GPR40 Family General

The GPR40 family of GPCRs consisting of GPR40-43 are tandemly located downstream of human gene CD22 on chromosomal locus 19q13.1 [27]. Phylogenetic analysis shows that this group is most closely related to the nucleotide, eicosanoid, protease-activated and lipid receptors in the Class A GPCR superfamily [31]. The family exhibits relatively limited similarity, 52% between FFA2 (GPR43) and FFA3 (GPR41) and 34 and 41% comparing FFA1 (GPR40) to FFA3 and FFA2 respectively. Ligand-pairing efforts by a number of groups identified free fatty acids as agonists for the GPR40 family, with short-chain fatty acids (SCFAs) selectively activating FFA2 and FFA3 [4,25] and longer chain saturated and unsaturated fatty acids (LCFAs) stimulating downstream signaling events via FFA1 [3,15,18]. For recent reviews on the GPR40 receptor family see [6,24]. Unbound LCFAs are thought to reach levels of 0.01-10μM in the circulation, mainly being products of dietary intake, adipose recycling and hepatic turnover of neutral fats, cholesteryl esters and phospholipids [28]. SCFAs are generated during fermentation of resistant starches and indigestible fiber by anaerobic gut flora and can reach millimolar concentrations in the hind-gut. The ratio of SCFAs in the colonic lumen is about 60% acetate, 25% propionate and 15% butyrate. SCFAs can also be generated as metabolic byproducts of anaerobic bacteria in the periodontal pocket and following alcohol ingestion. The concentration of total SCFAs in human peripheral blood is 50-100μM and 300-450μM in portal blood. With the central role that fatty acids play in metabolism and other physiological functions including regulation of the immune response, the GPR40 family currently provide a key focus for development of novel therapeutic agents.

FFA1

FFA1 mRNA expression occurs predominantly in human and rodent pancreatic islets [2,15,18] and has also been detected in a number of brain regions in human and monkey though not in rodent [3,23]. FFA1 expression is also present in a subset of human immune cells, predominantly monocytes. Amongst the many physiological roles of LCFAs, they are known to play a key role in the islet, regulating basal insulin secretion and that following a fast, although chronic exposure is detrimental to β-cell function. Conclusive evidence of a role of FFA1 in insulin secretion was achieved by Itoh et al. who demonstrated that attenuation of FFA1 expression in a mouse insulinoma line, MIN6, resulted in significant reduction in fatty-acid potentiation of insulin secretion. More recently, stimulation of FFA1 signalling using a selective small-molecule agonist GW9508 (100-fold selective against the fatty-acid activated receptor GPR120), resulted in potentiation of glucose stimulated insulin secretion in MIN6 cells which was completely attenuated following treatment with a FFA1 selective antagonist GW1100 [2]. FFA1 is coupled to G_αq resulting in elevation of intracellular Ca²⁺, a key component of insulin exocytosis. Evidence of fatty acid signalling via FFA1 through G_αs has been suggested in MIN6 cells [8]. Although several groups have reported that FFA1-mediated potentiation of insulin secretion is dependent on entry of Ca²⁺ via the voltage-gated Ca²⁺ channel following cell depolarization ¹⁴, it is also possible that elevations in cAMP may delay the ability of the delayed rectifier K⁺ channel from repolarising the cell and closing the voltage-gated Ca²⁺ channels. However, FFA1 does not account for all the effects of fatty acids on insulin secretion, since GW1100 could only partially reduce the insulin secreted following incubation with glucose and linoleic acid. FFA1 expression has also been reported in islet α-cells, with FFA1 specific anti-sense treatment preventing linoleic-stimulated glucagon secretion [9]. However, whereas FFA1 expression was increased in human insulinomas, it was undetectable in glucagonomas [30].

Deletion of the FFA1 gene in mice resulted in animals that were resistant to the detrimental effects of a high-fat diet on glucose homeostasis, insulin resistance and lipid levels, despite a reduction in fatty-acid potentiation of glucose-stimulated insulin secretion [29]. Moreover, mice overexpressing FFA1 in the β-cell developed diabetes shortly after birth. The role of FFA1 in the detrimental effects of fatty acids on islet function following chronic exposure remains controversial, since Poitout et al. reported that islets from FFA1 knockout mice remain sensitive to the inhibition of islet insulin secretion following chronic exposure to fatty acids [19]. Moreover oleate-mediated FFA1 activation was found to protect against lipoapoptosis in the mouse β-cell line NIT1 [36]. Two polymorphisms in FFA1 have been reported, the most prevalent being Arg²¹¹ -> His and a rare Asp¹⁷⁵ -> Asn. Although variations were not associated with type 2 diabetes or alterations in insulin release in a Danish cohort [11], studies in Japanese men suggested that the Arg²¹¹ -> His polymorphism may contribute to the variation of insulin secretory capacity [26].

The role of FFA1 in the brain and immune cells remain unclear. However, FFA1 has been suggested to play a role in the proliferation of breast-cancer cells by oleic acid and linoleic acid in-part via a G_αi signalling pathway [12]. These findings have led to suggestions by the authors that FFA1 may play a role in the link between dietary fat intake and cancer.

FFA2

FFA2 is mainly coupled to G_αq although it is capable of signalling through G_αi. Acetate and propionate are the most potent ligands for the receptor with pentanoate less active [4,25]. FFA2 is predominantly expressed in adipose, inflammatory cells and the GI-tract. FFA2 expression levels have been shown to increase with differentiation of 3T3-L1 adipocytes and acetate or propionate stimulated FFA2 signalling has been shown to inhibit isoproterenol-induced lipolysis and play a role in adipogenesis [14]. Furthermore, elevation of FFA2 expression was observed in adipose of mice fed a high-fat diet compared that of mice on a low-fat diet [22]. A role for FFA2 in GI gastrointestinal motility and secretion has also been suggested following the finding that the receptor co-localizes with PYY in mucosal epithelium and mast cells and with 5HT-containing mast cells in the distal colon [16]. This finding is consistent with the report that SCFAs release PYY [5] and 5-HT from the ileum and colon [10]. However, the ability of SCFAs to inhibit gastrointestinal motility appears independent of FFA2 since SCFAs remained able to inhibit neuronally mediated contractions of the colon in tissues from wild-type mice in FFA2 knockout mice [7]. Expression of FFA2 has also been described in mouse pancreas with increased expression in islets from db/db and ob/ob mice [22]. However, the expression of the receptor in many immune cell types including polymorphonuclear cells, monocytes, eosinophils and B-lymphocytes has led to suggestions that FFA2 may be responsible for some of the effects of SCFAs on the immune response such as leukocyte recognition, recruitment and migration [20]. A link between SCFAs and cancer through both FFA2 and FFA3 has been suggested, since both receptors are present in the MCF-7 human breast cancer line. Propionate stimulated phosphorylation of the p38 MAPK/HSP27 stress-response pathway was inhibited by FFA2-specific siRNA in these cells. The presence of the receptors on MCF-7 cells led authors to conclude that the receptors may mediate the cellular stress response to bacterial and alcohol-induced SCFAs [35].

GPR42

GPR42 appears to have evolved following recent gene duplication from FFA2 but is non-functional and is not detected by RT-PCR in normal human tissue. Although only varying from FFA2 by six amino acids, the receptor does not respond to SCFAs unless particular residues are changed to those in FFA2 [4].

FFA3

FFA3 differs from FFA2 most notably in its rank order potency of preferred ligands and G protein-coupling. Propionate is the most potent ligand for FFA3 followed by butyrate and pentanoate respectively. The expression of FFA3 in adipose is supported by expression of the receptor in differentiated human and mouse adipocytes, whilst little expression was observed in pre-adipocytes or brown fat [20,34]. Activation of FFA3 by propionate via a pertussis-toxin sensitive G_αi-coupled pathway, was reported to elevate leptin production from mouse adipocytes and primary adipocyte tissue cultures [34]. Furthermore, increases in circulating leptin were observed 8hrs after mice were dosed orally with propionate. However, controversy exists around the role of FFA3 in adipose since Hong et al. failed to detect FFA3 expression in adipocytes in vitro or ex-vivo, claiming instead that FFA2 played a role in adipogenesis [14]. There are currently no small-molecule tool compounds published for FFA3 nor has the phenotype of FFA3 knockout mice been described. A role for FFA3 in islet function remains a possibility following the report of islet expression of FFA3 and its upregulation in islets of db/db mice [21]. Metabolic effects of propionate have been reported although these have not been linked to FFA3. For example, propionate was found to decrease glucose-stimulated insulin secretion in rat islets [33] and treatment of fa/fa rats fed a high cholesterol and high fat diet with propionate, reduced glucose excretion and fasting blood glucose [1]. Moreover, expression of the receptor in various other cell-types including dendritic cells, lymphocytes, peripheral blood mononuclear cells and artery and arteriolar endothelial cells raises further questions as to the role of the receptor in immune cell function. A recent report has also suggested that FFA3 is involved in the apoptosis observed during reoxygenation and ischemic hypoxia in H9c2 cells [17].

Summary

The finding that free fatty acids were ligands for the GPR40 family not only introduced the concept that fatty acids could serve as signalling molecules at cell-surface receptors but initiated more recent discovery of other GPCRs activated by fatty acids such as GPR120 (activated by unsaturated fatty acids)[13] and GPR84 (putatively activated by medium-chain fatty acids) [32]. It is possible that, rather than being redundant, that receptor tissue distribution combined with local fatty acid levels and their carbon chain-lengths and degree of saturation will determine the physiological role of the receptors. The role of FFA1 is best understood due to the phenotyping of transgenic mice and identification of selective small-molecule agonists and an antagonist. Notably several thiazolidinediones, including Rosiglitazone which is currently marketed for Type 2 diabetes, have been demonstrated to activate FFA1 in vitro, although to what extent this property contributes to their clinical efficacy remains to be determined [18]. The importance of FFA1 in the brain and in immune cells remains to be elucidated. Fuller understanding of the physiological roles of FFA2 and FFA3, will await further phenotyping of knockout mice and identification and pharmacological characterisation of selective small-molecule tool compounds.

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