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

Abstract

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 GPCR-targeting 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.

Introduction

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 tissue-derived 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.

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 N-terminus 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 well-defined 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.

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 nucleotide-binding 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: G_q/G₁₁ activates phospholipase C to generate diacylglycerol and inositol 1,4,5 trisphosphate, which activate protein kinase C and mobilise intracellular Ca²⁺ respectively; G_s activates adenylate cyclase to stimulate cAMP production; and G_i/G₀ inhibits adenylate cyclase to decrease cAMP production; G_12/13 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 G_i/G₀ coupled GPCRs elevate Ca²⁺ by activating Ca²⁺ 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, G-proteins 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).

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.

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 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 (>65%), 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 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.

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 > mouse, rat adipose tissue and rodent in vivo experiments > adipose tissue and in vivo experiments from other species (dog, rabbit etc.) > 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 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 (Fig. 8, Fig. 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.

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 tri- hexa- 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 and 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), ET_A (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 non-selective 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 α_2A (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 G_s second messenger pathway, which leads to increased biosynthesis of cAMP, inhibit leptin secretion, whereas GPCRs that signal via G_i/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 G_s-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 V_1A (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 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).

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 G_i and G_s pathways (Section 1.3). Other GPCR-regulated signalling pathways also control lipolysis, including the G_q-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 lipolysis. Examples of receptors in this group include the well-known adrenergic α_1A (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 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 α_2A (ARDA2A), Y₁ (NPY1R), V_1A (AVPR1A), apelin (APLNR) and A₁ (ADORA1) receptors, all inhibit lipolysis, and a further ten receptors, including the highly expressed C5_a1 (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.

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 insulin-sensitising 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 C5_a1 (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 (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 GABA_B1 (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.