An atlas of G-protein coupled receptor expression and function in human subcutaneous adipose tissue.

JPT-06719; No of Pages 33 Pharmacology & Therapeutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate Editor: M. Curtis

An atlas of G-protein coupled receptor expression and function in human subcutaneous adipose tissue☆ Stefan Amisten a,b,⁎, Matt Neville b,c, Ross Hawkes a, Shanta J. Persaud a, Fredrik Karpe b,c,⁎⁎, Albert Salehi d a

Diabetes Research Group, Division of Diabetes & Nutritional Sciences, King's College London, Faculty of Life Sciences & Medicine, London, UK Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, UK NIHR Oxford Biomedical Research Centre, Churchill Hospital, Oxford OX3 7LE, UK d Department of Clinical Science, UMAS, Clinical Research Center, University of Lund, Sweden b c

a r t i c l e

i n f o

a b s t r a c t G-protein coupled receptors (GPCRs) are involved in the regulation of adipose tissue function, but the total number of GPCRs expressed by human subcutaneous adipose tissue, as well as their function and interactions with drugs, is poorly understood. We have constructed an atlas of all GPCRs expressed by human subcutaneous adipose tissue: the ‘adipose tissue GPCRome’, to support the exploration of novel control nodes in metabolic and endocrine functions. This atlas describes how adipose tissue GPCRs regulate lipolysis, insulin resistance and adiponectin and leptin secretion. We also discuss how adipose tissue GPCRs interact with their endogenous ligands and with GPCRtargeting drugs, with a focus on how drug/receptor interactions may affect lipolysis, and present a model predicting how GPCRs with unknown effects on lipolysis might modulate cAMP-regulated lipolysis. Subcutaneous adipose tissue expresses 163 GPCRs, a majority of which have unknown effects on lipolysis, insulin resistance and adiponectin and leptin secretion. These GPCRs are activated by 180 different endogenous ligands, and are the targets of a large number of clinically used drugs. We identified 119 drugs, acting on 23 GPCRs, that are predicted to stimulate lipolysis and 173 drugs, acting on 25 GPCRs, that are predicted to inhibit lipolysis. This atlas highlights knowledge gaps in the current understanding of adipose tissue GPCR function, and identifies GPCR/ligand/drug interactions that might affect lipolysis, which is important for understanding and predicting metabolic side effects of drugs. This approach may aid in the design of new, safer therapeutic agents, with fewer undesired effects on lipid homeostasis. © 2014 Elsevier Inc. All rights reserved.

Keywords: Adipose tissue Lipolysis Leptin Adiponectin Insulin resistance GPCR

Contents 1. 2. 3.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 Human abdominal and gluteofemoral subcutaneous adipose tissue G-protein coupled receptor ligand usage . . . . 0 Regulation of adipose tissue lipolysis, insulin resistance and secretion of adiponectin and leptin by G-protein coupled receptors expressed in human subcutaneous adipose tissue. . . . . . . . . . . . . . . . . . . . . 0 4. Adipose tissue G-protein coupled receptors as drug targets . . . . . . . . . . . . . . . . . . . . . . . . . . 0 5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Abbreviations: ABD, abdominal subcutaneous adipose tissue; GLUT, gluteofemoral subcutaneous adipose tissue; SAT, subcutaneous adipose tissue. ☆ This manuscript has not been published and is not under consideration for publication elsewhere. ⁎ Correspondence to: S. Amisten, 2.4N Hodgkin Building, King's College London, Guy's Campus, London SE1 1UL, UK. Tel.: +44 20 7848 6279. ⁎⁎ Correspondence to: F. Karpe, Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Churchill Hospital, Headington, OX3 7LE, UK. Tel.: +44 1865 857222; fax: +44 1865 857213. E-mail addresses: [email protected] (S. Amisten), [email protected] (F. Karpe).

http://dx.doi.org/10.1016/j.pharmthera.2014.09.007 0163-7258/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Amisten, S., et al., An atlas of G-protein coupled receptor expression and function in human subcutaneous adipose tissue, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.09.007

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S. Amisten et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

1. Introduction 1.1. Role of adipose tissue in energy storage and endocrine signalling In man, the main adipose tissue depots are located around internal organs (visceral fat (VAT)), in breast tissue, in bone marrow and as subcutaneous adipose tissue (SAT) in the upper and lower body. Adipose tissue is made predominantly of adipocytes, but other cell types contained in the stromal vascular fraction (SVF) are also present. The SVF cells are made up of adipose tissue macrophages, fibroblasts, endothelial cells and pre-adipocytes. An excess of VAT is unequivocally associated with increased risk of cardiovascular and metabolic disease (Grundy, 2004; Despres, 2007). SAT, however, constitutes the largest fat mass in the body and it can be divided into gluteofemoral (GLUT) and abdominal (ABD) depots. Increased GLUT adipose tissue is associated with a reduced risk of developing both diabetes and cardiovascular disease, whereas no such association has been found for ABD adipose tissue (Manolopoulos et al., 2010). Adipose tissue has long been seen exclusively as an energy storing organ, the main function of which is to store excess energy in the form of lipids. However, since the discovery of several adipose tissuederived hormones, such as adiponectin and leptin, it is now accepted that it is the body's largest endocrine organ, involved in the regulation of both cardiovascular and metabolic processes via complex networks of adipose-derived signalling molecules (Kershaw & Flier, 2004). The amount of energy stored in adipose tissue in the form of adipocyte lipid droplets is regulated through the balance of fat storage (mainly esterification of external fatty acids into triacylglycerol but also some de novo lipogenesis) and lipolysis. For its own energy provision, the white adipocyte is largely glycolytic with minimal fat oxidation whereas robust fat oxidation can be seen in brown adipocytes (Kersten, 2001). Two different responses can occur following increased demands for fat storage (positive energy balance): additional fat storage in existing cells (hyperplastic, with enlargement of lipid droplets) or in new cells (hyperproliferative, with increased number of cells recruited from adipocyte precursors resident in the tissue). Generation of new adipocytes is associated with poorer metabolic function, whereas additional fat storage in existing cells, as occurs in type 2 diabetes (T2D) and cardiovascular disease (CVD), is associated with an improved metabolic profile (Manolopoulos et al., 2010; Tchoukalova et al., 2010). Adipocyte lipid droplet turnover is strongly regulated by hormones. Thus, insulin, which is released from islet beta cells following postprandial increases in blood glucose, promotes fat storage through stimulation of lipogenesis and inhibition of lipolysis (Czech et al., 2013), whereas a number of stress hormones such as adrenaline, cortisol and growth hormone limit fat storage by stimulation of lipolysis, leading to the release of free fatty acids (FFA) and glycerol from adipocytes (Lafontan & Langin, 2009). Although the release of leptin from adipocytes can indirectly affect the balance between fat storage and lipolysis by its central effects on energy balance, it has also been reported to stimulate fatty acid oxidation and to inhibit lipogenesis (Bai et al., 1996; Wang et al., 1999). Another adipocyte-derived hormone, adiponectin, which is very abundant in serum, improves skeletal muscle and hepatic insulin sensitivity, with mostly indirect effects on adipose tissue. 1.2. Human subcutaneous adipose tissue G-protein coupled receptors The regulation of lipolysis is largely regulated by G-protein coupled receptors, both via direct effects on lipolysis by the tandem activation of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) stimulated by agonist-induced elevations in intracellular cAMP (Greenberg et al., 1991). A diverse range of agonists including neurotransmitters, neuropeptides and hormones exert their effects on adipose tissue cells by binding to specific cell surface receptors. The largest cell surface receptor class in man is the G-protein coupled receptor (GPCR) family (Fredriksson et al.,

2003), which is made up of integral membrane proteins with seven membrane spanning alpha helical domains and an extracellular Nterminus and an intracellular C-terminus. The main function of GPCRs is to sense changes in the local extracellular environment, and to transmit a signal into the cell, thus allowing the cell to respond in various ways to the detected changes. The roles of some adipocyte GPCRs, such as those responding to adrenaline and noradrenaline, with welldefined inhibitory or stimulatory effects on lipolysis via alpha- and beta-adrenergic receptors (Lafontan, 1994; Flechtner-Mors, et al., 2002; Langberg et al., 2013) respectively, as well as the inhibitory action of metabolites such as nicotinic acid acting on the receptor HCA2 (GPR109a) (Ren et al., 2009), are well known. However, the roles of most adipose tissue GPCRs in regulating lipolysis, adipose tissue insulin resistance and the secretion of adiponectin and leptin are unknown. Additionally, very little is known about human adipose tissue GPCR expression or how the integrated signalling via these GPCRs contributes to deliver the fine tuning of adipose tissue lipid turnover and hormone secretion. Moreover, a majority of studies investigating adipose tissue GPCR function have been carried out using rodent primary adipose tissue or a number of in vitro differentiated cell lines, such as murine 3T3 (Todaro & Green, 1963) and 3T3-L1 cells (H. Green & Kehinde, 1975) or their derivatives, which may not always be translatable to human physiology. This review provides a comprehensive atlas of the expression of mRNAs encoding all functional, non-olfactory GPCRs in human gluteofemoral and abdominal subcutaneous adipose tissue, and a summary of the roles of these GPCRs in regulating adipocyte function.

1.3. Human subcutaneous adipose tissue G-protein coupled receptor signalling In man, a large number of physiological functions are regulated by GPCRs, which is one of the reasons why GPCRs are such important drug targets in modern medicine. Upon receptor activation, GPCRs are able to couple to a large number of complex signalling pathways, which in turn elicit a number of different responses, such as the regulation of gene transcription, cytoskeletal rearrangements and the docking of secretory vesicles with the plasma membrane, which leads to vesicle degranulation (Bauer et al., 2007). The net downstream effect of GPCR activation on cellular function is determined by several factors such as constitutive receptor signalling, receptor dimerisation, presence and nature of agonists and antagonists that may interact with the receptor, availability of guanine nucleotidebinding proteins (G-proteins) and receptor internalisation (Baker & Hill, 2007). A majority of GPCRs transduce their signals via the α subunits of different classes of G-proteins: Gq/G11 activates phospholipase C to generate diacylglycerol and inositol 1,4,5 trisphosphate, which activate protein kinase C and mobilise intracellular Ca2+ respectively; Gs activates adenylate cyclase to stimulate cAMP production; and Gi /G 0 inhibits adenylate cyclase to decrease cAMP production; G12/13 activates the small GTPase Rho to regulate actin cytoskeleton remodelling. GPCRs are also able to signal via other pathways, including the modulation of ion channel activity, and many Gi /G 0 coupled GPCRs elevate Ca2+ by activating Ca 2+ flux channels (Billington & Penn, 2003). In summary, GPCR signalling is very complex, and often involves the simultaneous activation of several distinct second messenger signalling pathways, with the net effect being the activation or inhibition of cAMP generation and/or mobilisation of intracellular Ca2+ and/or effects on the actin cytoskeleton. To complicate things further, GPCR signalling may also vary between tissue types, depending on which receptors, Gproteins and downstream effectors are co-expressed in the different cell types. In addition, some GPCRs signal via non-G-protein mediated pathways, such as Wnt signalling by Frizzled receptors (Komiya & Habas, 2008).



1.4. Definition of the human G-protein coupled receptor repertoire and quantification of human subcutaneous adipose tissue G-protein coupled receptor expression The human GPCR repertoire has been defined as having 384 functional members excluding pseudogenes, olfactory and vomeronasal receptors (Amisten et al., 2013). The expression of mRNAs encoding all GPCRs in subcutaneous adipose tissue biopsies from ABD and GLUT depots from six healthy donors (see below) was quantified using quantitative real-time PCR (qPCR). qPCR has previously successfully been used to quantify GPCR expression in both mouse and human tissues (Regard et al., 2007; Regard et al., 2008; Amisten et al., 2013), as it is more suitable for accurate quantification of low-abundance genes than commonly used high-throughput technologies such as microarray and RNA-sequencing (Nagalakshmi et al., 2008). Quantification of the 384 genes that make up the human GPCRome was performed using Qiagen's QuantiTect qPCR primers and QuantiFast kits as described elsewhere (Amisten, 2012), with cDNA templates obtained from ABD and GLUT biopsies collected from the Oxford Biobank (www.oxfordbiobank.org.uk). The samples were harvested by needle biopsy from six healthy, normal weight to overweight individuals (40 ± 5 years, all men, BMI 27 ± 5 kg/m2) after infiltration of the subcutaneous adipose tissue by lignocaine. GPCR mRNA expression values in the biopsy samples were normalised against the house keeping gene peptidylprolyl isomerase A (PPIA) (Neville et al., 2011) using the ΔΔCt method (Pfaffl, 2001), and the risk of incorporating false negative results was minimised as described elsewhere (Amisten et al., 2013). The Mann–Whitney test (using GraphPad Prism 4.0) was used to identify GPCRs that were differentially expressed in the ABD and GLUT biopsies. 1.5. The human subcutaneous adipose tissue G-protein coupled receptor atlas Of the 384 GPCRs screened by qPCR, we detected the same 155 GPCR mRNAs in both the GLUT and the ABD adipose tissue biopsies, which accounts for 40.4% of all known functional, non-odorant GPCRs. Due to a

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lack of suitable qPCR primers, we were not able to quantify the expression of four GPCRs previously reported to be expressed in adipose tissue (HCA1 (HCAR1, formerly GPR81), HCA2 (HCAR2, formerly GPR109a), HCA3 (HCAR3, formerly GPR109b) and P2Y11 (P2RY11)) (Soga et al., 2003; Tunaru et al., 2003; Lee et al., 2005; Cai et al., 2008), as well as four receptors on which there is no published information on their expression in adipose tissue (GPR110, GPR137, NPSR1, UTS2R). The inability to quantify the mRNA expression of the P2RY11 gene is most likely due to its high GC content (N65%), which seems to be incompatible with the 60 °C annealing/extension temperature utilised by the QuantiFast qPCR enzyme used in this study. The adipose tissue GPCRs HCAR1 and HCAR3 all share a very high degree of sequence similarity with HCAR2 (65 and 97% sequence identity respectively) thus rendering it almost impossible to design primer pairs compatible with the QuantiFast qPCR enzyme that are able to specifically amplify transcripts originating from the individual HCAR1-3 genes. Other studies where qPCR has been used to quantify the expression of GPCRs have encountered similar difficulties (Regard et al., 2008). Thus, there are 159 GPCR mRNAs with known expression in ABD and GLUT subcutaneous adipose tissue, 4 GPCRs with unknown expression and 221 absent GPCRs. Of the 155 GPCRs that we detected in the ABD and GLUT biopsies (excluding the four known adipose tissue GPCRs HCA1, HCA2, HCA3 and P2Y11 for which no primers were available), we were able to quantify the relative mRNA expression of 95 GPCRs, but only trace mRNA levels of a further 60 GPCRs were detected, so it was not possible to quantify their expression. Given the established differences in the regulation of lipolysis in ABD and GLUT SAT (Manolopoulos et al., 2012), it would be expected that there would be differential expression of GPCRs. Surprisingly, however, we found that the relative expression of GPCRs in subcutaneous adipose tissue biopsies isolated from the ABD and GLUT depots was strikingly similar (r2 = 0.97) (Fig. 1), and only three GPCR mRNAs showed significantly higher expression in the GLUT biopsies than in the ABD biopsies: P2RY14 + 28.4 ± 8.1%, p = 0.026; PTAFR +37 ± 6.6%, p = 0.041, GPRC5C: +42.3 ± 9.7%, p = 0.0087 (Fig. 2, also highlighted with arrows in Fig. 1). The 159 different adipose tissue GPCRs expressed in the ABD and GLUT depots interact with a large number of endogenous ligands and

Fig. 1. Log mean mRNA expression of GPCRs relative the house keeping gene PPIA in subcutaneous abdominal and gluteofemoral adipose tissue biopsies. The mRNA expression of 384 GPCRs was quantified in paired samples originating from 6 age and gender matched donors. mRNAs encoding a total of 155 GPCRs were detected, and the expression of 95 GPCRs was quantified relative to PPIA, and mRNAs of a further 60 GPCRs were found to be present at trace levels (not shown in figure). 221 GPCRs were absent, and 8 GPCRs (GPR110, GPR137, HCAR1, HCAR2, HCAR3, NPSR1, P2RY11, UTS2R) were not quantified due to a lack of functional qPCR primers and are therefore not included in the figure. The expanded box highlights the relative expression of the 20 most abundant subcutaneous adipose tissue GPCRs in abdominal and gluteofemoral adipose tissue. Only three GPCR mRNAs (GPRC5C, P2RY14, PTAFR) were expressed at significantly higher levels in gluteofemoral compared to abdominal subcutaneous adipose tissue (black arrows, n = 6, p b 0.05).


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Fig. 2 (continued).


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Fig. 2 (continued).

with clinically relevant drugs, and the effects of signalling through these GPCRs on SAT function were identified using data manually extracted from PubMed.gov, GeneCards.org, Ingenuity Pathways Analysis (www.ingenuity.com), the IUPHAR GPCR database (Sharman &

Mpamhanga, 2011) and Drug-Bank (Knox et al., 2011) as described elsewhere (Amisten et al., 2013). A comprehensive analysis of the ligand usage of adipose tissue GPCRs and the presence in adipose tissue of these ligands is discussed in Section 2.



Fig. 2 (continued).


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Fig. 2 (continued).



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Fig. 2. The expression of 384 GPCRs in abdominal and gluteofemoral adipose tissue relative the house keeping gene PPIA. mRNAs encoding 155 GPCRs were detected, whereas 221 GPCR mRNAs were absent. 8 GPCRs (GPR110, GPR137, HCAR1, HCAR2, HCAR3, NPSR1, P2RY11, UTS2R) were not quantified due to a lack of functional qPCR primers. The effects of each GPCR on adipose tissue insulin resistance (IR), lipolysis (Li) and the secretion of adiponectin (A) and leptin (L) are indicated between the gene symbol and the Y axis. +: stimulatory effect; −: inhibitory effect; 0: no effect; ?: unknown effect; +/−: reports on both stimulatory and inhibitory effects; a: genetic association with GPCR; +/a: both stimulatory effect and genetic association reported; *: inferred effect where the exact identity of GPCR mediating effect on hormone secretion is not known due to the use of non-specific agonists/antagonists; #: effect reported due to activity of enzyme that synthesises GPCR ligand; diagonal line: GPCR absent. If a GPCR is a drug target, this is indicated with a symbol to the right of the expression bar, describing the mode of action of the drug(s) targeting the receptor: +: agonist; −: antagonist/inverse agonist; +/− both agonist and antagonist drugs target the same receptor. Expression data presented as mean ± SEM of n = 6 paired abdominal and gluteofemoral adipose tissue biopsy donors. * (inside graph area): p b 0.05. No GPCRs belonging to the beta-alanine, bombesin, calcium sensing, cholecystokinin, corticotrophin releasing factor, galanin, ghrelin, glucagon receptor family, gonadotrophin-releasing hormone, kisspeptin, melanin concentrating hormone, melanocortin, melatonin, motilin, neurotensin, opioid, orexin, peptide P518, prolactin-releasing peptide, relaxin family peptide, trace amine and zinc receptors were detected in ABD or GLUT, and are therefore not represented in the figure.

Manual PubMed data mining was used to identify publications in which GPCRs detected in the human ABD and GLUT adipose tissue biopsies have been implicated in the secretion of the adipose tissue hormones adiponectin (Fig. 3) and leptin (Fig. 4) and in the regulation of adipose tissue lipolysis (Fig. 5-b) and insulin resistance (Fig. 6). A majority of published functional studies on adipose tissue GPCRs has been performed using mouse or rat adipose tissue, and in some cases rodent immortalised cell lines such as the murine 3T3 (Todaro & Green, 1963) and 3T3-L1 cells (H. Green & Kehinde, 1975). When data on adipose tissue GPCR function was available from multiple sources,

the following experimental model preference was used: human adipose tissue and human in vivo experiments N mouse, rat adipose tissue and rodent in vivo experiments N adipose tissue and in vivo experiments from other species (dog, rabbit etc.) N rodent cell lines. Interestingly, most of the GPCRs that we identified in the ABD and GLUT biopsies currently have no documented roles in the regulation of adipose tissue insulin resistance, lipolysis or secretion of adiponectin and leptin. Based on our data on ABD and GLUT adipose tissue GPCR expression and publically available data on GPCR signalling pathways, we have constructed a model predicting how GPCRs with no documented impact


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on the regulation of lipolysis are expected to influence cAMP-mediated release of free fatty acids into the blood stream, which may be of importance for future studies on adipose tissue function (Fig. 7). However, it is important to mention a number of factors that may influence the accuracy of this lipolysis model: 1) only GPCRs that are expressed by adipocytes may directly influence adipocyte lipolysis; 2) GPCRs that are not traditionally known to couple via the stimulation or inhibition of cAMP may also be involved in the regulation of lipolysis, but are not accounted for in the cAMP based lipolysis model; 3) GPCRs may couple to different second messenger pathways in different tissues, resulting in the possibility that not all GPCRs that couple via cAMP are accounted for in this model. Nevertheless, this lipolysis model provides a useful summary of novel adipose tissue GPCRs that are potential novel regulators of lipolysis and such predictions may be experimentally validated. All GPCRs expressed in the ABD and GLUT subcutaneous adipose tissue biopsies were grouped according to the IUPHAR classification system into 73 subfamilies based on their ligand usage. A small number of GPCRs have not yet been assigned to any IUPHAR subfamily and, where possible, these GPCRs were added to existing subfamilies based on their ligand usage. The 159 GPCRs known to be present in SAT (including the four adipose tissue GPCRs (HCAR1-3 and P2RY11) and the four GPCRs with unknown expression in SAT (GPR110, GPR137, NPSR1, UTS2R) that we could not quantify due to a lack of suitable primers) were allocated to 51 of the 73 GPCR subfamilies, and their expression and a summary of their effects on adipose tissue insulin resistance, lipolysis, adiponectin and leptin secretion, and their status as drug targets is outlined in Section 3 (Figs. 8 and 3). Section 4 describes the drugs that interact with GPCRs expressed in ABD and GLUT adipose tissue biopsies, and how these drugs are predicted to affect lipolysis (Fig. 7). The predictions are based on what is known about how the GPCRs in question are known to regulate lipolysis and on the reported stimulatory or inhibitory effects drugs exert on their target GPCRs. Although there are currently no approved drugs targeting GPCRs that have the specific purpose of influencing adipose tissue lipolysis, drugs designed for other purposes may nevertheless interact with adipose tissue GPCRs and thereby influence lipid metabolism, which may in turn have consequences for cardiovascular and metabolic health. 1.6. Regulation of leptin and adiponectin secretion from white adipose tissue by G-protein coupled receptors Adipose tissue is an endocrine organ that secretes a large number of proteins (Lehr et al., 2012) including the peptide hormones leptin (E. D. Green et al., 1995; Zhang et al., 1994) and adiponectin (Maeda et al., 1996), both of which are important regulators of energy homeostasis. Dysregulation of these peptide hormones is associated with cardiovascular disease and metabolic disorders, such as obesity and type 2 diabetes (Considine et al., 1996; Stefan et al., 2003; Hopkins et al., 2007; Koh et al., 2008; Renaldi et al., 2009). Mouse adipocytes also secrete the hormone resistin, but in man, resistin is secreted primarily from the endothelium and from immune cells and not from adipocytes (Fain et al., 2003; Patel et al., 2003; Utzschneider et al., 2005). Adiponectin is secreted from the placenta (Chen et al., 2006) as well as from adipose tissue (Scherer et al., 1995). It self-assembles into trihexa- or dodecamers, and high-molecular weight adiponectin (HMWA) is associated with a reduced risk of developing T2D (Zhu et al., 2010). Adiponectin is abundant in the circulation, comprising 0.01% of the total plasma protein content (Arita et al., 1999), and its serum levels are inversely correlated with total body fat mass in adults (Peake et al., 2005). Raised plasma adiponectin concentrations are associated with protection against metabolic and cardiovascular disease (Hug & Lodish, 2005; Lara-Castro et al., 2007), but the relationship with cardiovascular disease is complex, possibly as a consequence of differing clinical definitions between studies. In addition, a U-shaped risk relationship with increased risk for the top quintile adiponectin concentrations has also been described (Kizer et al., 2013) demonstrating that

the beneficial effects of adiponectin occur within a relatively narrow concentration range. Besides its glucose-regulating effects (Diez & Iglesias, 2003), adiponectin exerts weight-reducing effects via adiponectin receptors expressed in the brain, and it may have synergistic effects on weight reduction with leptin. Adiponectin secretion from WAT is stimulated by insulin, which acts via its heterotetrameric tyrosine kinase receptor on adipocytes, and ligands that activate Gs-coupled GPCRs may also increase adiponectin release through elevations in adipocyte cAMP (Szkudelski et al., 2011). Surprisingly, an extensive literature search revealed that only five (A1 (ADORA1), apelin (APLNR), ETA (EDNRA), GPR116 and TSH (TSHR) receptors) of the 163 GPCRs present in SAT have been experimentally confirmed to stimulate adiponectin secretion and an additional seven GPCRs (such as the HCA2 (HCAR2, also known as the nicotinic acid receptor, GPR109a) and FFA4 (GPR120)) have inferred stimulatory effects on adiponectin release, based on experimental outcomes using nonselective agonists and antagonists so the exact identity of the receptor subtypes mediating the effects is not known. A further six GPCRs (such as the abundant AT1 (AGTR1), sst2 (SSTR2), and CGRP (CALCRL + RAMP1) receptors) have been experimentally confirmed to inhibit adiponectin secretion, and eight other SAT GPCRs (such as the abundant α2A (ADRA2A) and CCRL1 (also known as ACKR4) receptors), have inferred inhibitory effects. Seven GPCRs are reported to both have inferred stimulatory and inhibitory effects (see Section 3.39 below), seven additional GPCRs have no effect and 123 GPCRs (75.5%) have unknown effects on adiponectin secretion from WAT (Figs. 2c and 4, see Section 3 below for references). Leptin is a 16 kDa protein hormone that is primarily secreted from WAT (E. D. Green et al., 1995), and the level of circulating leptin is proportional to the total amount of body fat (Considine et al., 1996). A major function of leptin is to regulate appetite and metabolism through leptin receptors located in the hypothalamus (Baicy et al., 2007). Leptin receptors expressed elsewhere in the body mediate a plethora of non-metabolic functions, including regulation of the immune system, (Taleb et al., 2007), angiogenesis (Park et al., 2001), fetal lung function (Torday & Rehan, 2006), bone mass (Ducy et al., 2000) and a number of extra-hypothalamic neurological functions (Lieb et al., 2009). In mice and humans, absence of leptin signalling caused either by inactivating mutations of the leptin gene (ob/ob) or the leptin receptor (db/db) leads to severe obesity due to uncontrolled eating (Ingalls et al., 1950; Gibson et al., 2004). The release of leptin from WAT is tightly regulated by circulating hormones and other signalling molecules. One example is insulin, which stimulates leptin secretion via activation of the insulin receptor on WAT cells (Barr et al., 1997). A large number of other signalling molecules and hormones also participate in this regulation via interactions with GPCRs expressed in WAT. GPCRs that activate the Gs second messenger pathway, which leads to increased biosynthesis of cAMP, inhibit leptin secretion, whereas GPCRs that signal via Gi/G0, leading to inhibition of cAMP production, stimulate leptin secretion from adipose tissue. One of the most prominent stimuli to reduce leptin secretion is physical exercise, presumed to act through adrenaline acting via Gs-coupled beta-adrenergic receptors expressed in adipose tissue (Thompson et al., 2012). To date, nine adipose tissue GPCRs, including the abundant A1 (ADORA1), CGRP (CALCRL + RAMP1) and Y1 (NPY1R) stimulate leptin secretion. A further six GPCRs, including the highly expressed AT1 (AGTR1) and FFA4 (GPR120) receptors have an inferred stimulatory role on leptin secretion. Eight GPCRs, including the abundant apelin (APLNR) and V1A (AVPR1A) receptors, are confirmed to inhibit leptin release, while a further 13 (including the highly expressed S1P1 (S1PR1) and S1P3 (S1PR3) have inferred inhibitory effects. Seven GPCRs are reported to have both inferred stimulatory and inhibitory effects (see Section 3.39 below). One receptor, the membrane bound estrogen receptor GPER, has been reported to either stimulate or to have no effect



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Fig. 3. Mean expression relative to PPIA of GPCRs expressed in subcutaneous adipose tissue grouped according to their known effects on adiponectin secretion. Stimulation: GPCRs reported to stimulate adiponectin secretion; Inhibition: GPCRs reported to inhibit adiponectin secretion; Unknown effect: effect on adiponectin secretion by GPCRs not known; No effect: GPCRs have no effect on adiponectin secretion; Unclear effect: different studies have reported conflicting effects on adiponectin secretion; * Receptor subtype mediating effect has not been identified.

on leptin secretion (see Section 3.16 below). Furthermore, eight GPCRs have no effect on leptin release, and the remaining 111 SAT GPCRs (68.1%) have unknown effects on leptin secretion (Figs. 2d and 5, see Section 3 below for references). 1.7. Regulation of lipolysis by G-protein coupled receptors The hydrolysis of stored triacylglycerol (TAG) in adipocytes results in the release of free fatty acids and glycerol into the bloodstream.

Complete TAG hydrolysis depends on the activity of three enzymes, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol lipase. The classical pathway regulating lipolysis is controlled by the second messenger cAMP, the biosynthesis of which is regulated by GPCRs signalling via the Gi and Gs pathways (Section 1.3). Other GPCR-regulated signalling pathways also control lipolysis, including the Gq-coupled PLC and PKC signalling pathway as well as signalling through cGMP and MAPK pathways (Chaves et al., 2011). We found that 11 GPCRs have been reported to stimulate

Fig. 4. Mean expression relative to PPIA of GPCRs expressed in subcutaneous adipose tissue grouped according to their known effects on leptin secretion. Stimulation: GPCRs reported to stimulate leptin secretion; Inhibition: GPCRs reported to inhibit leptin secretion; Unknown effect: effect on leptin secretion by GPCRs not known; No effect: receptors have no effect on leptin secretion; * Receptor subtype mediating effect on leptin secretion has not been identified; # Effect on lipolysis dependent on which RAMP protein is associated with the receptor.


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a) Regulation of lipolysis

b) Predicted effects on lipolysis by GPCRs with no known effects on lipolysis

Fig. 5. a. Mean expression relative to PPIA of GPCRs expressed in subcutaneous adipose tissue grouped according to their known effects on lipolysis. Stim.: GPCRs reported to promote lipolysis; Inhib.: GPCRs reported to inhibit lipolysis; Unknown: effect on lipolysis by GPCRs not known; No effect: receptors have no effect on lipolysis; * Receptor subtype mediating effect on lipolysis has not been identified; # Effect on lipolysis dependent on which RAMP protein is associated with the receptor. b. Mean expression relative to PPIA of the 101 GPCRs expressed in subcutaneous adipose tissue that have unknown effects on lipolysis. The GPCRs are grouped according to how they activate major GPCR second messenger signalling pathways that stimulate (Stim.) or inhibit (Inhib.) the production of cAMP or that mobilise intracellular Ca2+ (Mobil. Of Ca2+). GPCRs with unknown signalling pathways are clustered into an unknown signalling group, and receptors that signal via pathways other than cAMP and Ca2+ are clustered into a miscellaneous signalling (Misc. signalling) group. Increased production of intracellular cAMP stimulates lipolysis, and this well characterised pathway can be used to predict how GPCRs with known signalling via stimulation or inhibition of cAMP production are likely to either stimulate or inhibit cAMP mediated lipolysis. By applying this model to the 101 GPCRs with unknown effects on lipolysis, we identified 8 GPCRs that are likely to stimulate and 19 GPCRs that are likely to inhibit cAMP mediated lipolysis. GPCRs that do not signal via cAMP, such as GPCRs that signal exclusively via Ca2+, are unlikely to have any effect on cAMP mediated lipolysis. With this model, it is not possible to predict how GPCRs that signal via miscellaneous other pathways, or that signal via unknown pathways, influence cAMP mediated lipolysis. See Table 1 for details on all GPCRs included in this lipolysis prediction analysis.



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Fig. 6. Mean expression relative to PPIA of GPCRs expressed in subcutaneous adipose tissue grouped according to their known effects on adipose tissue insulin resistance. Stimulation: GPCRs reported to promote insulin resistance; Inhibition: GPCRs reported to inhibit insulin resistance; Unknown effect: effect on insulin resistance by GPCRs not known; Genetic association: population genetics or gene expression studies have reported an association between GPCRs and insulin resistance; No effect: receptor has been reported to have no effect on adipose tissue insulin resistance. * Receptor subtype mediating reported effect not determined. # Effect on insulin resistance is dependent on which RAMP protein is associated with the receptor. # # Ligands that activate GPCRs are synthesised by enzymes whose expression and/or activity is reported to have direct effects on adipose tissue insulin resistance. These effects on insulin resistance are likely to be mediated at least partially via GPCRs expressed in adipose tissue.

lipolysis. Examples of receptors in this group include the well-known adrenergic α1A (ADRA1A) (Flechtner-Mors et al., 2002), β1 (ADRB1) and β2 (ADRB2) (Lafontan, 1994) receptors, as well as abundant, but less well known adipose tissue receptors such as the endothelin receptors ET-A (EDNRA) and ET-B (EDNRB). A further 16 receptors have inferred stimulatory effects on lipolysis in SAT, including the very

abundant H1 (HRH1), S1P1 (S1PR1), and S1P3 (S1PR3) receptors. The GPCR CALCRL, which is inactive on its own, both stimulates and inhibits lipolysis as it interacts with different RAMP proteins: lipolysis is inhibited by the peptide adrenomedullin (Harmancey et al., 2005), which is an agonist of the AM1 (CALCRL + RAMP2) AM2 (CALCRL + RAMP3) and CGRP (CALCRL + RAMP1) receptors and stimulated

Fig. 7. Model predicting how drugs targeting GPCRs that are expressed on SAT are likely to influence lipolysis under the control of the cAMP pathway. Predicted effects on lipolysis are modelled using data on known effects of GPCRs on lipolysis (see Section 3) in combination with known agonist, antagonist or inverse agonist effects of GPCR targeting drugs. 119 drugs, acting on 23 GPCRs, are predicted to stimulate and 173 drugs, acting on 25 GPCRs, are predicted to inhibit lipolysis. Mean SAT GPCR mRNA expression from 6 donors is presented relative the adipose tissue reference gene PPIA. GPCRs that are the targets of both agonist and antagonist/inverse agonist drugs may appear in both the ‘Stimulation’ and ‘Inhibition’ of lipolysis categories due to opposite effects on GPCR signalling by receptor agonists and antagonists/inverse agonists. Pred. stim: predicted stimulatory effects by drugs on lipolysis; Pred. inhib: predicted inhibitory effects by drugs on lipolysis.


14


Insulin resistance

a)

Lipolysis

Leptin

Adiponectin

b)

Inf.

c)

Inf.

Inf.

d)

Stimulation Inhibition

Inf. Inf.

57.1%

Unclear effect

Inf.

Inf.

62.6%

Inf.

75.5%

Genetic association

68.1%

No effect Unknown effect Inf.

Inferred effect

Fig. 8. Summary of the known effects of all GPCRs expressed in abdominal and gluteofemoral adipose tissue on adipose tissue insulin resistance (a), lipolysis (b) and the secretion of adiponectin (c) and leptin (d). Inf.: inferred effects based on experiments using non-specific agonists/antagonists. A majority of all adipose tissue GPCRs (light grey areas) have unknown effects on all of the above mentioned adipose tissue functions.

by alpha-CGRP (Danaher et al., 2008), an agonist of the CGRP (CALCRL + RAMP1), AM1 (CALCRL + RAMP2) and AM2 (CALCRL + RAMP3). Eleven GPCRs, including the abundant α2A (ARDA2A), Y1 (NPY1R), V1A (AVPR1A), apelin (APLNR) and A1 (ADORA1) receptors, all inhibit lipolysis, and a further ten receptors, including the highly expressed C5a1 (C5AR1) receptor, have inferred inhibitory effects on lipolysis. Five GPCRs have no effect on lipolysis, and a further eight GPCRs have both inferred stimulatory and inhibitory effects (see Section 3.39 below). The remaining 102 GPCRs (62.6%) in SAT have unknown effects on lipolysis (Figs. 2b and 6a). By combining existing knowledge on GPCR second messenger signalling (Sharman & Mpamhanga, 2011) and the ABD and GLUT SAT biopsy GPCR expression profiles with what is known about how cAMP regulates lipolysis (Chaves et al., 2011), we have constructed an in silico model predicting how the 102 GPCRs with unknown effects on lipolysis are likely to influence lipolysis regulated by cAMP (Fig. 5b, Table 1). However, these predictions need to be verified experimentally, as some GPCRs may signal via pathways other than those assigned to each receptor in the IUPHAR database (Sharman & Mpamhanga, 2011) due to both tissue- and species-specific factors.

(Hoda Y. Heneina et al., 2011), most likely via the activation of the AM1 (CRLR + RAMP2) or the AM2 (CRLR + RAMP3) receptors. Ten GPCRs, including the highly expressed A1 (ADORA1), apelin (APLNR), FFAR4 (GPR120) and GPR116 have been experimentally proven to inhibit insulin resistance, and a further four GPCRs, including the abundant FPR3 and GABAB1 (GABBR1) receptors, have inferred inhibitory effects on insulin resistance. Eight GPCRs have both inferred stimulatory and inhibitory effects (see Section 3.39 below), and one GPCR, BDKRB2, has no effect on insulin resistance. Furthermore, a number of population genetics and gene expression studies genetics are suggesting that eleven additional GPCRs, including the abundant α2A (ADRA2A) receptor, are associated with adipose tissue insulin resistance, but the mechanism linking these associations with insulin resistance have not been elucidated (Figs. 2a and 7). The remaining 93 SAT GPCRs (57.1%) have unknown effects on adipose tissue insulin resistance. Importantly, GPCRs with strong, experimentally proven effects on insulin resistance, such as the orphan receptor GPRC5B (Kim et al., 2012), are promising drug targets for the development of novel therapies aimed at reducing the risk of both T2D and CVD, as insulin resistance is a major risk factor for both conditions.

1.8. Adipose tissue G-protein coupled receptors and insulin resistance A clinical consequence of obesity is insulin resistance, which in itself is a major risk factor for the development of type 2 diabetes and cardiovascular disease. Adipose tissue is a central organ in the pathogenesis of insulin resistance, as excessive accumulation of triacylglycerol in adipocytes can lead to a deterioration of the insulin responsiveness of other energy storing organs such as skeletal muscle and the liver. The role of adipose tissue in the development of global insulin resistance is not entirely clear and is likely to depend on inappropriately low or ineffective fat storage in obesity leading to lipid overflow to other organs, failing release of insulinsensitising molecules such as adiponectin or an excess release of pro-inflammatory factors in response to overfilled adipose stores. Although the adipocyte per se is often considered to be one of the most insulin sensitive cells in the body, enlarged adipocytes often show reduced responsiveness to insulin. A number of GPCRs have been found to influence the emergence of insulin resistance, either in genetic association studies or in mouse models where selected GPCRs have been deleted or pharmacologically inhibited. Interestingly, only 19 SAT GPCRs, including the highly expressed C5a1 (C5AR1), AT1 (AGTR1), Y1 (NPY1R), P2Y14 (P2RY14) and GPRC5B receptors have been experimentally shown to promote insulin resistance. A further 17 GPCRs, including the abundant ET-A (EDNRA), ET-B (EDNRB),LPA1 (LPAR1), S1P1 (S1PR1) and S1P3 (S1PR3) receptors, have inferred stimulatory effects on insulin secretion. The GPCR CALCRL, which is inactive on its own, both stimulates and inhibits insulin resistance depending on which RAMP it is associated with: insulin resistance is promoted by the peptides CGRP and amylin (Molina et al., 1990) most likely acting via activation of the CGRP receptor (CALCRL + RAMP1) and inhibited by adrenomedullin

2. Human abdominal and gluteofemoral subcutaneous adipose tissue G-protein coupled receptor ligand usage Endogenous GPCR ligands can be classified according to their molecular structures into four main groups: small organic molecules (nucleotides, free fatty acids, amino acids, etc.), peptides/proteins, monatomic ions (H+, Zn2+, Ca2+) and large biological macromolecules (e.g. glycosaminoglycans). GPCRs with unknown endogenous ligands are collectively known as orphan receptors, and we have identified 45 receptors (including bitter taste receptors) in adipose tissue as belonging to this category. Ligands for the remaining 114 non-orphan GPCRs expressed in the ABD and GLUT biopsies are described below. 2.1. Small organic molecule ligands of human subcutaneous adipose tissue G-protein coupled receptors We define molecules such as amino acids, eicosanoids, free fatty acids, lipids, nucleotides and steroids, but not peptides, proteins or large biological macromolecules such as glycosaminoglycans, as small organic molecule ligands. Among the human adipose tissue GPCRs that respond to small organic molecule ligands, 59 receptors (37.1%) are activated by 67 different small organic molecule ligands, and 22 of these molecules are the designated ligands of two or more different GPCRs (Table 2). Of the 67 small organic molecules known to activate GPCRs expressed in adipose tissue, 35 (52.2%) have been reported as being present in adipose tissue. The presence in adipose tissue of an additional five (7.5%) small organic molecule ligands is inferred based on the expression in adipose tissue of the enzymes required for the synthesis of these ligands. Furthermore, two ligands (3%) are confirmed to reach adipose tissue via innervation and the circulation, two (3%) are



15

Table 1 Predicted effects on lipolysis by adipose tissue GPCRs with unknown effects on lipolysis. GPCR expression presented as mean expression relative to PPIA in abdominal (Abd.) and gluteofemoral (Glut.) adipose tissue biopsies. Effects on lipolysis are predicted based on known couplings of GPCRs to major GPCR second messenger signalling pathways (stimulation or inhibition of cAMP production and mobilisation of Ca2+) and the documented stimulatory effect of cAMP on lipolysis. ↑cAMP: stimulation of cAMP production; ↓cAMP: inhibition of cAMP production; ↑Ca2+: mobilisation of intracellular calcium; ?: unknown second messenger signalling; Misc.: activation of miscellaneous GPCR signalling pathways other than cAMP or Ca2+, including Wnt, small G-proteins etc. Predicted effects on lipolysis: ↑: stimulation of lipolysis; ↓: inhibition of lipolysis; ?: unknown effect on lipolysis. NQ: Expression not quantified. Gene symbol

Receptor name

ABD.

GLUT.

GPCR sign. Pathway

Pred. effect on lipolysis

ELTD1 GPRC5B LPHN1 GPR124 GPR21 TAS2R45 GPR146 GPR116 TAS2R19 FPR3 LPHN2 GPR125 TAS2R14 GPR34 GPR52 GPR135 TAS2R7 CD97 GPR160 TAS2R46 GPR126 GPRC5C TAS2R3 TAS2R43 EMR2 TAS2R31 OPN3 P2RY8 TAS2R20 CELSR1 BAI2 CELSR2 CXCR6 EMR3 GPR114 GPR141 GPR148 GPR174 GPR45 GPR82 GPR85 LPHN3 TAS2R10 TAS2R13 TAS2R39 TAS2R5 GPR110 GPR137 GPR133 GPR65 GPBAR1 PTAFR GRM5 GPR4 PROKR1 NPSR1 GPR37 ADRA2C LPAR1 P2RY14 GABBR1 GPR183 P2RY13 HTR1F LPAR2 CXCR2 CXCR4 CCR1 CX3CR1 GPR75

ELTD1 GPRC5B LPHN1 GPR124 GPR21 TAS2R45 GPR146 GPR116 TAS2R19 FPR3 LPHN2 GPR125 TAS2R14 GPR34 GPR52 GPR135 TAS2R7 CD97 GPR160 TAS2R46 GPR126 GPRC5C TAS2R3 TAS2R43 EMR2 TAS2R31 OPN3 P2RY8 TAS2R20 CELSR1 BAI2 CELSR2 CXCR6 EMR3 GPR114 GPR141 GPR148 GPR174 GPR45 GPR82 GPR85 LPHN3 TAS2R10 TAS2R13 TAS2R39 TAS2R5 GPR110 GPR137 GPR133 GPR65 GPBA PAF mGlu5 GPR4 PKR1 NPS GPR37 α2C LPA1 P2Y14 GABAB1 GPR183 P2Y13 5-HT1F LPA2 CXCR2 CXCR4 CCR1 CX3CR1 GPR75

0.595 0.196 0.154 0.141 0.073 0.065 0.054 0.045 0.030 0.021 0.019 0.019 0.018 0.016 0.013 0.012 0.011 0.011 0.011 0.010 0.0099 0.0083 0.0073 0.0065 0.0052 0.0052 0.0017 0.0005 0.0005 0.0002 trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace NQ NQ 0.120 0.052 0.026 0.009 trace trace trace NQ 0.035 0.030 0.025 0.015 0.013 0.011 0.0068 0.0052 0.0032 0.0021 0.010 0.0083 0.0049 0.0039

0.662 0.196 0.151 0.081 0.124 0.071 0.039 0.032 0.051 0.018 0.013 0.017 0.029 0.019 0.018 0.018 0.007 0.010 0.009 0.015 0.010 0.012 0.0069 0.0074 0.0052 0.0083 0.0019 0.0008 0.0005 0.0002 trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace trace NQ NQ 0.128 0.072 0.021 0.012 Trace Trace Trace NQ 0.050 0.027 0.016 0.020 0.012 0.0088 0.011 0.0065 0.0028 0.0022 0.012 0.0065 0.0084 0.0047

? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ↑cAMP ↑cAMP ↑cAMP ↑cAMP↑Ca2+ ↑cAMP↑Ca2+ ↑cAMP↑Ca2+ ↑cAMP↑Ca2+ ↑cAMP↑Ca2+ 2 ↓cAMP ↓cAMP↑Ca2+ ↓cAMP↑Ca2+ ↓cAMP↑Ca2+ ↓cAMP↑Ca2+ ↓cAMP ↓cAMP ↓cAMP ↓cAMP↑Ca2+ ↓cAMP↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+

? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ? ? ? ? (continued on next page)


16


Table 1 (continued) Gene symbol

Receptor name

ABD.

GLUT.

GPCR sign. Pathway

Pred. effect on lipolysis

CCR7 CYSLTR2 P2RY2 CYSLTR1 CCR3 CXCR3 F2RL3 FPR2/ALX FZD1 GPR27 LPAR5 NMUR1 UTS2R FZD4 GPR56 CXCR7 FZD5 FZD6 LGR4 CCRL1 FZD7 DARC FZD8

CCR7 CysLT2 P2Y2 CysLT1 CCR3 CXCR3 PAR4 FPR2/ALX FZD1 GPR27 LPA5 NMU1 UT FZD4 GPR56 CXCR7 FZD5 FZD6 LGR4 CCRL1 FZD7 FY FZD8

0.0032 0.0021 0.0011 Trace Trace trace trace trace trace trace trace trace NQ 0.092 0.079 0.026 0.022 0.018 0.0102 0.0081 0.0062 trace trace

0.0037 0.0021 0.0017 trace trace Trace Trace Trace Trace Trace Trace Trace NQ 0.082 0.083 0.027 0.022 0.023 0.0070 0.013 0.0049 Trace Trace

↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ ↑Ca2+ Misc. Misc. Misc. Misc. Misc. Misc. Misc. Misc. Misc. Misc.

? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

confirmed absent, and the presence in adipose tissue of the remaining 23 small organic molecule GPCR ligands (34.3%) is currently unknown (Table 2). 2.2. Peptide and protein ligands of human subcutaneous adipose tissue Gprotein coupled receptors Of the 159 GPCR mRNAs known to be expressed in subcutaneous adipose tissue, 57 (35.8%) are activated by 112 different peptides or proteins, and 32 (28.6%) of these peptides or proteins are the designated ligands of two or more different GPCRs (Table 3). There is a large capacity for autocrine and paracrine signalling within adipose tissue, as 53 of the 112 peptide/protein ligands (47.3%) are reported to be present in adipose tissue. Four peptide/protein ligands are reported absent, and the expression in adipose tissue of the remaining 55 peptide/protein ligands (49.1%) is unknown. 2.3. Monatomic ion ligands of human subcutaneous adipose tissue Gprotein coupled receptors Human subcutaneous adipose tissue expresses mRNAs encoding two pH sensitive GPCRs (GPR4 and GPR65) that are activated by protons (H+), which are ubiquitously present in all living cells. 2.4. Biological macromolecule ligands of human subcutaneous adipose tissue G-protein coupled receptors Subcutaneous adipose tissue expresses mRNA encoding EMR2, a GPCR that is activated by the biological macromolecules dermatan sulphate and chondroitin sulphates, which are both present in adipose tissue (Koshiishi et al., 1999), suggesting the existence of autocrine or paracrine activation of this receptor. 3. Regulation of adipose tissue lipolysis, insulin resistance and secretion of adiponectin and leptin by G-protein coupled receptors expressed in human subcutaneous adipose tissue In the sub-sections below, the expression in ABD and GLUT subcutaneous tissue biopsies of the 159 adipose tissue GPCRs has been placed alphabetically into 51 different receptor subfamilies based on the IUPHAR classification system (Sharman & Mpamhanga, 2011). Bar

graphs of GPCRs belonging to an additional 22 subfamilies are not displayed in Fig. 2, as no mRNAs encoding the 52 GPCRs belonging to these subfamilies were detected in the examined adipose tissue biopsies. The remaining 169 absent GPCRs have been assigned to their respective GPCR families below, and the expression of four GPCRs (GPR110, GPR137, NPSR1, UTS2R) was not determined here due to a lack of suitable qPCR primers (indicated in Fig. 2), bringing the total number of GPCRs accounted for to the 384 known functional members (excluding pseudogenes, olfactory and vomeronasal receptors). A small number of recently deorphanised GPCRs have not yet been assigned to any subfamily by IUPHAR and, where possible, these receptors have been added to established subfamilies based on their ligand usage. Where no such match could be made, the receptors were placed into an ‘unclassified receptors’ subfamily. Orphan receptors, for which no ligands are known, are grouped into an ‘orphan receptors’ subfamily. Taste receptors, although mostly orphans, are grouped into a ‘taste receptors’ subfamily, separate from the other orphan receptors. In the sections below we have summarised all available published information on the effects of GPCR activation on the regulation of insulin resistance, lipolysis and the secretion of adiponectin and leptin. Where differences in nomenclature between gene and receptor names occur, the GPCR gene symbol is given in brackets after the receptor's pharmacological name. Since the majority of the functional data used to create this adipose tissue GPCR atlas is derived from experiments performed on rodents, rodent tissue or isolated rodent adipocytes, it is possible that some GPCRs may have different functions in man. Future experiments using human tissues and cells are needed to identify these species differences.

3.1. Adenosine receptors The A1 receptor (ADORA1) is the only adenosine receptor expressed in SAT. Activation of the Gi coupled A1 receptor by adenosine stimulates adiponectin (Szkudelski et al., 2011) and leptin (Rice et al., 2000) secretion and inhibits lipolysis (Johansson et al., 2007) and insulin resistance (Green, 1987; Dong et al., 2001; Dhalla et al., 2007; Dhalla et al., 2007). The A1 receptor is blocked by caffeine, which results in stimulation of lipolysis (Verdy, 1967). Interestingly, consumption of coffee has been associated with a reduced risk of developing T2D, but this effect is unlikely to be dependent on the inhibitory effects of caffeine on A1 adenosine receptors, as the same protective effect has also been observed in consumers of decaffeinated coffee (Natella & Scaccini, 2012).



17

Table 2 Small organic molecules (SOMs) GPCRs expressed in subcutaneous adipose tissue and the known presence in adipose tissue of the SOM ligands. 42 (62.7%) of the 67 SOM ligands known to interact with GPCRs expressed in SAT have previously been reported in SAT. *: ligand of more than one adipose tissue GPCR; # reaches adipose tissue via innervation and the circulation; ## converted into active ligand by enzymes present in adipose tissue. Gene symbol

Receptor name

Small organic molecule GPCR ligand

Ligand previously reported in adipose tissue

PTGDR2 LTB4R, LTB4R2 LTB4R2 LTB4R2 FPR2/ALX LTB4R2 GPER LTB4R, LTB4R2 CNR1 HCAR3 HTR1F, HTR2A, HTR2B P2RY12

DP2 BLT1, BLT2 BLT2 BLT2 FPR2/ALX BLT2 GPER BLT1, BLT2 CB1 HCA3 5-HT1F, 5-HT2A, 5-HT2B, P2Y12

? Yes Yes Yes Yes## Yes Yes ? Yes Yes## Yes ?

GPR183 FFAR3 CHRM3 ADORA1 P2RY12, P2RY13 ADRA1B, ADRA2A, ADRA2C, ADRB1, ADRB2, ADRB3 FFAR4 CNR1 FPR2/ALX P2RY2, P2RY11, P2RY12, P2RY13 FFAR3 GPBAR1 GPBAR1 P2RY12 GPBAR1 FFAR4 DRD1 GABBR1 HRH1, HRH2 FFAR3 LTB4R, LTB4R2 GRM5, GRM6, GRM7

GPR183 FFA3 M3 A1 P2Y12, P2Y13 α1B, α2A, α2C, β1, β2, β3 FFA4 CB1 FPR2/ALX P2Y2, P2Y11, P2Y12, P2Y13 FFA3 GPBA GPBA P2Y12 GPBA FFA4 D1 GABAB1 H1, H2 FFA3 BLT1, BLT2 mGlu5, mGlu6, mGlu7

11-dehydro-thromboxane B2 12R-HETE* 12S-HETE 12S-HPETE 15-deoxy-Lipoxin A4 15S-HETE 17β-estradiol 20-hydroxy-Leukotriene B4* 2-arachidonoylglycerol 3-hydroxyoctanoic acid 5-HT* 5-phosphoribosyl 1-pyrophosphate (PRPP) 7α, 25-dihydroxycholesterol Acetic acid Acetylcholine Adenosine ADP* Adrenaline* Alpha-linolenic acid Anandamide Aspirin triggered lipoxin A4 ATP* Butyric acid Chenodeoxycholic acid Cholic acid CysLTE4 Deoxycholic acid Docosahexaenoic acid Dopamine Gamma-aminobutyric acid Histamine* Isobutyric acid Leukotriene B4*

FFAR4 GPBAR1 HCAR1

FFA4 GPBA HCA1

CYSLTR1, CYSLTR2, FPR2/ALX CYSLTR1, CYSLTR2, FPR2/ALX CYSLTR1, CYSLTR2 FPR2/ALX LPAR1, LPAR2, LPAR5, P2RY10, S1PR1

CycLT1, CycLT2, FPR2/ALX CycLT1, CycLT2, FPR2/ALX CycLT1, CycLT2 FPR2/ALX LPA1, LPA2, LPA5, P2RY10, S1P1 GPR174, P2RY10 PAF FFA4 GPR18 α1A, α1B, α2A, α2C, β1, β2, β3

GPR174, P2RY10 PTAFR FFAR4 GPR18 ADRA1A, ADRA1B, ADRA2A, ADRA2C, ADRB1, ADRB2, ADRB3 FFAR4 SMO PTAFR FFAR4 FFAR3 PTGDR2, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, TBXA2R PTGDR2, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, TBXA2R PTGDR2, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, TBXA2R PTGDR2, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, TBXA2R PTGDR2 FFAR3 GPR32 CMKLR1 S1PR1, S1PR3, S1PR4, P2RY10 S1PR1, S1PR3, S1PR4 SUCNR1 TBXA2R

L-glutamic

? ? ? Yes Yes Yes# Yes Yes ? Yes ? ? ? Yes ? Yes Yes## Yes Yes ? Yes Yes

L-lactic

Yes ? Yes

acid* Linoleic acid Lithocholic acid

acid Leukotriene C4* Leukotriene D4* Leukotriene E4* Lipoxin A4 Lysophosphatidic acid*

Yes No ? ? Yes

Lysophosphatidylserine* mc-PAF myristic acid N-arachidonyl glycine Noradrenaline*

? ? Yes ? Yes

FFA4 SMO PAF FFA4 FFA3 DP2, EP2, EP3, EP4, FP, IP1, TP

Oleic acid Oxysterols Platelet activating factor (PAF) Palmitoleic acid Pentanoic acid Prostaglandin D2*

Yes Yes Yes Yes ? Yes##

DP2, EP2, EP3, EP4, FP, IP1, TP

Prostaglandin E2*

Yes


Prostaglandin F2α*

Yes


Prostaglandin I2*

Yes

DP2 FFA3 GPR32 Chemerin receptor S1P1, S1P3, S1P4, P2RY10 S1P1, S1P3, S1P4 Succinate receptor TP

Prostaglandin J2 Propanoic acid Resolvin D1 Resolvin E1 Sphingosine 1-phosphate* Sphingosyl phosphorylcholine* Succinate Thromboxane A2

Yes## ? Yes Yes No ? ? Yes (continued on next page)


18



Receptor name

Small organic molecule GPCR ligand


P2RY14 P2RY2, P2RY11 HCAR2

P2Y14 P2Y2, P2Y11 HCA2

UDP-glucose UTP* β-D-hydroxybutyric acid (nicotinic acid)

Yes ? Yes#

3.2. Adrenergic receptors The α2A adrenoreceptor (ADRA2A) is the most abundant adrenergic receptor found in SAT, followed by β2 (ADRB2), α1A (ADRA1A), α2C (ADRA2C), β1 (ADRB1) and trace expression of α1B (ADRA1B) and β3 (ADRB3). The α1D (ARDA1D) and α2B (ADRA2B) receptors were not detected in SAT. Adrenergic receptor agonists (adrenaline and noradrenaline) inhibit the secretion of adiponectin (Imai et al., 2006; Szkudelski et al., 2011), but it has not been established which receptor(s) are mediating this effect. Due to the overlapping ligand usage but opposing second messenger signalling generated from α and β adrenergic receptors, adrenergic stimulation has been reported to have different effects on lipolysis in ABD and GLUT subcutaneous adipose tissue (Manolopoulos et al., 2012). The β1, β2 and β3 receptors all inhibit leptin secretion (Scriba et al., 2000; Cammisotto & Bukowiecki, 2002) whereas noradrenaline has been reported to stimulate leptin secretion through activation of α1 adrenergic receptors (Ihara et al., 2006). Also, both adrenaline and noradrenaline stimulate lipolysis via activation of the α1A and β1, β2 and β3 receptors (Lafontan, 1994; Flechtner-Mors et al., 2002) and inhibit lipolysis via activation of the α2A receptor (Langberg et al., 2013). Based on the expression of the α1A, α1B and α1C receptors in SAT, it is most likely that experiments demonstrating a stimulation of lipolysis using α1 agonists (Flechtner-Mors et al., 2002) are mediated by the α1A receptor, but effects by the low abundance α1B receptor, although unlikely, cannot be excluded. Single nucleotide polymorphisms reported to affect the function of the human ADRA2A gene are associated with increased risk of T2D, but it is not known if these polymorphisms also influence the regulation of lipolysis by α2A (Talmud et al., 2011). Noradrenaline promotes insulin resistance, and this effect may be mediated by the α1A receptor (Andersson et al., 1994). Associations with insulin sensitivity have also been suggested for several other adrenergic receptors (ADRA1B (Grassi et al., 2011), ADRA2A (Talmud et al., 2011), ADRB1(Mottagui-Tabar et al., 2008), ADRB2 (Prior et al., 2011), ADRB3 (Huang et al., 2013)), but these proposed associations have not yet been experimentally validated. 3.3. Anaphylatoxin receptors Anaphylatoxin receptors are activated by the complement peptides C3a, C4a and C5a. In vivo experiments in rats have demonstrated that C3a and C5a inhibit lipolysis (Lim et al., 2013), but the receptor mediating this inhibition has not been identified. In human SAT, high levels of mRNA encoding the complement receptor C5a1 (C5AR1), as well as a moderate abundance of C3a (C3AR1) and a low abundance of C5a2 (GPR77) were detected, suggesting that the C5a1 and C3a receptors are most likely mediating inhibitory effects on lipolysis in man. Activation of C3a, C5a1 and C5a2 all promote adipose tissue inflammation and insulin resistance (Mamane et al., 2009; Gauvreau et al., 2013; Phieler et al., 2013; Roy et al., 2013). The effects of anaphylatoxin receptor activation on adiponectin and leptin secretion are unknown. 3.4. Angiotensin receptors The peptide hormone angiotensin exists as several isoforms, derived from proteolytic cleavage of the precursor protein angiotensinogen into

the peptides Ang I, Ang II, Ang III and Ang IV (also known as Ang 1–7). Human SAT expresses high levels of the AT1 receptor (AGTR1) but no AT2 (AGTR2). There are also trace levels of the two Ang IV (Ang 1–7) receptors MAS1 and MAS1L. Lipolysis is inhibited by Ang II acting via the AT1 receptor (Goossens et al., 2007) and stimulated by Ang IV (Oh et al., 2012; Passos-Silva et al., 2013) acting via an unknown Ang IV receptor. Activation of AT1 inhibits adiponectin secretion in the rat (Ran et al., 2006), and angiotensin II stimulates leptin secretion from 3T3-L1 cells and human adipocytes (Kim et al., 2002). Genetics as well as functional studies have demonstrated that the AT1 receptor promotes insulin resistance (Kouyama et al., 2005; Akasaka et al., 2006). The effects of MAS1 and MAS1L on insulin resistance are unknown. 3.5. Apelin receptor The peptide apelin exists in several different isoforms, and apelin is the natural ligand of the apelin receptor (APLNR), which is highly expressed in SAT. Activation of the apelin receptor stimulates adiponectin release (Higuchi et al., 2007) and inhibits leptin secretion (Yue et al., 2011), lipolysis (Than et al., 2012) and insulin resistance (Yue et al., 2010; Attane et al., 2011). 3.6. Bile acid receptor Human SAT expresses high levels of mRNA coding for the bile acid receptor GPBA (GPBAR1), also known as TGR5 (D. P. Kumar et al., 2012), which is activated by circulating bile acids. The effect of GPBA activation on SAT function is unknown. 3.7. Bradykinin receptors We detected trace levels of bradykinin B2 receptor mRNA (BDKRB2) and no expression of B1 (BDKRB1) in SAT. Bradykinin receptors are activated by kallidin and bradykinin peptides, and the B2 receptor has been reported to inhibit insulin resistance (Duka et al., 2001) and stimulate leptin secretion in mice (Mori et al., 2008). The BT receptor has no effect on lipolysis (Axelrod et al., 1985) and unknown effects on adiponectin secretion. 3.8. Calcitonin receptors The gene encoding the calcitonin receptor-like receptor CRLR (CALCRL) is expressed by SAT whereas CTR (CALCR) is absent. The CRLR receptor is non-functional on its own, but interacts with members of the membrane protein receptor activity-modifying proteins (RAMPs) to generate distinct receptors that are preferentially activated by the calcitonin gene-derived peptides adrenomedullin and CGRP. Thus, CRLR forms complexes with RAMPs to form the CGRP receptor (CRLR + RAMP1), the AM1 receptor (CRLR + RAMP2) and the AM2 receptor (CRLR + RAMP3). The CGRP receptor is activated primarily by the peptides alpha- and beta-CGRP, but also by the related peptides adrenomedulin, adrenomedullin 2, amylin and calcitonin. The AM1 and AM2 receptors are activated predominantly by adrenomedullin but also by alpha- and beta-CGRP, adrenomedullin 2, amylin and calcitonin. Activation of the CGRP receptor inhibits adiponectin and stimulates leptin secretion in the rat (Liao et al., 2013), whereas lipolysis is inhibited by the



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Table 3 Presence in adipose tissue of peptide and protein ligands activating GPCRs that are expressed in subcutaneous adipose tissue. 53 (48.2%) of the 110 GPCR peptide and protein ligands known to interact with GPCRs expressed in SAT have previously been reported in SAT. $: functional CGRP receptor formed by association with RAMP1; $$: functional AM1 receptor formed by association with RAMP2; $$$: functional AM2 receptor formed by association with RAMP3; *: ligand of more than one adipose tissue GPCR; #: expression in SAT induced by proinflammatory stimuli; ## isoform not specified; ### expression confirmed only in murine 3T3-L1 cells. Gene symbol

Receptor name

Peptide/protein ligand of adipose tissue GPCR


APLNR BDKRB2 BDKRB2 CALCRL C3AR1, C5AR1 GPR77 C3AR1, C5AR1, GPR77 AGTR1 AGTR1 AGTR1 AGTR1 FPR2/ALX APLNR APLNR APLNR AVPR1A BDKRB2 GPR77 F2RL3 CCR3 CCR1, CCR3 CCR1 CCR1, CCR3 CCR1 CCRL1, CCRL2, CCR7 CCR3 CCRL1, CCR7 CCR1 CCR3 CCRL1 CCR3 CCR3 CCR1 CCR1, CCR3, CCRL2, GPR75 CCR1, CCR3 CCR1, CCR3 CD97 CALCRL CMKLR1, CCRL2, GPR1 GPR56 SSTR2 SSTR2 CX3CR1 CXCR1, CXCR2 CXCR3 CXCR3, CXCR7 CXCR4, CXCR7 CXCR6 CXCR2 CXCR2 CXCR3 CXCR2 CXCR1, CXCR2 CXCR2 CXCR1, CXCR2 CXCR3 EDNRA, EDNRB EDNRA, EDNRB EDNRA, EDNRB LPHN3 FPR3 VIPR1, VIPR2 LHCGR BDKRB2 LHCGR BDKRB2 BDKRB2 DARC TACR1 TACR1 GPR37

Apelin receptor B2 B2 $$$ CGRP$, AM$$ 1 , AM2 C3a, C5a1 C5a2 C3a, C5a1, C5a2 AT1 AT1 AT1 AT1 FPR2/ALX Apelin receptor Apelin receptor Apelin receptor V1A B2 C5a2 PAR4 CCR3 CCR1, CCR3 CCR1 CCR1, CCR3 CCR1 CCRL1, CCRL2, CCR7 CCR3 CCRL1, CCR7 CCR1 CCR3 CCRL1 CCR3 CCR3 CCR1 CCR1, CCR3, CCRL2, GPR75 CCR1, CCR3 CCR1, CCR3 CD97 $$$ CGRP$, AM$$ 1 , AM2 Chemerin receptor, CCRL2, GPR1 GPR56 sst2 sst2 CX3CR1 CXCR1, CXCR2 CXCR3 CXCR3, CXCR7 CXCR4, CXCR7 CXCR6 CXCR2 CXCR2 CXCR3 CXCR2 CXCR1, CXCR2 CXCR2 CXCR1, CXCR2 CXCR3 ETA, ETB ETA, ETB ETA, ETB LPHN3 FPR3 VPAC1, VPAC2 LH B2 LH B2 B2 FY NK1 NK1 GPR37

(Pyr1)apelin-13 [des-Arg9]-bradykinin [Hyp3]-bradykinin Adrenomedullin Anaphylatoxin C3a* Anaphylatoxin C3a Anaphylatoxin C5a* Ang 1–7 Angiotensin I Angiotensin II Angiotensin III Annexin I Apelin-13 Apelin-17 Apelin-36 AVP Bradykinin C4a Cathepsin G CCL11 CCL13* CCL14 CCL15* CCL16 CCL19* CCL2 CCL21* CCL23 CCL24 CCL25 CCL26 CCL28 CCL3 CCL5* CCL7* CCL8* CD55 CGRP Chemerin* Collagen type III alpha-1 CST-14 CST-17 CX3CL1 CXCL1* CXCL10 CXCL11* CXCL12* CXCL16 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6* CXCL7 CXCL8* CXCL9 Endothelin 1* Endothelin 2* Endothelin 3* FLRT3 Humanin GHRH* hCG Kallidin LH Lys-[des-Arg9]-bradykinin Lys-[Hyp3]-bradykinin n.a. Neurokinin A Neurokinin B neuropeptide head activator

Yes## ? ? Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes## Yes## Yes## ? ? Yes Yes Yes Yes ? Yes Yes Yes Yes ? Yes Yes Yes Yes Yes Yes Yes Yes Yes ? Yes Yes Yes ? ? Yes Yes Yes Yes Yes Yes Yes ? Yes Yes Yes Yes ? Yes Yes ? ? Yes No ? ? ? ? ? ? n.a. ? ? ? (continued on next page)


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Receptor name

Peptide/protein ligand of adipose tissue GPCR


NPSR1 NMUR1 NMUR1 NMUR1 NPY1R, NPY5R, PPYR1 NPY1R, NPY5R, PPYR1 AVPR1A VIPR1, VIPR2 VIPR1, VIPR2 NPY1R, NPY5R, PPYR1 PROKR1 PROKR1 PROKR1 PTH1R, PTH2R PTH1R PTH1R, PTH2R LGR4 LGR4 LGR4 LGR4 VIPR1, VIPR2 FPR2/ALX SSTR2 SSTR2 TACR1 F2R, F2RL3 TRHR PTH2R BDKRB2 F2RL3 TSHR UTS2R UTS2R VIPR1, VIPR2 FZD4, FZD7, FZD8 FZD1 FZD1 FZD1, FZD6 FZD6 FZD1, FZD5, FZD6 FZD1

NPS NMU1 NMU1 NMU1 Y1, Y5, Y4 Y1, Y5, Y4 V1A VPAC1, VPAC2 VPAC1, VPAC2 Y1, Y5, Y4 PRK1 PRK1 PRK1 PTH1, PTH2 PTH1 PTH1, PTH2 LGR4 LGR4 LGR4 LGR4 VPAC1, VPAC2 FPR2/ALX sst2 sst2 NK1 PAR1, PAR4 TRH1 PTH2 B2 PAR4 TSH UT UT VPAC1, VPAC2 FZD4, FZD7, FZD8 FZD1 FZD1 FZD1, FZD6 FZD6 FZD1, FZD5, FZD6 FZD1

Neuropeptide S NMS NMU-23 NMU-25 NPY* NPYY* oxytocin PACAP-27* PACAP-38* PP* Prokineticin 1 Prokineticin 2 Prokineticin 2β PTH* PTHrP PTHrP 1–36* R-spondin 1 R-spondin 2 R-spondin 3 R-spondin 4 Secretin* Spinorphin SRIF-14 SRIF-28 Substance P Thrombin* Thyrotropin-releasing hormone TIP39 T-kinin Trypsin (serine proteases) TSH Urotensin II Urotensin-related peptide VIP* Wnt proteins* Wnt-1 Wnt-2 Wnt-3a* Wnt-4 Wnt-5a* Wnt-7b

? ? Yes Yes Yes ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? No# No# ? ? ? ? ? ? ? Yes ? No n.a. ? ? ? Yes### Yes ?

peptide adrenomedullin (Harmancey et al., 2005) and stimulated by alpha-CGRP (Danaher et al., 2008). Insulin resistance is promoted by the peptides CGRP and amylin (Molina et al., 1990) and inhibited by adrenomedullin (Hoda Y. Heneina et al., 2011). 3.9. Cannabinoid receptors The cannabinoid receptor CB1 (CNR1) is expressed in SAT, whereas the cannabinoid receptors CB2 (CNR2), GPR55 and GPR119 are all absent. The anandamide analogue N-palmitoylethanolamine, which is the most abundant cannabimimetic compound produced by human adipocytes, has no effect on adiponectin and leptin secretion (Gonthier et al., 2007), and the CB1 antagonist rimonabant has no direct effect on lipolysis in the rat (Molhoj et al., 2010). In obese mice, an inverse agonist targeting the CB1 receptor reduces leptin hypersecretion and restores peripheral leptin sensitivity (Tam et al., 2012). There is also experimental evidence demonstrating that CB1 promotes insulin resistance in rodents (Aller et al., 2012; Tam et al., 2012; Y. Tang et al., 2012). Trace levels of the receptor GPR18, which has been proposed to be activated by is activated by Δ9-tetrahydrocannabinol (McHugh et al., 2012), were also detected in SAT (See Section 3.29 Narachidonyl glycine receptor below). 3.10. Chemerin receptors Chemerin is a 14 kDa protein encoded by the RARRES2 gene in adipocytes following proteolytic cleavage of prochemerin by serine proteases

(Zabel et al., 2005). Surprisingly, we found that the non-signalling chemerin receptor CCRL2, which binds chemerin without initiation of signal transduction (Zabel et al., 2008), is by far the most abundant chemerin receptor in gluteofemoral and abdominal SAT biopsies, whereas the two other chemerin receptors, ChemR23 (CMKLR1) and GPR1, are present only at trace levels. Chemerin has been implicated in adipocyte differentiation and function in both rodent and human adipose tissue (Bozaoglu et al., 2007; Goralski et al., 2007), and stimulates lipolysis in differentiated murine 3T3-L1 adipocytes (Roh et al., 2007), but the receptor mediating this effect has not been identified. Chemerin does not have any known effects on adiponectin and leptin secretion. Ablation of CMKLR1 has been found to reduce signs of adipose tissue inflammation, but this also reduced glucose-mediated insulin secretion, with a potentially diabetogenic effect in mice (Ernst et al., 2012), whereas the chemerin receptors GPR1 and CCRL2 have not been studied in this respect. 3.11. Chemokine receptors Human SAT expresses high levels of the mRNA encoding the CXC chemokine receptor CXCR7, as well as moderate levels of mRNAs encoding the chemokine receptors CXCR1, CXCR2, CXCR4, CX3CR1, GPR75, CCR1 and CCR7 and trace levels of CXCR3, CXCR6 and CCR3. Chemokines are relatively small (8-10 KDa) secreted proteins whose main functions centre around the regulation of immunity and inflammation, processes implicated in both diabetes and CVD (Fantuzzi, 2005).



Surprisingly, the effects of chemokines on SAT function are largely unknown, despite the high levels of expression of several chemokine receptors in SAT. Chemokines are often produced by invading immune cells and may also have non-immune functions by providing signals to adipocytes. CCL3 stimulates leptin secretion from human adipocytes via an unknown receptor (Gerhardt et al., 2001). This effect is likely to be mediated by CCR1, as this is the only CCL3 receptor present in SAT. CCL2, which is the ligand of the chemokine receptors CCR2, CCR3 and CCR5 has no effect on leptin secretion (Gerhardt et al., 2001). The effects of the remaining chemokines on leptin secretion remain to be elucidated. Similarly, the only cytokine with a reported effect on lipolysis is chemerin, which is discussed above (see 3.10 Chemerin receptors), and surprisingly no known effects on adiponectin secretion have been reported for any chemokine receptor. The chemokine receptors CXCR2, CXCR3 and CXCR4 promote insulin resistance (Chavey et al., 2009; Deiuliis et al., 2013; Gupta et al., 2013) in both mouse and man (Huber et al., 2008), and activation of the CX3CR1 receptor has no effect on insulin resistance (Morris et al., 2012). The remaining chemokine receptors in subcutaneous adipose tissue (GPR75, CCR7, CXCR1, CXCR6, CXCR7) have not been studied in relation to insulin resistance. 3.12. Collagen receptor The mRNA encoding the collagen receptor GPR56, which interacts with type III, alpha-1 collagen and signals via activation of the RhoA pathway (Luo et al., 2011), is one of the most abundant GPCR mRNAs identified in human SAT. The role of GPR56 in the regulation of adipose tissue function is currently not known but the signalling from matrix to adipocytes is an emerging area (Divoux & Clement, 2011). 3.13. Dihydroxycholesterol receptor 7α,25-dihydroxycholesterol has recently been identified as an endogenous ligand for GPR183, also known as Epstein–Barr virusinduced molecule-2 (EBI2) (C. Liu et al., 2011). GPR183 signals constitutively via the Gi pathway (Rosenkilde et al., 2006), and plays an important role in the regulation of B lymphocyte migration. We detected intermediate levels of GPR183 mRNA in SAT, where it has unknown effects on SAT function. 3.14. Dopamine receptors We detected trace levels of the D1 dopamine receptor (DRD1) in SAT. Low expression of the D2, D4 and D5 dopamine receptors (DRD2, DRD4, DRD5) has also been reported in SAT (Borcherding et al., 2011), but we only detected mRNA encoding the D1. Administration of a D1/ D5 agonist stimulates adiponectin and inhibits leptin secretion from isolated human adipocytes in vitro (Borcherding et al., 2011), and dopamine stimulates lipolysis in vivo in man (Pernet et al., 1984). The effect of D1 receptor activation on adipose tissue insulin resistance is unknown. 3.15. Endothelin receptors Endothelin-1, -2 and -3 (ET-1, -2, -3) are vasoactive peptides primarily secreted from the vascular endothelium. Endothelin peptides are ligands of the endothelin receptors ETA (EDNRA) and ETB (EDNRB), which are both highly expressed in SAT. Activation of ETA stimulates lipolysis (Eriksson et al., 2009) and adiponectin secretion (Clarke et al., 2003) but has unknown effects on the secretion of leptin, whereas ETB stimulates lipolysis (Eriksson et al., 2009; Garciafigueroa et al., 2013) but its effects on adiponectin and leptin secretion are unknown. Chronic endothelin-1 administration promotes adipose tissue insulin resistance, but it is not known if this effect is mediated by the ETA or ETB receptor (Wilkes et al., 2003).

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3.16. Estrogen receptor The membrane estrogen receptor GPER (also known as GPR30), is expressed at intermediate levels in SAT. Estrogen, which is the natural ligand of both GPER and nuclear estrogen receptors, has no effect on adiponectin release (Babaei et al., 2010) and it is reported to have either stimulatory or no effect on leptin secretion (Shimizu et al., 1997; Nilsson et al., 2011), and inhibitory effects on lipolysis (Gormsen et al., 2012). However, it has not been established whether these effects of estrogen are mediated by GPER or by the nuclear estrogen receptors. Activation of GPER has inhibitory effects on adipose tissue insulin resistance (Sharma et al., 2013). 3.17. Formylpeptide receptors The formylpeptide receptor FPR3 is expressed at high levels in SAT. Formylpeptide receptors are activated by formylated peptides, but a range of other endogenous agonists has also been identified, including the peptides PACAP27 and β-amyloid 1–42 (Dufton & Perretti, 2010), both of which are ligands of the FPR2/ALX receptor. We detected trace levels of FPR2/ALX expression in SAT, whereas FPR1 was absent. The peptide humanin, a FPR3 agonist, reduces insulin resistance (Muzumdar et al., 2009), but the receptor mediating this effect has not been identified. The effects of the FPR3 receptor on lipolysis and on adiponectin and leptin secretion are unknown. The FPR2/ALX receptor is also a receptor for Resolvin D1 (RvD1) (see Section 3.42 below). As RvD1 exogenous RvD1 stimulates adiponectin secretion and marginally inhibits leptin secretion (Claria et al., 2012), and also reduces adipose tissue inflammation and insulin resistance (Hellmann et al., 2011) via an unknown mechanism, it is also possible that formylated peptides activating the FPR2/ALX receptor may have similar effects on SAT function, as FPR2/ALX is a receptor for both RvD1 and formylated peptide. 3.18. Free fatty acid receptors mRNA encoding the FFA4 receptor (FFAR4, also known as GPR120) is the most abundant free fatty acid receptor mRNA in SAT. Ablation of GPR120 indicates a role for circulating omega-3 poly-unstaurated fatty acids in protecting against global insulin resistance, most likely mediated by reduced adipose tissue inflammation (D. Y. Oh et al., 2010). It has also been reported that omega-3 polyunsaturated fatty acids stimulate adiponectin (Gonzalez-Periz et al., 2009) and leptin (Peyron-Caso et al., 2002) secretion and inhibit lipolysis (Lorente-Cebrian et al., 2012), but it has not been conclusively demonstrated that these effects are directly mediated by activation of GPR120. A second free fatty acid receptor, FFA3 (FFAR3, also known as GPR41), is expressed at low levels in SAT. FFA3 is activated by short chain free fatty acids (SCFA) such as acetic acid, propanoic acid,.isobutyric acid, butyric acid and pentanoic acid. FFA3 activation stimulates leptin secretion (Xiong et al., 2004) but has unknown effects on adiponectin secretion and insulin resistance. There is no reason to believe these very short chain fatty acids are particularly abundant in adipose tissue, but colonic fermentation of fibre increases production of circulating SCFAs (Wong et al., 2006) that may act at adipocyte FFA3. The FFA3 ligand butyric acid inhibits lipolysis (Ohira et al., 2013), but it is not known if this effect is mediated by FFA3. FFA2 (Gpr43) has been reported to be present in murine adipocytes, where it inhibits lipolysis (Ge et al., 2008), but we failed to detect FFAR2 (GPR43) expression in human SAT, which suggests that there are species differences in the expression of this receptor. 3.19. Frizzled receptors Human SAT expresses high levels of mRNAs coding for the frizzled receptors FZD4, intermediate levels of FZD5, FZD6 and FZD7, and trace levels of FZD1, FZD8 and SMO. Frizzled receptors are activated by Wnt


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proteins and signal either via the classical β-catenin pathway or via complex β-catenin-independent signalling pathways, which involve both Gi and Ca2+-dependent signalling cascades (Slusarski et al., 1997; Schulte, 2010; Kilander et al., 2011). Gene expression studies in adipose tissue and skeletal muscle suggest an association between FZD1 receptor expression and insulin resistance in man (X. Yang et al., 2004). However, given the very low expression of FZD1 in human subcutaneous adipose tissue, it is possible that this effect is mainly mediated by FZD1 receptor activation in other tissues. The effects of the remaining FZD receptors on SAT function remain unknown. Secreted frizzled-related proteins (SFRPs) are soluble proteins that act as endogenous inhibitors of Wnt signalling. SFRP1, 2, 4 and 5 are expressed in human adipose tissue, and SFRP1, SFRP2 and SFRP4 are adipokines that are secreted from human adipose tissue (Ehrlund et al., 2013). SFRP1 increases adiponectin secretion and is downregulated in obesity, and SFRP4 is up-regulated in obesity and is associated with insulin resistance and reduced insulin secretion from pancreatic islets (Mahdi et al., 2012; Ehrlund et al., 2013). The frizzled receptor SMO is expressed at trace levels in SAT, and oxysterols, which are ligands of the SMO receptor, are known to stimulate lipolysis (Lau et al., 1995), but it is not known if this effect is mediated via the SMO receptor. The R-spondin receptor LGR4, which is present at intermediate levels in SAT and signals at least partially via the Wnt pathway, has unknown effects on SAT function. The R-spondin receptor LGR5 is not expressed in SAT.

3.20. GABAB receptors GABAB1 receptor (GABBR1) mRNA is highly expressed in SAT, but we did not detect any mRNA expression of the GABAB2 receptor (GABBR2). Gamma-aminobutyric acid (GABA), the ligand of GABA receptors, inhibits insulin resistance (Tian et al., 2011) and stimulates adiponectin secretion (Ohara et al., 2011). These effects of GABA may occur through the GABAB1 receptors that we have identified in SAT, but it is possible that GABA exerts its actions through the GABAA receptors, which are ligand-gated ion channels rather than GPCRs. GABAB1 has unknown effects on lipolysis and leptin secretion.

3.21. Glycoprotein hormone receptors Glycoprotein hormone receptors are heavily glycosylated GPCRs that are activated by glycoprotein hormones. Intermediate levels of the thyroid-stimulating hormone receptor (TSHR) mRNA and trace levels of the luteinizing hormone (LH) receptor (LHCGR) are present in SAT. The TSH receptor, which is activated by thyroid-stimulating hormone (TSH), stimulates lipolysis (Elgadi et al., 2010) as well as adiponectin and leptin secretion (Santini et al., 2010; Kumar et al., 2011). Experiments in TSHR null mice have demonstrated that the TSHR receptor promotes insulin resistance (T. Wang et al., 2013), and these data are supported by a genetic association study in man (Peeters et al., 2007). Activation of the LH receptor stimulates adiponectin release (Y. H. Liu et al., 2006), has no effect on lipolysis (Tell et al., 1977) and has unknown effects on leptin secretion and insulin resistance.

3.22. Glycosaminoglycan receptor The glycosaminoglycans dermatan sulphate and chondroitin sulphate signal through the receptor EGF-like module-containing mucinlike hormone receptor-like 2 (EMR2) to regulate cell attachment (Stacey et al., 2003). SAT expresses intermediate levels of EMR2, and its role in regulating adipose tissue function is unknown.

3.23. Histamine receptors SAT expresses high levels of mRNAs encoding the H1 (HRH1) and intermediate levels of mRNAs coding for the H2 (HRH2) histamine receptors. Experiments using Hrh1 and Hrh2 receptor KO mice have shown that the H1 receptor moderately reduces insulin resistance, whereas the H2 receptor has very strong inhibitory effects on insulin resistance (K. Y. Wang et al., 2010). Histamine has unknown effects on adiponectin and leptin secretion, and has been found to weakly stimulate lipolysis (Carpene et al., 2001), but the receptor mediating this effect has not been identified. 3.24. Hydroxycarboxylic acid and Krebs cycle intermediate receptors There are several studies reporting expression of the lactic acid receptor HCA1 (HCAR1, also known as GPR81), the nicotinic acid receptor HCA2 (HCAR2, also known as GPR109a) and the 3-hydroxyoctanoic acid receptor HCA3 (HCAR3, also known as GPR109b) in SAT (Soga et al., 2003; Tunaru et al., 2003; Cai et al., 2008). However, we were unable to quantify the expression of these receptors in SAT due to a lack of suitable qPCR primers. The succinic acid receptor SUCNR1 is also expressed in SAT, whereas the OXGR1 receptor is absent. Activation of HCA1, HCA3 and SUCNR1 have unknown effects on adiponectin and leptin secretion, and inhibit lipolysis (Ahmed et al., 2009; C. Liu et al., 2012; Regard et al., 2008). Nicotinic acid, acting on HCA2, inhibits lipolysis (Ren et al., 2009) and stimulates adiponectin and leptin secretion (Wang-Fisher et al., 2002; Westphal et al., 2007; Linke et al., 2009; Digby et al., 2010). Nicotinic acid has also been shown to promote insulin resistance (Andersson et al., 1994), but it has not been experimentally proven that these effects of nicotinic acid are exclusively attributed to activation of HCA2. Activation of the 3-hydroxyoctanoic acid receptor HCA3 inhibits insulin resistance (Jeninga et al., 2009), whereas the effects on insulin resistance by the HCA1 and SUCNR1 receptors are unknown. 3.25. Leukotriene receptors Leukotrienes are eicosanoid inflammatory mediators, produced mainly by dedicated immune system cells, but also by cells that make up subcutaneous adipose tissue (Claria et al., 2013). Intermediate levels of the leukotriene receptor CysLT2 (CYSLTR2) and trace levels of CysLT1 (CYSLTR1), FPR2/ALX, BLT1 (LTB4R) and BLT2 (LTB4R2) are present in SAT. The effects of activation of leukotriene receptors on lipolysis and the secretion of adiponectin and leptin have not been identified. The BLT1 receptor promotes insulin resistance in mice (Spite et al., 2011), whereas the effects of the remaining leukotriene receptors on insulin resistance are unknown. 3.26. Lysophospholipid receptors Lysophospholipids, the natural ligands of the LPA1–6 receptors, are phospholipids that are missing one of their two O-acyl chains, and include the extracellular signalling molecules lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). High levels of LPA1 (LPAR1) were detected in SAT, as well as intermediate levels of LPA2 (LPAR2) and trace levels of LPA5 (LPAR5). SAT also expresses high levels of the S1P receptors S1P1 (S1PR1) and S1P3 (S1PR3), as well as trace amounts of S1P4 (S1PR4) and the novel lysophospholipid receptors GPR174, which is activated by lysophosphatidylserine, and P2RY10, which is activated by S1P, LPA and lysophosphatidylserine. S1P promotes insulin resistance (Wang et al., 2014), and has also been reported to stimulate lipolysis and inhibit leptin secretion (Jun et al., 2006), but the receptors mediating these effects have not been identified. The effects of S1P on adiponectin secretion are unknown. LPA has no effect on leptin secretion (Jun et al., 2006), unknown effects on adiponectin secretion and lipolysis, and it promotes insulin resistance (Rancoule et al., 2014) through interactions with unidentified



LPA receptor subtypes. The lysophosphatidylserine receptor GPR174 has unknown effects on SAT function. 3.27. Metabotropic glutamate receptors We identified mRNAs encoding trace levels of the metabotropic glutamate receptors mGlu5, mGlu6 and mGlu7 (GRM5, GRM6 and GRM7) in human SAT. Activation of metabotropic glutamate receptors have unknown effects on SAT function.

23

receptor by NPY and PYY inhibits lipolysis and stimulates leptin secretion (Serradeil-Le Gal et al., 2000), but has no effect on adiponectin secretion in man (Kos et al., 2007) . The roles of Y4 and Y5 receptors in the regulation of lipolysis and the secretion of leptin and adiponectin are unknown. A common genetic variant in the Y5 receptor has been associated with insulin resistance (Coletta et al., 2007), but this has not been confirmed in statistically more powerful genome-wide association studies. 3.33. Non-signalling receptors

3.28. Muscarinic acetylcholine receptors Human SAT expresses low levels of mRNA encoding the M3 (CHRM3) muscarinic cholinergic receptor. We did not detect any expression of M1 (CHRM1), M2 (CHRM2), M4 (CHRM4) and M5 (CHRM5) transcripts in SAT. The neurotransmitter acetylcholine has been shown to inhibit lipolysis (T. T. Yang et al., 2009) and stimulate leptin secretion (Yamada et al., 2001) via activation of the M3 receptor. It is currently unknown whether M3 receptor activation regulates adiponectin secretion or insulin resistance. 3.29. N-arachidonyl glycine receptor We detected trace levels of mRNA encoding receptor GPR18 in SAT. GPR18 has been proposed to be activated by N-arachidonyl glycine and Δ9-tetrahydrocannabinol (McHugh et al., 2012). Δ9tetrahydrocannabinol inhibits lipolysis in 3T3-L1 cells (Teixeira et al., 2010), but it is not known if this effect is mediated by GPR18. The roles of GPR18 activation in the regulation of insulin resistance and in the secretion of adiponectin and leptin are unknown. 3.30. Neuromedin U receptors SAT expresses trace levels of mRNA encoding the neuromedin U receptor NMU1 (NMUR1), whereas NMU2 (NMUR2) is absent. There are no reported studies on the effects of neuromedin U on SAT function. 3.31. Neuropeptide AF/FF, B/W, S and HA receptors Human SAT expresses high levels of GPR37, which is a high-affinity receptor of the neuropeptide head activator (HA), which was first isolated from hydra (Rezgaoui et al., 2006). A peptide identical to HA has also been isolated from human hypothalamus, bovine hypothalamus and rat intestine (Bodenmuller & Schaller, 1981), but no precursor gene encoding this peptide has so far been found in any sequenced genome. It is not known how GPR37 regulates SAT function. We failed to detect any expression of receptors activated by the neuropeptides B/W (NPBWR1, NPBWR2) and AF/FF (NPFFR1, NPFFR2) and we were unable to quantify expression of the neuropeptide S receptor (NPSR1) due to a lack of specific primers. 3.32. Neuropeptide Y, YY, PP and Y-like receptors Neuropeptide Y (NPY) exerts diverse metabolic and cardiovascular functions (Angelone et al., 2011; Parker & Bloom, 2012) through acting as an agonist at the Y1 (NPY1R), Y2 (NPY2R), Y4 (PPYR1), and Y5 (NPY5R) family of receptors. NPY is also a low-affinity agonist at GPR83 (Sah et al., 2007) and an antagonist of the PRRP receptor (Lagerstrom et al., 2005). Other NPY receptor ligands are peptide YY (PYY), which is an agonist at Y1, Y2, Y4, Y5 and GPR83 (Sah et al., 2007), and pancreatic polypeptide (PP), which is a potent agonist of the Y4 receptor, with lower agonistic effects at Y1, Y2 and Y5 receptors. High levels of Y1 receptor mRNA were detected in SAT, as well as trace mRNA expression of the Y5 (NPY5R) and Y4 (PPYR1) receptors, whereas Y2 (NPYR2) and GPR83 were absent. Activation of the Y1

SAT expresses intermediate levels of the non-signalling receptors CCRL1 and CCRL2 as well as trace levels of the FY receptor (DARC). CCRL1, CCRL2 and FY bind to a large number of cytokines without initiating any intracellular signalling (Mantovani et al., 2006). Nevertheless, it is possible that these receptors influence adipose tissue function by accumulation (through binding and re-release of ligands to the local microenvironment) or depletion (though high-affinity binding) of ligands of many chemokine receptors in the local microenvironment, which may influence the ability of chemokines to interact with other receptors in SAT. CCRL1, CCRL2 and FY have unknown effects on SAT function, but the chemokine CCL2, which is a ligand of CCRL1, is reported to inhibit adiponectin secretion through an unidentified CCL2 receptor and to have no effect on leptin secretion (Gerhardt et al., 2001). In a similar fashion, the CCRL2 ligand chemerin has been found to stimulate lipolysis in differentiated murine 3T3-L1 adipocytes (Roh et al., 2007) through an unknown receptor. CCRL1 has unknown effects on lipolysis and insulin resistance. 3.34. Nucleotide receptors SAT expresses high levels of the UDP-glucose receptor P2Y14 (P2RY14) as well as intermediate levels of the ADP receptor P2Y13 (P2RY13), low levels of the ATP and UTP receptor P2Y4 (P2RY4) and trace levels of the ADP receptor P2Y12 (P2RY12). The ATP and UTP receptor P2Y11 (P2RY11), which we were unable to quantify due to the lack of suitable primers, stimulates lipolysis (Lee et al., 2005). ATP and UTP have also been reported to inhibit leptin secretion (Lee et al., 2005), but the receptor mediating this effect has not been identified. The effects on insulin resistance, lipolysis and on adiponectin and leptin secretion by the remaining nucleotide receptors remain largely unknown, although it has been reported that P2Y14 promotes insulin resistance (Xu et al., 2012). 3.35. Orphan receptors Orphan GPCRs are receptors for which no endogenous ligands are known. Human SAT expresses high levels of mRNAs encoding the orphan GPCRs ELTD1, GPR21, GPR34, GPR52, GPR116, GPR124, GPR133, GPR146, GPRC5B and LPHN1, intermediate levels of GPR125, GPR126, GPR135, GPR160, GPRC5C and LPHN2, and low levels of 14 other orphan receptors. SAT also expresses a number of orphan taste receptors, discussed below (see Section 3.46). Due to a lack of pharmacological tools, the effects on SAT function of orphan receptors are largely unknown, with the exception of GPR21, GPR116 and GPRC5B. Adipose tissue-specific Gpr116 knockout mice present with insulin resistance and reduced levels of adiponectin, suggesting that GPR116, which signals via mobilisation of Ca2+ (X. Tang et al., 2013), is involved in both insulin receptor signalling and adiponectin secretion (Nie et al., 2012). The gene encoding the orphan receptor GPRC5B, which has been proposed to be activated by L-glutamate (Soni, Amisten et al., 2013), contains an intronic single nucleotide polymorphism that is associated with body mass index in man (Speliotes et al., 2010), and Gprc5bdeficient mice are protected from insulin-resistance and diet-induced obesity due to reduced adipose tissue inflammation (Y. J. Kim et al., 2012). Another orphan receptor, GPR21, promotes insulin resistance


24


via stimulation of the migration of pro-inflammatory macrophages into adipose tissue in both mouse and man (Gardner et al., 2012; Osborn et al., 2012). The remaining orphan receptors expressed in SAT have unknown effects on insulin resistance.

3.36. Parathyroid hormone receptors The parathyroid hormone (PTH) receptors PTH1 (PTH1R) and PTH2 (PTH2R) are expressed at trace levels in SAT. PTH1 and PTH2 are activated by the peptides TIP39 (tuberoinfundibular peptide of 39 residues), PTH, PTHrP (parathyroid hormone-related protein) and PTHrP(1–36) (Hoare & Usdin, 2001). PTH stimulates lipolysis (Taniguchi et al., 1987) in SAT, but the receptor subtypes mediating this effect have not been identified. In a small study of subjects undergoing curative parathyroid surgery for primary hyperparathyroidism, no change in serum leptin and adiponectin levels were seen, suggesting that PTH has no effect on the secretion of these hormones (Bhadada et al., 2011). There are some associations between Vitamin D, systemic PTH concentration and insulin resistance, (Alvarez et al., 2010; Frost et al., 2010), but it is not known which PTH receptor(s) are mediating this effect.

3.37. Platelet activating factor receptor The platelet activating factor receptor (PTAFR) is expressed at intermediate levels in human SAT. Its ligand, the phospholipid platelet activating factor (PAF), is synthesised mainly, but not exclusively, by immune system cells (Zimmerman et al., 2002). Activation of the PAF receptor promotes insulin resistance (Menezes-Garcia et al., 2014), but the effects of PAF on lipolysis and the secretion of adiponectin and leptin are unknown.

3.38. Prokineticin receptors Human SAT expresses trace levels of the mRNA encoding the PKR1 receptor (PROKR1), and activation of PKR1 has unknown effects on SAT function.

3.39. Prostanoid receptors Human SAT expresses high levels of transcripts encoding the EP3 receptor (PTGER3), as well as low levels of mRNAs encoding the receptors EP2 (PTGER2), EP4 (PTGER4), FP (PTGFR), IP1 (PTGIR) and trace levels of TP (TBXA2R) and D2 (PTGDR2, also known as GPR44). All of the above prostanoid receptors are activated by the prostaglandins PGD2, PGE2, PGF2α, PGI2 and thromboxane A2, whereas PGJ2 is an agonist of the DP1 and DP2 receptors. PGI2, likely derived from endothelial cells, stimulates lipolysis in isolated rat adipocytes, whereas PGE2 has a potent anti-lipolytic effect (Chatzipanteli et al., 1992). Based on the expression levels and pharmacological profiles of SAT prostanoid receptors, it is likely that the antilipolytic effects of PGE2 are mediated by the EP3 receptor, whereas the lipolytic effects of PGI2 are likely mediated by the IP1 receptor. PGE2 stimulates secretion of leptin from primary mouse adipocytes (Fain et al., 2000), and PGD2 and PGJ2 inhibit adiponectin and leptin secretion from differentiated murine 3T3-L1 adipocytes (Peeraully et al., 2006). PGI2 inhibits insulin resistance, whereas PGE2 promotes it (Hsieh et al., 2009; Sato et al., 2010; Inoue et al., 2012; Iyer et al., 2012). The specific receptor subtypes involved in the above processes have not been determined due to the extensive overlapping ligand preferences of the different prostanoid receptors expressed in SAT. Further experiments using more selective agonists and antagonists and/or knock-out mouse models are needed to elucidate the roles of individual prostaglandin receptors in the regulation of SAT function.

3.40. Protease activated receptors Trace amounts of mRNAs encoding the protease activated receptors PAR1 (F2R) and PAR4 (F2RL3), but not PAR2 (F2RL1) and PAR3 (F2RL2) were detected in SAT. The effects of PAR1 and PAR4 activation on lipolysis and the secretion of adiponectin and leptin are unknown. Thrombin, which activates PAR1 and PAR4, promotes insulin resistance (Mihara et al., 2010), but it is not known if this effect is mediated by PAR1 and PAR4 receptors expressed in SAT.

3.41. Proton receptors High levels of the proton receptor GPR65, as well as trace levels of the proton receptor GPR4, were detected in SAT. GPR4 and GPR65 have unknown effects on lipolysis, insulin resistance and adiponectin secretion. It is not yet known whether GPR65 plays a role in regulating leptin secretion, but it has been reported that serum leptin concentrations are increased in Gpr4 null mice (Giudici et al., 2013). Based on the very low expression of GPR4 in human SAT, further experiments are needed to clarify the role of GPR4 in leptin secretion in man.

3.42. Resolvin receptors Trace mRNA levels of the resolvin receptors CMKLR1, GPR32 and FPR2/ALX (also a formylpeptide receptor, see Section 3.17 above) were detected in SAT. Resolvins are anti-inflammatory lipids synthesised from the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Serhan et al., 2002). Resolvin D1 (RvD1), which is an agonist of the resolvin receptors FPR2/ALX and GPR32, is present at significantly reduced levels in adipose tissue from obese mice, and addition of exogenous RvD1 stimulates adiponectin secretion and marginally inhibits leptin secretion (Claria et al., 2012). It has also been reported that RvD1 reduces adipose tissue inflammation and insulin resistance (Hellmann et al., 2011), but there is no information of whether it regulates lipolysis. The receptors involved in these effects of RvD1 have not been identified. Resolvin E1 (RvE1) acts as an agonist at the receptor CMKLR1, which is also activated by the protein chemerin (see Section 3.10 above). Since chemerin stimulates lipolysis (Roh et al., 2007) and the CMKLR1 receptor promotes insulin resistance (Ernst et al., 2012), it is likely that RvE1 binding to the CMKLR1 receptor has a similar mode of action, but this remains to be tested experimentally.

3.43. Serotonin receptors The monoamine neurotransmitter serotonin is primarily found in the gastrointestinal tract, but it is also present in platelets and in the central nervous system (Berger et al., 2009). SAT expresses intermediate levels of mRNAs encoding the serotonin receptors 5-HT1F (HTR1F) and 5-HT2B (HTR2B) as well as trace levels of 5-HT2A (HTR2A). In vivo studies in rats using the serotonin reuptake inhibitor fluoxetine lowers serum leptin levels (Dryden et al., 1999), but the exact mechanism mediating this effect is poorly characterised. The 5-HT2B receptor stimulates lipolysis (Sumara et al., 2012), whereas the effects of 5-HT1F and 5-HT2A on lipolysis are unknown. 5-HT2A inhibits adiponectin secretion from 3T3-L1 adipocytes (Uchida-Kitajima et al., 2008), but there is currently no published information on the role of serotonin receptors in adiponectin secretion from native adipose tissue. Experiments in rats with the oral 5-HT2A and 5-HT2B receptor antagonist Terguride have demonstrated that the 5-HT2A and/or 5-HT2B receptors promotes insulin resistance, but it is not known which of the two receptors is mediating this effect (Golda et al., 2001). 5-HT1F has unknown effects on insulin resistance.



25

3.44. Somatostatin receptors

3.48. Unclassified receptors

The peptide hormone somatostatin is secreted by pancreatic islet δcells as well as by neuroendocrine neurons in the hypothalamus and by paracrine cells in the gastrointestinal tract (Florio & Schettini, 2001). In SAT, we detected low mRNA levels of one somatostatin receptor, sst2 (SSTR2). Somatostatin inhibits lipolysis (Endroczi et al., 1988) and decreases serum adiponectin and leptin concentrations in vivo in man (Donahoo et al., 1997; Faraj et al., 2008). The receptor(s) mediating these effects has not been identified, but it is likely that sst2 is responsible in SAT, as it appears to be the only somatostatin receptor expressed in this tissue. A population genetics study has also found an association between the SST2 gene and insulin resistance (Sutton et al., 2009), but it is not known if this effect is mediated by SAT sst2 receptors.

CD97,a receptor that is activated by a protein ligand, CD55 (Hamann et al., 1996), is expressed in SAT but it has unknown effects on adipose tissue function. 3.49. Urotensin receptor The cyclic peptide hormone urotensin II is predominantly expressed in the CNS and at lower levels also in the cardiovascular system, kidney and pituitary (Ames et al., 1999), and it is an agonist at the UT receptor (UTS2R). Due to the unavailability of suitable primers, we were unable to quantify the expression of UTS2R in SAT. UTS2R has unknown effects on lipolysis and the secretion of adiponectin and leptin, and a population genetic study has demonstrated an association between the urotensin receptor and insulin resistance (Saez et al., 2011).

3.45. Tachykinin receptors 3.50. Vasopressin and oxytoxin receptors Two tachykinin peptide genes encoding several tachykinin peptides of different isoforms are known in man. The TAC1 gene encodes the peptides neurokinin A, neuropeptide K, neuropeptide gamma and substance P, and the TAC3 gene encodes neurokinin B. SAT express trace levels of one tachykinin receptor, NK1 (TACR1), and the NK1 agonist substance P stimulates lipolysis (Miegueu et al., 2013) and leptin secretion and inhibits the secretion of adiponectin (Karagiannides et al., 2011; Karagiannides et al., 2011). Activation of the NK1 receptor also promotes insulin resistance (Karagiannides et al., 2011).

Human SAT expresses mRNA encoding the V1A receptor (AVPR1A), which is activated by the peptides vasopressin and oxytocin. Vasopressin inhibits lipolysis (Hiroyama et al., 2007), but it has unknown effects on adiponectin secretion and adipose tissue insulin resistance. V1A null mice exhibit elevated leptin serum levels, but it is not known if this effect is attributed to the removal of a direct inhibitory effect of V1A receptors on leptin secretion, or if this is just a consequence of an increased overall adipocity in the null mice (Aoyagi et al., 2007). 3.51. VIP and PACAP receptors

3.46. Taste receptors The human genome encodes three sweet taste receptors (TAS1–3) and 24 bitter taste receptors (TAS2R1–60). In total, 13 taste receptors, all belonging to the bitter taste subfamily, were detected in SAT. High mRNA levels were found of TAS2R14, TAS2R19, TAS2R45 and TAS2R46 as well as intermediate levels of TAS2R3, TAS2R7, TAS2R31 and TAS2R43, low levels of TAS2R20 and trace levels of TAS2R5, TAS2R10, TAS2R13 and TAS2R39. To date no endogenous ligands have been identified for bitter taste receptors, and there are no experimentally verified effects of signalling through these receptors on SAT function. While no endogenous ligands are known, members of the bitter taste receptor subfamily can be activated by a range of bitter tasting exogenous compounds (Dotson et al., 2008; Le Neve et al., 2010; Hayes et al., 2011). Interestingly, the artificial sweeteners saccharin and acesulfame K stimulate adipogenesis and suppress lipolysis independently of sweet taste receptors in both mouse and man (Simon et al., 2013) and it is known that they activate the bitter taste receptors TAS2R31 and TAS2R43 (Kuhn et al., 2004), both of which are expressed in SAT. There is some suggestion that artificial sweeteners influence adiposity and energy metabolism in mice (Mitsutomi et al., 2014), but the exact mechanism underlying these effects on whole body metabolism remains to be determined. In addition, an association between increased consumption of artificially sweetened soft drinks and T2D has been reported (Fagherazzi et al., 2013), and it is possible that these effects are mediated by saccharin and/or acesulfame K activation of TAS2R31 and TAS2R43.

3.47. Thyrotropin-releasing hormone receptor Thyrotropin-releasing hormone (TRH) is a three amino acid peptide that binds to the TRH1 receptor (TRHR). Trace levels of TRHR mRNA were detected in SAT, and experiments carried out using rabbit adipose tissue found no effect of TRH on lipolysis (Murthy & Modesto, 1974). The effects of TRH on insulin resistance and adiponectin and leptin secretion are unknown.

The peptides VIP (vasoactive intestinal peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) are agonists at VPAC1 (VIPR1) and VPAC2 (VIPR2) receptors. SAT expresses low levels of mRNA encoding the VPAC2 receptor and trace levels of VPAC1 mRNA. Both VIP and PACAP stimulate lipolysis in rat adipose tissue via activation of VIPR2, whereas VIPR1 activation has no effect on lipolysis (Akesson et al., 2005). PACAP inhibits adiponectin and leptin release (Bik et al., 2007; Yu et al., 2009) via activation of VPAC2. PACAP also promotes insulin resistance (Tomimoto et al., 2008), but it is not known if this effect is mediated via VPAC1 or VPAC2. 4. Adipose tissue G-protein coupled receptors as drug targets Targeting GPCRs to modulate adipose tissue function is aimed primarily at altering lipid storage through the induction of lipolysis, releasing adipose tissue-derived signals such as adipokines or modifying the structural/functional features of the tissue by impacting on cellularity, recruitment of new adipocytes or reducing immune cell invasion. To date, there has not been a dedicated pharmacological approach towards any of these targets. However, nicotinic acid (vitamin B2), which is an agonist at HCA2 (GPR109A), shows interesting features as it is both strongly anti-lipolytic and it increases the plasma concentration of adiponectin. It has been assumed that the anti-lipolytic action of nicotinic acid is causally related to its plasma triglyceride-lowering effect, but this has recently been questioned using a mouse model where HCAR2 (GPR109A) was ablated (Lauring et al., 2012). Also, the clinical utility of nicotinic acid as a drug has been questioned recently due to an absence of reducing cardiovascular events in a large randomised controlled trial (Group, 2013). 4.1. Predicted effects on adipose tissue function by drugs targeting GPCRs expressed in adipose tissue In the absence of drugs designed to target adipose tissue GPCRs to modulate adipose tissue function, drugs designed to target GPCRs for other purposes may nevertheless influence aspects of adipose tissue


Receptor name

Effect of GPCR on lipolysis

Drug(s) targeting GPCR

Drug mechanism of action

Pred. net effect of drug on lipolysis

Mean ABD expression rel. PPIA

Mean GLUT expression rel. PPIA

ADRA2C

α2C

?

Agonist/antagonist

?

0.0299

0.027459

PTAFR CXCR4 GABBR1 HTR1F CXCR2

PAF CXCR4 GABAB1 5-HT1F CXCR2

? ? ? ? ?

Agonists: alpha-methyl dopa, apraclonidine, betanidine, clonidine, dexmedetomidine, dihydroergotamine, dipivefrin, epinephrine, ergotamine, guanfacine, labetalol, mephentermine, methyldopa, naphazoline, norepinephrine, phenylpropanolamine, propylhexedrine, pseudoephedrine, UK 14304; Antagonists: apomorphine, asenapine, dapiprazole, dexefaroxan, ephedrine, fenoldopam, iloperidone, paliperidone, phenoxybenzamine, phentolamine, quetiapine, quinidine, risperidone, tolazoline, yohimbine Lexipafant Plerixafor Baclofen Eletriptan, almotriptan, fenfluramine, ergotamine Repertaxin, SB-265610

? ? ? ? ?

0.009007 0.009945 0.013295 0.005187 0.002109

0.012341 0.01233 0.012251 0.00654 0.002207

CYSLTR2 CXCR1

CysLT2 CXCR1

? ?

Montelukast, zafirlukast Repertaxin, SB-265610

? ?

0.002098 0.001448

0.002062 0.001883

HTR2A

5-HT2A

?

?

Trace

Trace

GRM7 GRM6 GRM5 LTB4R2 P2RY12 LTB4R CYSLTR1 F2R CNR1

mGlu7 mGlu6 mGlu5 BLT2 P2Y12 BLT1 CysLT1 PAR1 CB1

? ? ? ? ? ? ? ? No effect

? ? ? ? ? ? ? ? No effect

Trace Trace Trace Trace Trace Trace Trace Trace 0.007126

Trace Trace Trace Trace Trace Trace Trace Trace 0.005771

LHCGR BDKRB2 ADRA2A

LH B2 α2A

No effect No effect Inhib.

Agonist Antagonist Agonist

No effect No effect Inhib.

Trace Trace 0.193421

Trace Trace 0.256062

PTGER3 EDNRB ADRB2

EP3 ETB β2

Inhib.* Stim. Stim.

Agonist Antagonist Antagonist

Inhib.* Inhib. Inhib.

0.122649 0.079456 0.057334

0.115118 0.07315 0.041858

EDNRA

ETA

Stim.

Antagonist

Inhib.

0.049931

0.052282

AVPR1A ADRA1A

V1A α1A

Inhib. Stim.

Agonist Antagonist

Inhib. Inhib.

0.044306 0.040094

0.047432 0.033706

AGTR1 HRH1

AT1 H1

Stim. Stim.

Agonists: dihydroergotamine, ergotamine, psilocybine; Antagonists / inv. agonists: APD125, apomorphine, aripiprazole, asenapine, blonanserin, cyproheptadine, eplivanserin, flibanserin, lurasidone, mirtazapine, nefazodone, olanzapine, opipramol, paliperidone, quetiapine, risperidone, trazodone, ziprasidone Fasoracetam Fasoracetam Fasoracetam Etalocib Prasugrel, ticagrelor, clopidogrel Etalocib Zeneca ZD 3523, montelukast, zafirlukast Chrysalin, argatroban, bivalirudin Agonists: trans-(±)-nabilone, BAY 38-7271, delta-8-tetrahydrocannabinol, delta-9-tetrahydrocannabinol; Antagonists: Ibipinabant, rimonabant ORG 41841, menotropin Anatibant, icatibant Alpha-methyl dopa, apraclonidine, betanidine, brimonidine, clonidine, dexmedetomidine, dihydroergotamine, dipivefrin, ephedrine, epinastine, epinephrine, ergotamine, gabapentin, guanabenz, guanfacine, labetalol, mephentermine, methyldopa, naphazoline, norepinephrine, oxymetazoline, propylhexedrine, phenylpropanolamine, pseudoephedrine, tizanidine, UK 14304 Prostaglandin E1 Bosentan, sitaxsentan, atrasentan Carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, nebivolol, olanzapine, pindolol, propranolol, sotalol, timolol Bosentan, avosentan, clazosentan, ambrisentan, sitaxsentan, ZD4054, SB 234551, TBC 3214, BSF 302146, fandosentan, atrasentan Felypressin, vasopressin, lypressin Alfuzosin, aripiprazole, asenapine, carvedilol, dapiprazole, dihydroergotamine, DL 017, Doxazosin, iloperidone, KMD-3213, Labetalol, nefazodone, olanzapine, paliperidone, phenoxybenzamine, phentolamine, prazosin, promethazine, quetiapine, quinidine, risperidone, tamsulosin, terazosine, tolazoline, UK-294315 Azilsartan medoxomil, candesartan, eprosartan, irbesartan, losartan, olmesartan medoxomil, telmisartan, valsartan Acrivastine, alcaftadine, antazoline, aripiprazole, asenapine, astemizole, azatadine, azelastine, bepotastine, bilastine, bromodiphenhydramine, brompheniramine, buclizine, carbinoxamine, cetirizine, chlorpheniramine, clemastine, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, diphenhydramine, doxylamine, emedastine, epinastine, fexofenadine, hydroxyzine, ketotifen, levocabastine, levocetirizine, loratadine, meclizine, olanzapine, olopatadine, opipramol, paliperidone, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, risperidone, terfenadine, triprolidine, ziprasidone

Antagonist Antagonist Agonist Agonist Antagonist/inv. agonist Antagonist Antagonist/inv. agonist Agonist/ antagonists/inv. agonist Agonist Agonist Agonist Antagonist Antagonist Antagonist Antagonist Antagonist Agonist/antagonist

Antagonist Antagonist/inv. agonist

Inhib. Inhib.

0.033718 0.016599

0.031958 0.021965


Gene symbol

26


Table 4 Model predicting how drugs that interact with GPCRs expressed in SAT are likely to influence lipolysis. Drugs that interact with GPCRs expressed by SAT are classified by their mode of action as agonists or antagonists/inverse agonists. Their predicted effects of drugs on lipolysis is modelled using data on how GPCRs are known to regulate lipolysis combined with how drugs alter normal GPCR signalling. 119 drugs, acting on 23 GPCRs, are predicted to stimulate and 173 drugs, acting on 25 GPCRs, are predicted to inhibit lipolysis. Stim.: stimulation of lipolysis; Inhib.: inhibition of lipolysis. * Due to extensive overlapping ligand usage within the prostanoid receptor family, the activation of different second messenger signalling pathways by different prostanid receptors and a lack of selective prostanoid receptor agonists/antagonists it is difficult to determine what effects prostanoids and their receptors, as well as drugs that agonise or antagonise prostanoid receptors, have on the regulation of lipolysis.

A1 FP β1

Inhib. Inhib.* Stim.

PTGER4 PTGER2 HRH2 HTR2B PTGIR SSTR2 CHRM3 TACR1 TBXA2R ADRB3 DRD1

EP4 EP2 H2 5-HT2B IP1 sst2 M3 NK1 TP β3 D1

Inhib.* Inhib.* Stim. Stim. Inhib.* Inhib. Inhib. Stim. Stim.* Stim. Stim.

SMO ADRA1B

SMO α1B

Stim. Stim.

ADRA2A

α2A

Inhib.

S1PR1 PTGER3 AVPR1A ADRB2

S1P1 EP3 V1A β2

Stim. Stim.* Inhib. Stim.

ADRA1A

α1A

Stim.

S1PR3 ADORA1 PTGFR ADRB1

S1P3 A1 FP β1

Stim. Inhib. Stim.* Stim.

PTGER4 PTGER2 TSHR HTR2B PTGIR CHRM3

EP4 EP2 TSH 5-HT2B IP1 M3

Stim.* Stim.* Stim. Stim. Stim.* Inhib.

ADRB3

β3

Stim.

DRD1 S1PR4 PTH2R TBXA2R PTH1R ADRA1B

D1 S1P4 PTH2 TP PTH1 α1B

Stim. Stim. Stim. Inhib.* Stim. Stim.

Adenosine, tecadenoson Tafluprost, travoprost, isopropyl unoprostone, bimatoprost, latanoprost Acebutolol, alprenolol, amiodarone, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, esmolol, labetalol, levobetaxolol, levobunolol, metipranolol, metoprolol, nadolol, nebivolol, pindolol, propranolol, sotalol, timolol Misoprostol, ONO-4819, prostaglandin E1, prostaglandin E2 CP 533536, misoprostol, prostaglandin E1, prostaglandin E2 Asenapine, cimetidine, epinastine, famotidine, nizatidine, ranitidine Apomorphine, asenapine, mirtazapine, opipramol, risperidone Treprostinil, epoprostenol Pasireotide, lanreotide, octreotide ABT-089, acetylcholine, arecoline, bethanechol, carbamylcholine, cevimeline, methacholine, pilocarpine MK-0517, AV608, aprepitant S-18886, torsemide Carteolol, carvedilol, esmolol, labetalol, pindolol, propranolol, sotalol, timolol Asenapine, fluphenazine, haloperidol, iloperidone, lisuride, olanzapine, perphenazine, pimozide, prochlorperazine, promazine, quetiapine, thioridazine, thiothixene, trifluoperazine, ziprasidone Vismodegib Alfuzosin, aripiprazole, asenapine, carvedilol, dapiprazole, DL 017, Iloperidone, labetalol, nefazodone, olanzapine, paliperidone, phenoxybenzamine, phentolamine, prazosin, promethazine, quetiapine, quinidine, risperidone, tamsulosin, terazosine, tolazoline, UK-294315, ziprasidone Apomorphine, asenapine, dapiprazole, dexefaroxan, dihydroergotamine, ephedrine, epinastine, fenoldopam, mirtazapine, paliperidone, phenoxybenzamine, phentolamine, quetiapine, quinidine, risperidone, tolazoline, trazodone, yohimbine Fingolimod Prostaglandin E1 Conivaptan Albuterol, arbutamine, arformoterol, bitolterol, dipivefrin, dobutamine, ephedrine, epinephrine, formoterol, indacaterol, isoetharine, isoproterenol, isoxsuprine, levalbuterol, metaproterenol, pirbuterol, ritodrine, salmeterol, terbutaline Apraclonidine, arbutamine, clonidine, dipivefrin, dopamine, D-pseudoephedrine, Ephedrine, epinephrine, ergotamine, mephentermine, metaraminol, methoxamine, midodrine, naphazoline, norepinephrine, phenylephrine, phenylpropanolamine, Pseudoephedrine, ziprasidone Fingolimod Aminophylline, caffeine, clofarabine, dyphylline, theophylline Tafluprost, travoprost, isopropyl unoprostone, bimatoprost, latanoprost Arbutamine, dipivefrin, dobutamine, ephedrine, epinephrine, isoetharine, isoproterenol, isoxsuprine, levalbuterol, norepinephrine, pindolol, pirbuterol, terbutaline Misoprostol, ONO-4819, prostaglandin E1, prostaglandin E2 CP 533536, misoprostol, prostaglandin E1, prostaglandin E2 ORG 41841 Eletriptan Treprostinil, epoprostenol Anisotropine methylbromide, atropine, benztropine, biperiden, brompheniramine, buclizine, carbinoxamine, cisatracurium, cyclopentolate, darifenacin, dicyclomine, diphenhydramine, doxacurium, fesoterodine, flavoxate, glycopyrrolate, homatropine, homatropine methylbromide, hyoscyamine, ipratropium, methantheline, methscopolamine, olanzapine, orphenadrine, oxybutynin, pipecuronium, procyclidine, propantheline, quinidine, scopolamine, solifenacin, tiotropium, tolterodine, tridihexethyl, trihexyphenidyl, tropicamide, trospium Arbutamine, dipivefrin, dobutamine, ephedrine, epinephrine, isoproterenol, isoxsuprine, KUL 7211, Levalbuterol, metaproterenol, pirbuterol, terbutaline Apomorphine, bromocriptine, clozapine, dihydroergocryptine, dopamine, fenoldopam, pergolide, rotigotine Fingolimod PTH S-18886, torsemide PTH Apraclonidine, clonidine, dihydroergotamine, dipivefrin, dobutamine, dopamine, D-pseudoephedrine, Epinephrine, ergotamine, mephentermine, metaraminol, methoxamine, midodrine, naphazoline, norepinephrine, phenylephrine, phenylpropanolamine, pseudoephedrine, PYM-50018

Agonist Agonist Antagonist

Inhib. Inhib.* Inhib.

0.013431 0.010574 0.009969

0.011049 0.007631 0.005455

Agonist Agonist Antagonist Antagonist Agonist Agonist Agonist Antagonist Antagonist Antagonist Antagonist

Inhib.* Inhib.* Inhib. Inhib. Inhib.* Inhib. Inhib. Inhib. Inhib.* Inhib. Inhib.

0.007665 0.005537 0.002939 0.002069 0.001301 0.000881 0.000875 Trace Trace Trace Trace

0.007686 0.004681 0.004622 0.002439 0.001582 0.000905 0.000523 Trace Trace Trace Trace

Antagonist Antagonist

Inhib. Inhib.

Trace Trace

Trace Trace

Antagonist

Stim.

0.193421

0.256062

Agonist Agonist Antagonist Agonist

Stim. Stim.* Stim. Stim.

0.268022 0.122649 0.044306 0.057334

0.238651 0.115118 0.047432 0.041858

Agonist

Stim.

0.040094

0.033706

Agonist Antagonist Agonist Agonist

Stim. Stim. Stim.* Stim.

0.016455 0.013431 0.010574 0.009969

0.015115 0.011049 0.007631 0.005455

Agonist Agonist Agonist Agonist Agonist Antagonist

Stim.* Stim.* Stim. Stim. Stim.* Stim.

0.007665 0.005537 0.004616 0.002069 0.001301 0.000875

0.007686 0.004681 0.00501 0.002439 0.001582 0.000523

Agonist

Stim.

Trace

Trace

Agonist Agonist Agonist Antagonist Agonist Agonist

Stim. Stim. Stim. Stim.* Stim. Stim.

Trace Trace Trace Trace Trace Trace

Trace Trace Trace Trace Trace Trace

S. Amisten et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx 27


ADORA1 PTGFR ADRB1

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function such as the regulation of lipolysis. Using both public and private databases (see Section 1.4 above), we have identified 51 adipose tissue GPCRs as being targets of 282 different drugs (Table 4). Although many of the targeted receptors are expressed at low levels in adipose tissue, mRNAs encoding the S1P1 (S1PR1), ETA (EDNRA), ETB (EDNRB), AT1 (AGTR1), EP3 (PTGER3), V1A (AVPR1A), α2a (ADRA2A) and β2 adrenergic receptors are abundantly expressed in adipose tissue and their effect on lipolysis is therefore more likely to be influenced by drugs targeting the above receptors. Based on our knowledge of adipose tissue GPCRs that are targeted by drugs regulating lipolysis and the mechanism of action of each drug (i.e. agonist, antagonist, inverse agonist) targeting these receptors, we have constructed a model predicting how these drugs might affect lipolysis (Fig. 7, Table 4). The effects on lipolysis of some of these drugs, such as agonists and antagonists of the α2A receptor, are well known, whereas the predicted effects of other drugs may merit further investigation, especially those drugs that are prescribed for long-term administration, such as agonists and antagonists at the 5-HT2A, 5-HT2B, V1A and α1B receptors. The receptors identified using this model, as well as the drugs targeting them, merit further investigation, as pharmacological modulation of the above receptors may influence the likelihood of disease progression for individuals already at high risk of developing CVD and T2D. 5. Conclusions and perspectives Adipose tissue has come a long way from being considered as a passive energy storing tissue to its current status as the body's largest endocrine organ (Kershaw & Flier, 2004). It is a principal energy storing organ, and the influx of glucose and efflux of lipids from adipose tissue, as well as the secretion of a number of hormones, such as adiponectin and leptin, is tightly regulated. This regulation is, at least in part, achieved through the signalling via a large number of GPCRs that are present on different cell types within adipose tissue. In this study, we have quantified mRNAs encoding GPCRs found in adipose tissue, and not in purified adipocytes. Therefore, it is likely that some of the GPCRs that we have detected are also, and perhaps exclusively, expressed in non-adipocyte cells, such as macrophages, endothelial cells, fibroblasts, adipocyte precursor cells, etc. Interestingly, whole mount histological staining of human SAT obtained either by needle aspiration or by excision of intact SAT suggests that only 12–15% of all cells present in adipose tissue are adipocytes. It is important to take into account that adipocytes are larger (57–100 μm) (Jernas et al., 2006) than any other cell types present in SAT, and also have the ability to expand and contract to accommodate for required lipid storage needs (Eto et al., 2009). It should be borne in mind that the potential differences in cellular expression pattern within the tissue may introduce uncertainties for our model predicting how GPCRs are likely to affect lipolysis, as only receptors expressed in adipocytes are likely to be relevant. This ‘noise’ from other cell types could be eliminated by quantifying the expression of GPCRs in collagenase purified human SAT adipocytes (Jernas et al., 2006) instead of intact SAT, which may provide a clearer picture of which GPCRs are involved in lipolysis and the regulation of adiponectin and leptin secretion. However, by using isolated adipocytes, it is also likely that several GPCRs, that may be important for the regulation of SAT inflammation and insulin resistance, would be overlooked, as they may be expressed in non-adipocyte SAT cell types such as adipose tissue macrophages. It has long been known that GPCRs are important regulators of adipose tissue function, but very little is known about how a majority of GPCRs participate in this regulation. The way different GPCRs interact as an ‘adipose tissue GPCRome’ at the systemic level is also unknown, and we have reported here that the human adipose tissue GPCRome is made up of 159 functional, non-odorant GPCRs. Using a very similar approach to the one used here, we previously determined the complete GPCRome in human islets of Langerhans, the only other human tissue for which a GPCRome has been defined (Amisten et al., 2013). We

observed that the human islet GPCRome is made up of 293 GPCRs, which is nearly twice as many GPCRs as detected in adipose tissue. Both tissues are metabolically active tissues that are made up of several different cell types, so it is difficult to attribute the difference only to cell heterogeneity within the two tissue types. We found a remarkable resemblance in the GPCR mRNA expression profiles between subcutaneous adipose tissue originating from abdominal (ABD) and gluteofemoral (GLUT) depots, with only three GPCRs being significantly up-regulated in the GLUT compared to the ABD tissue biopsies. These findings suggest that, at least when it comes to GPCR expression, ABD and GLUT adipose tissue can be considered as rather similar. However, this tissue comparison is made across a wide range of gene expression levels, and more subtle differences in for example highly expressed alpha and beta-adrenoreceptors may have significant implications for the difference in the regulation of lipolysis between ABD and GLUT adipose tissues (Wahrenberg et al., 1989). One surprise finding from this study is how little we know about how GPCRs regulate adipose tissue function: more than 50% of all GPCRs have undefined roles in the regulation of insulin resistance, lipolysis and in the secretion of the adipose tissue hormones adiponectin and leptin. A lot more research will be needed to determine the impact of GPCRs on adipose tissue function—especially insulin resistance, as this phenomenon will have direct consequences for identifying mechanisms for intervention to delay or stop the development of T2D and CVD. Our analysis of the adipose tissue GPCRome revealed extensive autoand paracrine signalling networks in SAT, and illustrate how interconnected adipose tissue cells are with both their local and systemic environment. Surprisingly, peptides seem to have a greater impact on SAT GPCR signalling than small organic molecule ligands: 59 SAT GPCRs are activated by 67 different small organic molecules and a further 57 SAT GPCRs are activated by 112 different peptides or proteins. The remaining GPCRs with known ligands are activated by protons or biological macromolecules. It is also worth noting that a large number of SAT GPCRs remain orphans, such that endogenous ligands have not been identified for these receptors. The abundant adipose tissue orphan receptor GPRC5B has many properties suggesting that it might be an interesting novel drug target candidate for metabolic disease. Thus, GPRC5B has been reported to inhibit insulin secretion from islets (Soni et al., 2013) and promote inflammation and adipose tissue insulin resistance in mice (Y. J. Kim et al., 2012). Moreover, a genetic variant in close proximity of the GPRC5B receptor gene is associated with changes in BMI in man (Speliotes et al., 2010), but further research is needed to elucidate the underlying mechanism behind this association. In this study, we have measured the mRNA expression of a large number of GPCRs. qPCR was chosen for the quantification of the SAT GPCRome due to its ease of use, high specificity, sensitivity and cost effectiveness compared to other gene expression quantification platforms such as microarray and RNA-sequencing. It is technically more challenging to quantify and localise the GPCR proteins encoded by the 159 GPCRs whose mRNAs we detected in subcutaneous adipose tissueand successful detection is limited by the availability of validated antiGPCR antibodies for western blot or immunohistochemical studies. However, it is reasonable to assume that the vast majority of the GPCR mRNAs we have detected in SAT reflect the expression of GPCR proteins in this tissue, as there is a very good correlation between our mRNA expression profiles and published data on how GPCRs regulate SAT function, described in Section 3 above. In conclusion, a detailed analysis of the subcutaneous adipose tissue GPCRome has revealed that SAT expresses a large number of GPCRs that are part of a complex adipose tissue GPCRome, where the majority of all receptors have unknown roles in regulating adipose tissue function. A significant number of GPCRs expressed by SAT may be susceptible to off-target effects of current drug therapies aimed at non-metabolic disorders, and further understanding of their expression and function in SAT may help to reduce unwanted side effects and also identify hitherto unknown beneficial side effects of current medications targeting GPCRs



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Growth hormone receptor expression in human gluteal versus abdominal subcutaneous adipose tissue: Association with body shape.

Folate receptor expression on murine and human adipose tissue macrophages.

Characterisation and comparison of adipose tissue macrophages from human subcutaneous, visceral and perivascular adipose tissue.

Human omental and subcutaneous adipose tissue exhibit specific lipidomic signatures.

Microarray based gene expression analysis of murine brown and subcutaneous adipose tissue: significance with human.

Keratinocyte progenitor cells reside in human subcutaneous adipose tissue.

Hexokinase isozymes of normal human subcutaneous adipose tissue.

Characteristic expression of extracellular matrix in subcutaneous adipose tissue development and adipogenesis; comparison with visceral adipose tissue.

Noradrenaline reversal in canine subcutaneous adipose tissue.

Human adipose tissue accumulation is associated with pro-inflammatory changes in subcutaneous rather than visceral adipose tissue.

Visceral adipose tissue but not subcutaneous adipose tissue is associated with urine and serum metabolites.

Triacylglycerol biosynthesis in bovine liver and subcutaneous adipose tissue.

Erythropoietin does not activate erythropoietin receptor signaling or lipolytic pathways in human subcutaneous white adipose tissue in vivo.

Differential Expression of Cell Cycle Regulators During Hyperplastic and Hypertrophic Growth of Broiler Subcutaneous Adipose Tissue.

Castration-induced changes in microRNA expression profiles in subcutaneous adipose tissue of male pigs.

Global comparison of gene expression between subcutaneous and intramuscular adipose tissue of mature Erhualian pig.

Epicardial Adipose Tissue Is Nonlinearly Related to Anthropometric Measures and Subcutaneous Adipose Tissue.

Subcutaneous adipose tissue zinc-α2-glycoprotein is associated with adipose tissue and whole-body insulin sensitivity.

TNF-α gene expression in subcutaneous adipose tissue associated with HOMA in Asian Indian postmenopausal women.

Characterization of stromal vascular fraction and adipose stem cells from subcutaneous, preperitoneal and visceral morbidly obese human adipose tissue depots.

Lipoprotein lipase and hormone-sensitive lipase activities in human subcutaneous lipomas: comparison with normal subcutaneous adipose tissue.

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy.

Central sympathetic innervations to visceral and subcutaneous white adipose tissue.

Comparative gene array analysis of progenitor cells from human paired deep neck and subcutaneous adipose tissue.