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  • br Conclusions br Acknowledgements The authors thank Matt JM


    Acknowledgements The authors thank Matt JM Wood and colleagues (AstraZeneca, UK) for permission to use logD and protein binding data presented in Sup. Table S2.
    Introduction The number of people suffering from type 2 diabetes mellitus (T2DM) has escalated worldwide as a consequence of unhealthy lifestyle and obesity [1]. T2DM is characterized by insulin-resistance, decreased insulin secretion from the pancreatic β-cells and loss of intestinal glucagon-like-peptide (GLP)-1 release in response to diet; deficits that are even more pronounced in obese individuals [2]. The G protein-coupled receptor (GPR) 119 acts as a lipid-sensor in the gut, where it induces the release of incretin hormones, GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) from the intestinal L and K-cells, respectively, in response to dietary lipids [3], [4], [5], [6]. Besides its expression in intestinal enteroendocrine p2y receptor (EEC), GPR119 is also expressed in pancreatic β-cells, where it leads to insulin secretion in a glucose-dependent manner [7], [8], [9]. GPR119 in pancreas may possibly be stimulated by 2-monoacylglycerol (MAG) generated by lipoprotein lipase [10]. GPR119 couples to Gαs with high constitutive activity and in response to lipid-based agonists like lysophosphatidylcholines (LPCs), oleic-acid containing N-acyl-amino compounds like N-oleoyldopamine (OLDA) and N-acylethanolamines (NAEs) [3], [8], [9], [11], [12], [13], [14]. Of these, the oleic-acid containing OEA is the most potent lipid-based GPR119 agonist [3]. It stimulates cAMP accumulation in GPR119 transfected cells with high potency (EC50 of 0.2–2.9μM) [3], [12] and releases GLP-1 in vitro from GLUTag-cells, an EEC cell-line with endogenous GPR119 expression, and in vivo after intraluminal injection in mice [15]. Monoacylglycerols (MAGs) such as 2-oleoyl glycerol (2-OG), which is generated in high amounts during digestion of the diet, also activate GPR119 as shown in recent in vivo studies in humans, where ingested 2-OG stimulated GLP-1 release [3], [4]. Similar experiments in wild type and GPR119-deficient mice proved that the GLP-1 secretion induced by 2-OG (and the more stable ether analog of 2-OG) indeed is mediated via GPR119 activation [5]. The massive interest in GPR119 as a pharmaceutical target for the treatment of T2DM and obesity has resulted in numerous synthetic GPR119 ligands, for recent reviews see [14], [16], [17]. In 2007, the prototypical GPR119 agonist, AR231453, was published as a GPR119-specific glucose-dependent insulin secretagog with similar efficacy as GLP-1 [8], and shortly thereafter was shown to stimulate incretin hormone release in mice [13]. This inspired many companies to create new GPR119 agonists, though only few have reached clinical trials [16], [18], [19]. GPR119 antagonists and inverse agonists have also been described, like AR436352, used in the present study. The signaling through Gαs is well established for GPR119 [3], [8], [9], [11], [12], [13], [14], [20], [21], [22], [23] as measured by cAMP accumulation and/or downstream activation of the transcription factor CREB (cAMP response element binding protein) in response to both lipids (e.g. OEA, OLDA, LPC, 2-OG) and multiple synthetic GPR119 agonists [3], [8], [9], [11], [12], [20], [24], [25]. In addition, calcium release has been described for ligand-mediated GPR119 activation [24], [26], whereas G protein-independent signaling via β-arrestin recruitment has only been sparsely described in the patent literature [27]. Here, we present an in depth analysis of the signaling profile of GPR119 with and without ligand addition. We compare the activity of the endogenous lipid OEA and the synthetic non-lipid-based agonist AR231453 through G protein-dependent and -independent pathways to search for signaling bias. Given their structural differences, and only partly overlapping binding sites [28], these two ligands were furthermore combined in order to study allosteric modulation properties. The synthetic GPR119 inverse agonist AR436352 [28] was included to study ligand-modulation of the constitutive activity at the transcriptional activity level (CRE, SRE and NFAT). As the activity of lipid-receptors may be under influence of endogenous lipids revealing apparent constitutive activity, as shown for GPR40 and EBI2 (also known as GPR183) [29], [30], [31], [32] – we tested whether this was the case for GPR119 by addition of two inhibitors of the enzymes that degrade the endogenous GPR119 agonists MAGs and NAEs. Our studies expand the basic pharmacological knowledge of GPR119, and introduce signaling bias, allosteric actions and apparent constitutive activity as important traits for this receptor.