AEA and 2-AG trigger hyperphagia through activation of CB1, a GPCR that is highly expressed in the brain and is a major regulator in the hypothalamic reward system controlling food intake (Williams et al., 1998; Williams & Kirkham, 1999). Thus, the CB1 antagonist rimonabant (SR141716A) was developed as an antiobesity agent, and although it was effective in reducing food intake and body weight it was withdrawn from clinical use after only two years due to its adverse psychological effects. 1.2. CB1- and CB2-independent effects of cannabinoids Although there is a good understanding of the consequences of CB1 and CB2 activation, some ligands that were originally thought to be specific for CB1 or CB2 can also act independently of these receptors, suggesting the existence of a third cannabinoid receptor (Derocq et al., 1998; Jarai et al., 1999; Nieri et al., 2003; Curran et al., 2005; Kaplan et al., 2005). GPR55 was first suggested as a novel cannabinoid receptor when in silico screening of patents from GlaxoSmithKline and AstraZeneca revealed that it interacts with some cannabinoid receptor agonists and antagonists (Brown, 2007). GPR55 is still listed as an orphan on the IUPHAR database, but a range of endogenous and pharmacological cannabinoid ligands have been identified following transient expression of this receptor in HEK293 cells (Ryberg et al., 2007). This review will consider the expression and signalling pathways downstream of GPR55 and the emerging evidence suggesting its importance in metabolism, with a focus on its role in tissues involved in energy homeostasis. 2. GPR55 expression and signalling Since the cloning of GPR55 in 1999 and its identification as a novel G-protein coupled receptor (GPCR) highly expressed in the human brain (Sawzdargo et al., 1999), mRNA encoding GPR55 has been detected in various regions of the brain and spinal cord including the hippocampus, caudate and putamen (Sawzdargo et al., 1999), brain stem,

cerebellum, frontal cortex, hypothalamus, striatum (Ryberg et al., 2007) and dorsal root ganglion (DRG) neurons (Lauckner et al., 2008). Confirmation that the mRNA is translated into protein has been demonstrated by immunohistochemistry in DRG neurons using an antibody whose specificity was determined by immunostaining of GPR55expressing HEK293 cells but not native HEK293 cells (Lauckner et al., 2008). Intriguingly, in mouse striatum, hypothalamus and brain stem, expression levels of GPR55 mRNA are comparable to those of CB1, suggestive of an important role for GPR55 in these regions (Ryberg et al., 2007; Henstridge et al., 2011). Consistent with this, it has recently been reported that GPR55 plays a role in motor co-ordination (Wu et al., 2013). 2.1. GPR55 pharmacology The current literature describing the comparative pharmacology of CB1, CB2 and GPR55 is conflicting and confusing. GPR55 is a seven transmembrane spanning GPCR that shares only approximately 14% sequence identity with CB1 and CB2 (Baker et al., 2006) and lacks their typical ‘cannabinoid binding pocket’ (Kotsikorou et al., 2011). It is therefore rather surprising that GPR55 was considered to be a third cannabinoid receptor, and this low homology and altered cannabinoid binding site might underlie some of the controversy surrounding the interactions between cannabinoids and GPR55, as described below. Detailed pharmacology of GPR55 has been reviewed by others (Ross, 2009; Sharir & Abood, 2010) and in this section we highlight a selection of cannabinoid and non-cannabinoid compounds that have been reported to be ligands for GPR55 (see Table 1). The abilities of AEA and 2-AG, endocannabinoids that are agonists at CB1 and CB2, to activate GPR55 were first demonstrated in GTPγS binding assays, which indicated that AEA was equipotent in activating GPR55, CB1 and CB2, while 2-AG was up to 200-fold more potent in activating GPR55 than it was in stimulating either CB1 or CB2 (Ryberg et al., 2007). However, other experiments using GPR55-expressing HEK293 cells demonstrated that AEA, but not 2-AG, triggered GPR55dependent increases in intracellular calcium ([Ca2+]i) and activation of Rho small GTPases (Lauckner et al., 2008), casting doubt on 2-AG exerting its effects via GPR55. Further evidence that AEA acts via GPR55 was provided by observations that elevations in [Ca2+]i in primary human endothelial cells in response to this cannabinoid were absent following siRNA-mediated down-regulation of GPR55 (WaldeckWeiermair et al., 2008; Henstridge et al., 2009). Conversely, other studies using GPR55-HEK293 cells reported that AEA did not increase

Table 1 Agonist and antagonist ligands for GPR55. The table is a non-exhaustive list of compounds that are reported to have significant pharmacological effect at GPR55, either as agonists or antagonists. Effects on targets other than GPR55 are also listed for each ligand (– indicates that the ligands are reported to be highly selective for GPR55). More detailed information is provided in the main text. Compounds

Activity at GPR55 (ref.)

Other activities (ref.)

Anandamide (AEA)

Agonist (Ryberg et al., 2007; Lauckner et al., 2008; Waldeck-Weiermair et al., 2008; Henstridge et al., 2009) Agonist (Ryberg et al., 2007) Agonist (Oka et al., 2007; Lauckner et al., 2008; Waldeck-Weiermair et al., 2008; Henstridge et al., 2009; Oka et al., 2009) Agonist (Johns et al., 2007; Ryberg et al., 2007; Waldeck-Weiermair et al., 2008; Romero-Zerbo et al., 2011) Antagonist (Ryberg et al., 2007; Lauckner et al., 2008; Whyte et al., 2009)

CB1 and CB2 agonist (Li et al., 2011); TRPV1 agonist (Ross, 2003) CB1 and CB2 agonist (Li et al., 2011) Ca2+-activated K+ channel agonist (Bondarenko et al., 2011a,b)

2-Arachidonoylglycerol (2-AG) Lysophosphatidylinositol (LPI) O-1602 Cannabidiol (CBD)

CID16020046 PSB-SB-258 SR141716A AM251 CID1792197 CID1172084 CID2440433

Antagonist (Kargl et al., 2013; Kotsikorou et al., 2013) Antagonist (Rempel et al., 2013) Agonist (Kapur et al., 2009; Yin et al., 2009) Agonist (Kapur et al., 2009; Yin et al., 2009) Agonist (Kotsikorou et al., 2011) Agonist (Kotsikorou et al., 2011) Agonist (Kotsikorou et al., 2011)

GPR18 agonist? (McHugh et al., 2010) Weak CB1 and CB2 antagonist (Petitet et al., 1998); TRPV2 agonist (Qin et al., 2008); PPARγ agonist (O'Sullivan et al., 2009); 5HT1A agonist (Campos & Guimaraes, 2008) – – CB1 antagonist (Li et al., 2011) CB1 antagonist (Li et al., 2011) – – –

Please cite this article as: Liu, B., et al., GPR55: From orphan to metabolic regulator? Pharmacology & Therapeutics (2014), http://dx.doi.org/ 10.1016/j.pharmthera.2014.06.007

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[Ca2+]i (Oka et al., 2007, 2009), and it did not act as a GPR55 agonist in β-arrestin recruitment assays (Sharir & Abood, 2010), and it is clear that AEA has promiscuous receptor selectivity with agonist actions at TRPV1 receptors (Ross, 2003) as well as at cannabinoid receptors (Table 1). Thus, although some studies support activation of GPR55 by AEA and 2-AG, there is currently no consensus between published studies on whether these cannabinoids are bona fide GPR55 agonists and their actions at other receptors makes it difficult to draw firm conclusions on their cellular mode of action. Several studies have demonstrated that a bioactive lipid, L-αlysophosphatidylinositol (LPI), has agonist effects in GPR55-HEK293 cells leading to suggestions that LPI is an endogenous noncannabinoid ligand of the receptor (Oka et al., 2007; Lauckner et al., 2008; Waldeck-Weiermair et al., 2008; Henstridge et al., 2009; Oka et al., 2009). In addition, loss of LPI-induced cellular responses following GPR55 knockdown has been observed in human endothelial cells (Kargl et al., 2013), cancer cell lines (Pineiro et al., 2011) and in rodent synaptic cells (Sylantyev et al., 2013). Despite these findings, GPR55-independent effects of LPI occurring via activation of Ca2+-activated K + channels have also been reported (Bondarenko et al., 2011a,b). The phytocannabinoid cannabidiol (CBD) is a major component of cannabis and a synthetic CBD analogue, O-1602, is reported to be a potent and selective GPR55 agonist that lacks significant binding affinity for either CB1 or CB2 (Johns et al., 2007; Ryberg et al., 2007; Waldeck-Weiermair et al., 2008). However, a recent study has cast doubt on the requirement of GPR55 for transducing O-1602-mediated signalling since it was able to induce similar feeding behaviour in both wild-type and GPR55 knockout mice (Diaz-Arteaga et al., 2012). GPR18 has been proposed as an alternative target for O-1602 (McHugh et al., 2010), although O-1602 did not act as an agonist at GPR18 expressed in rat sympathetic neurons (Lu et al., 2013). In contrast to the proposed GPR55 agonist effects of O-1602, native CBD is an effective GPR55 antagonist (Ryberg et al., 2007), which has been widely used to inhibit GPR55 agonist-induced biological effects (Ryberg et al., 2007; Lauckner et al., 2008; Whyte et al., 2009). However, a recent study has proposed that CBD may have GPR55 agonist activity since it provided protection against acute pancreatitis in mice to a similar extent to that seen using O-1602 (Yu et al., 2013). To further complicate interpretation of these studies, CBD has also been implicated in the activation of a range of receptors including TRPV2 (Qin et al., 2008), 5HT1A (Campos & Guimaraes, 2008; De Petrocellis et al., 2008; Resstel et al., 2009) and PPARγ (O'Sullivan et al., 2009). The multiple targets of CBD has highlighted the requirement for novel, selective GPR55 antagonists, and promising candidates have recently been identified using β-arrestin recruitment assays for highthroughput screening of compound libraries (Kotsikorou et al., 2011, 2013) or of coumarin derivatives (Rempel et al., 2013). These competitive GPR55 antagonists, which have negligible pharmacological effects on CB1 and CB2 receptors, have much potential for investigating whether GPR55 may be a suitable drug target, but their effects on biological functions are largely untested. However, promising data have been obtained with a novel antagonist, CID16020046, which effectively reduced LPI-mediated physiological responses in primary human platelets and endothelial cells (Kargl et al., 2013). A divergence in the effects of some ligands at CB1 and GPR55 has been observed, such that the synthetic CB1 antagonists AM251 and SR141716A (rimonabant) have agonist effects in β-arrestin and luminescence reporter assays in cell lines engineered to express GPR55 (Kapur et al., 2009; Yin et al., 2009). Subsequent functional studies revealed that AM251 promoted GPR55-dependent Ca2+ mobilisation and both AM251 and SR141716A activated ERK1/2 mitogen-activated protein (MAP) kinases and phosphorylation of the transcription factors CREB and NF-κB via GPR55 (Henstridge et al., 2010). The concentrations of these ligands that activate GPR55 are higher than those required to antagonise CB1 (Pertwee, 2005; Ryberg et al., 2007), but it is possible

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that rimonabant may reach sufficient concentrations when administered in vivo to both inhibit CB1 and activate GPR55. The activation of GPR55 by rimonabant, may contribute to the off-target effects associated with its therapeutic use in obesity (Kapur et al., 2009). 2.2. GPR55 signalling cascades The G protein subunits with which GPR55 associates and the signalling cascades downstream of its activation have been examined in detail using GPR55-HEK293 cells. Pretreatment with pertussis toxin to ADP ribosylate Gαi or with peptides to block specific Gα protein subunits indicated that GPR55 is coupled to Gα13, but not to Gαs, Gαi or Gαq (Ryberg et al., 2007). This was confirmed in a study where overexpression of catalytically inactive Gα13 abolished LPI-induced calcium mobilisation (Henstridge et al., 2009). In contrast, involvement of both Gαq and Gα13 was suggested by another report that co-transfection of GPR55-HEK293 cells with dominant negative Gαq or Gα13 reduced calcium responses to GPR55 ligands (Lauckner et al., 2008). Experiments in cell lines and primary cells have shown that pharmacological activation of GPR55 by cannabinoids and phospholipidderived agonists induced elevations in [Ca2+]i (Oka et al., 2007; Waldeck-Weiermair et al., 2008; Henstridge et al., 2010; Pineiro et al., 2011; Kargl et al., 2013; Sylantyev et al., 2013; Yu et al., 2013), and these responses were a consequence of calcium release from the endoplasmic reticulum following activation of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) generation (Lauckner et al., 2008; Waldeck-Weiermair et al., 2008). Both Gαq and Gα13 were found to be involved in GPR55-mediated PLC activation (Lauckner et al., 2008). In addition, Rho small GTPases, including RhoA, Cdc42 and Rac1, are another class of second messengers that are considered to be activated by GPR55 via Gα13 (Ryberg et al., 2007; Whyte et al., 2009; Obara et al., 2011). These small GTPases in turn activate Rhoassociated protein kinase (ROCK), which can increase [Ca2+]i both via PLC-mediated phosphatidylinositol bisphosphate hydrolysis and IP3 generation, and through actin cytoskeleton remodelling (Lauckner et al., 2008). Activation of GPR55 also leads to phosphorylation and consequent activation of the MAP kinases ERK1/2 via the RhoA-ROCK pathway and/or increases in [Ca2+]i (Waldeck-Weiermair et al., 2008; Henstridge et al., 2009; Oka et al., 2010; Pineiro et al., 2011; Anavi-Goffer et al., 2012). The transcription factors NF-κB and CREB, which are downstream of the ERK pathway, are activated by GPR55 (Henstridge et al., 2010), as is another transcription factor (ATF-2), although this occurs via p38 MAPK, not through ERK1/2 (Oka et al., 2010). In addition, GPR55 can also activate the transcription factor NFAT in a RhoA-dependent manner (Henstridge et al., 2009). Once activated, these transcription factors regulate expression of key genes that are responsible for transducing the effects of GPR55 in a diverse range of cellular functions including tumorigenesis, bone resorption and neuropathic pain (Henstridge, 2012). It is interesting to note that some of the GPR55-mediated signalling is ligand-specific. For instance, AM251 and SR141716A, but not LPI, were able to induce CREB phosphorylation (Henstridge et al., 2010). It is clear that further research is needed to fully elucidate the GPR55 signal transduction cascades, but our current understanding of the key pathways through which this receptor is reported to act are summarised in Fig. 1. 3. Action of GPR55 in metabolically active tissues In addition to being highly expressed by discrete brain regions, as described in Section 2, GPR55 is also expressed in a wide range of peripheral tissues, including spleen, adrenals and bone (Sawzdargo et al., 1999; Ryberg et al., 2007; Whyte et al., 2009), and also in metabolically important cells such as adipocytes (Moreno-Navarrete et al., 2012), and those of the gastrointestinal tract (Ryberg et al., 2007; Lin et al., 2011; Schicho et al., 2011) and islets (Romero-Zerbo et al., 2011; McKillop

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Fig. 1. Signalling pathways downstream of GPR55 activation. The diagram shows the main pathways through which GPR55 signals to exert its effects on cellular function. It is widely accepted to be coupled to the Gα13 subunit, which activates Rho small GTPases and their downstream effector Rho-associated protein kinase (ROCK). Activated ROCK can elevate intracellular Ca2+ either through phospholipase C (PLC)-induced phosphatidylinositol bisphosphate (PIP2) hydrolysis and inositol 1,4,5-tris phosphate (IP3)-mediated Ca2+ mobilisation from the endoplasmic reticulum or cytoskeleton remodelling. In addition, ROCK can modulate a range of gene transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activating transcription factor 2 (ATF-2), nuclear factor of activated T-cells (NFAT) and cAMP response element-binding protein (CREB), through activation of mitogenactivated protein kinases (MAPKs) such as ERK1/2 and p38 MAPK. GPR55 can also couple to Gαq, which increases intracellular Ca2+ through PLC activation. Ca2+ and DAG activate protein kinase C (PKC), which also regulates transcription factor activity through MAPKs.

et al., 2013). As for identification of GPR55 in the brain, much of the work on the presence of GPR55 in peripheral tissues has focused on mRNA expression, but some studies have made use of GPR55 antibodies to identify protein expression. For example, GPR55 expression has been identified by western blotting in human visceral adipose tissue, and shown to be increased in obese individuals and those with type 2 diabetes (Moreno-Navarrete et al., 2012). In addition, GPR55 has been detected in mouse osteoclasts by fluorescence immunohistochemistry, and specificity of the antibody used was confirmed by the lack of immunoreactivity in osteoclasts that were generated from bone marrow macrophages from GPR55 knockout mice (Whyte et al., 2009). Similarly, it has been demonstrated by immunohistochemistry that GPR55 is expressed by islet β-cells, but not by neighbouring α- or δ-cells, in wild type pancreas but not in pancreas from GPR55 null mice (Romero-Zerbo et al., 2011). Our current understanding of the contribution of this receptor to the maintenance of energy balance is discussed below and summarised in Fig. 2. 3.1. A role for GPR55 in weight management? Recent identification of the CB1 receptor antagonists SR141716A and AM251 as agonists for GPR55 (Table 1) points towards a possibility that activation of GPR55 could play a role in the decreased food consumption and weight loss associated with these agents, so it is worth considering the influence of GPR55 in the regulation of appetite and body weight. However, in contrast to the predicted effect of GPR55 activation with rimonabant reducing food intake, there is evidence for a link between GPR55 activation and increased calorific load. For example, in a recent study acute central and peripheral administration of O-1602

induced hyperphagia, which was accompanied by decreased expression of the anorexigenic neuropeptide cocaine- and amphetamine-regulated transcript (CART), and chronic O-1602 administration increased adiposity (Diaz-Arteaga et al., 2012). Consistent with this, the GPR55 antagonist CBD reduced food consumption and body weight gain in the absence (Sofia & Knobloch, 1976; Ignatowska-Jankowska et al., 2011; Farrimond et al., 2012) or presence (Klein et al., 2011; Le Foll et al., 2013) of Δ9-tetrahydrocannabinol (Δ9-THC), a CB1 agonist. Perhaps the most compelling evidence supporting a role for GPR55 in weight gain was provided by a recent study investigating the role of the endogenous GPR55 ligand LPI in human obesity, which found that circulating LPI levels were significantly elevated in obese patients, and they correlated positively with body weight, BMI and fat percentage in female subjects (Moreno-Navarrete et al., 2012). GPR55 expression in visceral adipose tissue (VAT) was enhanced in obese individuals and even more so in obese people with T2DM and, as with circulating LPI levels, expression of GPR55 in VAT also correlated with body weight, BMI and fat percentage. In the same study, the authors also found that treatment of VAT explants with LPI elevated the expression of lipogenic enzymes, and peroxisome proliferator activated receptor γ (PPARγ), a key regulator of adipocyte differentiation and lipid storage, was also up-regulated (Moreno-Navarrete et al., 2012). Additional support for a role for GPR55 in appetite regulation comes from studies of anorexia nervosa (AN), an ‘eating disorder’ characterised by excessive food restriction. In a study conducted in a female Japanese population, a genetic association was detected between increased vulnerability to AN and dysfunctional alteration of the GPR55 gene by its nonsynonymous SNP, Gly195Val (Ishiguro et al., 2011). This observation that the Val195 allele of GPR55, which had a

Please cite this article as: Liu, B., et al., GPR55: From orphan to metabolic regulator? Pharmacology & Therapeutics (2014), http://dx.doi.org/ 10.1016/j.pharmthera.2014.06.007

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Fig. 2. Expression and function of GPR55 in tissues involved in energy homeostasis. The diagram summarises our current knowledge of GPR55 expression in the key tissues responsible for controlling fuel homeostasis, and its main roles in these tissues.

reduced signalling capacity in vitro, was linked to an increased risk of AN is consistent with inactive GPR55 being associated with reductions in food intake. The studies summarised above support a role for GPR55 activation in promoting food intake but this is likely to be an oversimplification. For example, it is difficult to define whether the reduced food intake observed with CBD is a consequence of GPR55 inhibition since CBD interacts with a range of receptors to exert its pharmacological effects (Table 1). Furthermore, other studies have reported that CBD had no significant effect on food intake (Wiley et al., 2005; Riedel et al., 2009; Scopinho et al., 2011; Farrimond et al., 2012), and this might be a consequence of it regulating both orexigenic and anorexigenic cascades through interactions with multiple effectors. In addition, while O-1602 induced hyperphagia in rats, it was also able to induce food intake in GPR55−/− mice, suggesting that these effects of O-1602 can occur independently of GPR55 activation (Diaz-Arteaga et al., 2012). Furthermore, GPR55−/− mice do not differ significantly in body weight compared to their wild-type littermates when fed on standard chow (Wu et al., 2013), although this could reflect compensatory mechanisms allowing normalisation of food intake in mice following GPR55 deletion, perhaps via CB1 up-regulation. Further characterisation of the role of GPR55 in the regulation of food intake and body weight is clearly warranted, with the need to identify the brain circuitry of GPR55 expression in relation to appetite control in different species, and these studies will require the availability of specific GPR55 agonists/antagonists and of tissue-specific conditional GPR55 knockout mice. In particular, examining the effect of LPI on lipogenesis in VAT when GPR55 is down-regulated and in GPR55−/− mice is necessary to provide further information on the role of LPI signalling via GPR55 in obesity.

3.2. Role of GPR55 in gastrointestinal motility GPR55 mRNA has been detected throughout the GI tract and its expression has been confirmed in myenteric neurons of ileum and/or colon in humans and rodents by immunohistochemistry (Ryberg et al., 2007; Lin et al., 2011; Schicho & Storr, 2012; Li et al., 2013). A role for GPR55 in regulating the passage of digested food through the GI tract is supported by observations that O-1602 reduced intestinal contractility and delayed gut transit time, effects that were lost in GPR55−/− mice (Ross et al., 2012). Although to date there have not been any reports on a possible role of GPR55 in intestinal secretion its expression in the colonic mucosa, a region that controls secretory processes via intrinsic neurons (Schicho & Storr, 2012), suggests that this is an area worthy of further investigation. In this context it is worth noting that peptide YY (PYY), an anorectic peptide that exerts its actions via central and peripheral effects, is highly concentrated in the distal ileum and colon and is also known to stimulate intestinal contractility (Ferrier et al., 2000). Given the emerging role of GPR55 in food intake and energy regulation, it is intriguing to speculate that GPR55 activation in the gut represses PYY secretion, which may contribute to the reported effects of GPR55 agonists to stimulate food intake and reduce contraction of the GI tract.

3.3. Regulation of islet function by GPR55 Islets of Langerhans play a crucial role in fuel homeostasis with the secretion of insulin during the absorptive state and glucagon during times of fuel restriction. GPR55 mRNA and protein have been identified in rat and mouse β-cell lines (BRIN-BD11and MIN6) and in rat, mouse and human islets (Li et al., 2011). Immunohistochemical analyses have

Please cite this article as: Liu, B., et al., GPR55: From orphan to metabolic regulator? Pharmacology & Therapeutics (2014), http://dx.doi.org/ 10.1016/j.pharmthera.2014.06.007

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indicated that GPR55 is expressed by β-cells in mouse and rat islets, but it was not detected in α- or δ-cells (Romero-Zerbo et al., 2011; McKillop et al., 2013), suggesting that it might play a physiological role in regulating glucose homeostasis through its effects on insulin secretion. Indeed, O-1602 has been reported to potentiate glucose-stimulated insulin secretion from mouse, rat and human islets, an effect that was reduced in GPR55−/− mice, indicative of a direct stimulatory role for GPR55 in β-cells (Romero-Zerbo et al., 2011; Song et al., 2012). A recent evaluation of the effects of various GPR55 ligands on insulin secretion found that all agonists used — AM251, O-1602, abnormal CBD (a synthetic GPR55-activating CBD analogue) and the phospholipid-derived endocannabinoids OEA and PEA — stimulated insulin secretion from BRIN-BD11 cells and mouse islets, and their effects were inhibited by CBD (McKillop et al., 2013), suggesting that they acted via GPR55 activation. In vivo studies have also confirmed that short-term administration of GPR55 agonists to rats or mice increased plasma insulin levels and improved glucose tolerance (Romero-Zerbo et al., 2011; McKillop et al., 2013). These observations imply that GPR55 agonists have acute, beneficial effects on insulin secretion and glucose handling, which contrast with their chronic effects to induce lipogenesis and weight gain, as described above. LPI has long been known to be an insulin secretagogue (Metz, 1986), and although LPI-induced insulin secretion was suggested to be at least partly mediated by the mobilisation of [Ca2+]i in β-cells (Metz, 1988), its precise mode of action in islets has never been established. The identification of LPI as an endogenous ligand of GPR55 (Oka et al., 2007) points to a possibility that the insulinotropic effect of LPI could be mediated through activation of this receptor, but our studies have shown that LPI potentiates glucose-stimulated insulin secretion from both WT and GPR55−/− mice (Song et al., 2013), consistent with earlier reports of GPR55-independent effects of LPI (Bondarenko et al., 2011a,b). The effects of O-1602 on insulin secretion, however, were lost following deletion of GPR55 (Song et al., 2013), providing further support for this compound exerting its effects in islets via GPR55 activation. Development of specific GPR55 agonists is clearly required given the off-target effects in some studies of the two main GPR55 agonists, LPI and O1602, and promising candidates have recently been identified by high throughput screening (Kotsikorou et al., 2011) (Table 1). There has been some investigation of the signalling cascades downstream of GPR55 activation in β-cells and studies to date suggest that elevations in [Ca2+]i are important in transducing the stimulatory effects of GPR55 agonists. Thus, O-1602 induced transient increases in [Ca2+]i in rat islets (Romero-Zerbo et al., 2011), and AM251, O-1602, abnormal CBD, OEA and PEA all caused rapid increases in [Ca2+]i in MIN6 and BRIN-BD11 insulin-secreting cells (Ning et al., 2008; McKillop et al., 2013), consistent with GPR55 signalling via PLC activation in β-cells (see Fig. 1). These agonists also stimulated small increases in intracellular cyclic AMP levels in BRIN-BD11 cells (Ning et al., 2008; McKillop et al., 2013), which are likely to be secondary to activation of calciumsensitive isoforms of adenylate cyclase since there is no evidence that GPR55 is directly coupled to Gs to promote cyclic AMP accumulation. GPR55-mediated activation of PLC can occur both via conventional Gαq coupling and also by the Gα13 pathway (Fig. 1) so it is possible that GPR55 also regulates β-cells via Gα13 signalling since this Gprotein has previously been identified in islets (Skoglund et al., 1999; Hammar et al., 2009). However, inhibition of the Rho kinase ROCK causes increased insulin release from rat β-cells purified from islets by fluorescence-activated cell sorting (Hammar et al., 2009) and this negative role for RhoA on insulin release is inconsistent with the observed stimulatory effects of GPR55 agonists summarised above. It is therefore possible that there are both stimulatory and inhibitory cascades activated by GPR55 agonists in islets, via separate G-proteins and further work is required to define the signalling cascades that regulate insulin exocytosis downstream of GPR55 activation. There is some evidence that GPR55 may also play a role in maintaining functional insulin-producing β-cells, since OEA can protect β-cells

from palmitate-induced apoptosis, an effect that was not mediated via another OEA receptor, GPR119 (Stone et al., 2012), and islets isolated from GPR55−/− mice exhibited significantly higher levels of apoptosis than islets from wild-type mice (Song et al., 2012; Liu et al., 2013). These observations are consistent with the reported effects of O-1602 to promote phosphorylation of ERK1/2 and CREB in various cell types (Whyte et al., 2009; Henstridge et al., 2010; Andradas et al., 2011; Pineiro et al., 2011) (Fig. 1), effectors that are known to regulate β-cell mass (Burns et al., 2000; Jhala et al., 2003). 3.4. Expression of GPR55 in other metabolically relevant tissues While a picture of the role of GPR55 in adipocytes, islets and the GI tract is starting to develop, as described above, very little is known about its expression and function in other tissues involved in energy homeostasis. GPR55 mRNA and protein expression have been detected in the liver in rats, mice and humans (Sawzdargo et al., 1999; Romero-Zerbo et al., 2011; Moreno-Navarrete et al., 2012). However, no significant differences in hepatic expression of GPR55 were found between obese patients with normal and impaired glucose tolerance, T2DM patients and lean control subjects, and the functional role of GPR55 in the liver remains unclear (Moreno-Navarrete et al., 2012). In addition, there have been suggestions that GPR55 is expressed in rat skeletal muscle but functional analyses have not been carried out to address its potential physiological role (Simcocks et al., in press). 4. Conclusions and perspectives Although GPR55 was initially classified as a third cannabinoid receptor its sequence and ligand profile (Table 1) demonstrate that it does not sit neatly in this classification, and it has yet to find a suitable GPCR family. In some respects its effects in tissues involved in energy homeostasis mirror those of CB1, given that it has been implicated not only in the potentially deleterious effects of weight gain and fat storage, but also in beneficial action at β-cells to stimulate insulin secretion. However, full interpretation of studies is limited by the lack of specificity of many of the agonists and antagonists used to date, and future work in this area would benefit from the use of GPR55−/− mice and newly developed ligands with improved specificity (Table 1). It is worth noting that under physiological conditions GPR55−/− mice do not have a metabolic phenotype so investigating glucose tolerance in GPR55−/− mice in both normal and pathological conditions such as diet-induced obesity would therefore provide further insights into defining the role of GPR55 in maintaining energy homeostasis. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments Our research on GPR55 is funded by Diabetes UK (11/0004307), King's College London (KINGS award) and The Henry Lester Trust (2011-2013). References Anavi-Goffer, S., Baillie, G., Irving, A. J., Gertsch, J., Greig, I. R., Pertwee, R. G., et al. (2012). Modulation of L-alpha-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids. J Biol Chem 287, 91–104. Andradas, C., Caffarel, M. M., Perez-Gomez, E., Salazar, M., Lorente, M., Velasco, G., et al. (2011). The orphan G protein-coupled receptor GPR55 promotes cancer cell proliferation via ERK. Oncogene 30, 245–252. Baker, D., Pryce, G., Davies, W. L., & Hiley, C. R. (2006). In silico patent searching reveals a new cannabinoid receptor. Trends Pharmacol Sci 27, 1–4. Bluher, M., Engeli, S., Kloting, N., Berndt, J., Fasshauer, M., Batkai, S., et al. (2006). Dysregulation of the peripheral and adipose tissue endocannabinoid system in human abdominal obesity. Diabetes 55, 3053–3060. Bondarenko, A. I., Malli, R., & Graier, W. F. (2011a). The GPR55 agonist lysophosphatidylinositol acts as an intracellular messenger and bidirectionally

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Glossary N-acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD): a phospholipase that is responsible for the synthesis of the endocannabinoid N-arachidonoylethanolamide (AEA) from N-arachidonoyl-phosphatidylethanolamine.

2-Arachidonoylglyercol (2-AG): an endocannabinoid arachidonic acid derivative that is synthesised on demand by diacylglycerol lipases. It activates CB1 and CB2 and has also been reported to be a GPR55 agonist. Abnormal-cannabidiol (Abn-CBD): a synthetic CBD analogue that acts as a selective agonist for GPR55. Activating transcription factor 2 (ATF-2): a transcription factor that is activated by phosphorylation in response to stress and growth stimuli. AM251: an analogue of SR141716A that also acts as an antagonist at CB1 and an agonist at GPR55. Anorexigenic: an agent that reduces food intake and results in weight loss. N-arachidonoylethanolamide (AEA; anandamide): an endocannabinoid arachidonic acid derivative that is synthesised upon demand by N-acyl-phosphatidylethanolamide phospholipase D. It activates CB1 and CB2 and has also been reported to be a GPR55 agonist. Cannabinoids: compounds that act as ligands for cannbinoid receptors. Cannabidiol (CBD): a phytocannabinoid GPR55 antagonist that is also a low affinity antagonist for CB1 and CB2. Cannabinoid receptor 1 (CB1): a GPCR identified initially in rat brain, which plays key roles in energy homeostasis. Cannabinoid receptor 2 (CB2): a GPCR that is highly expressed in immune tissues and cells where it regulates immune and inflammatory reactions. Low expression levels have also been detected in peripheral tissues. Cocaine- and amphetamine-regulated transcript (CART): an anorexigenic neuropeptide that reduces food intake through interaction with appetite circuits in the brain. Cyclic AMP response element-binding protein (CREB): a transcription factor that regulates gene expression by binding to DNA sequences called cyclic AMP response elements. Diacylglycerol lipases (DAG-lipases): Two isoforms of this enzyme (α and β) hydrolyse diacylglycerol to form the endocannabinoid 2-arachidonoylglyercol (2-AG). Endocannabinoid system (ECS): a signalling cascade consisting of the two classical cannabinoid receptors, CB1 and CB2, their endogenous ligands, and enzymes that synthesise and degrade these ligands. Fatty acid amide hydrolase (FAAH): an enzyme that catalyses the degradation of the endocannabinoid N-arachidonoylethanolamide (AEA) to form arachidonic acid and ethanolamine. G-protein coupled receptors (GPCRs): seven transmembrane spanning cell surface receptors, which transduce their effects following ligand binding by interacting with intracellular heterotrimeric GTP-binding proteins that couple to downstream effectors. G-protein coupled receptor 55 (GPR55): a GPCR that has been classified as a novel cannabinoid receptor due to its sensitivity to distinct cannabinoid ligands in a variety of tissues, but which shows only low sequence homology with CB1 (13.5%) and CB2 (14.4%). Hyperphagia: excessive food intake. L-α-lysophosphatidylinositol (LPI): a bioactive lipid that is formed following phospholipase A2-mediated hydrolysis of the membrane lipid phosphatidylinositol. It is the only known endogenous ligand of GPR55, although GPR55-independent effects of LPI have been reported. Mitogen-activated protein kinases: a family of protein kinases that are activated by phosphorylation on tyrosine and theronine residues. They regulate the activity of a range of transcription factors to control cell proliferation and survival. Monoacylglycerol lipase (MGL): an enzyme responsible for inactivating the endcannabinoid 2-arachidonoylglyercol (2-AG) by degrading it to arachidonic acid and glycerol. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB): a family of transcription factors that regulate expression of genes involved in a range of cellular processes including proliferation and apoptosis. Nuclear factor of activated T-cells (NFAT): a family of transcription factors that are activated by calcium-dependent phosphatase-induced dephosphorylation. Oleoylethanolamine (OEA): phospholipid-derived fatty acid analogue of AEA that activates GPR55 and is also a GPR119 agonist. Orphan: a GPCR that is known to exist but for which ligands have not been identified. Palmitoylethanolamine (PEA): phospholipid-derived fatty acid analogue of AEA that activates GPR55 and is also a GPR119 agonist. Peptide YY (PYY): an anorectic peptide secreted from gastrointestinal L-cells that exerts its actions via central and peripheral effects. Peroxisome proliferator-activated receptor γ (PPARγ): a nuclear receptor that regulates gene transcription by forming heterodimers with retinoid X receptors. It is a key regulator of adipocyte differentiation and lipid storage. Phytocannabinoids: plant-derived cannabinoids. Rho small GTPases: a family of low molecular weight GTPases that are involved in regulating actin cytoskeleton dynamics. Rho-associated protein kinase (ROCK): a serine threonine kinase with two isoforms [ROCKI (ROKβ) and ROCK-II (ROKα)] that is a downstream effector of the Rho family of small GTPase proteins, which are key regulators of cell migration, proliferation and apoptosis. ROCK transduces Rho signalling by phosphorylating downstream substrates. SR141716A (rimonabant): a synthetic CB1 antagonist that was approved in some countries as an anorectic anti-obesity drug (Acomplia), but was later withdrawn due to its deleterious psychotropic effects. SR141716A also acts as an agonist at GPR55. Transcription factors: proteins containing DNA-binding domains that allow them to interact with specific DNA sequences to regulate gene transcription. Type 2 diabetes mellitus: the more common form of diabetes in which insulin resistance and insufficient insulin secretion from β-cells results in hyperglycaemia.

Please cite this article as: Liu, B., et al., GPR55: From orphan to metabolic regulator? Pharmacology & Therapeutics (2014), http://dx.doi.org/ 10.1016/j.pharmthera.2014.06.007