br Experimental br Acknowledgments The

Experimental
Acknowledgments The authors are grateful to Fundamental Scientific Research Fund of Chinese Academy of Medical Sciences (No. 2016ZX350030) for financial support.
Introduction 2-Lysophosphatidylcholines (1-acyl-glycero-3-phosphocholines, 2-LPC) are blood derived factors involved in glucose homeostasis. Oleoyl LPC (18:1) was shown to enhance glucose stimulated insulin secretion (GSIS) in NIT-1 insulinoma Alsterpaullone and perfused rat pancreas [1]. LPC was also found to stimulate glucose uptake in 3T3-L1 adipocytes and this activity was sensitive to variations in acyl chain lengths. Whereas palmitoyl and myristoyl LPC derivatives (16:0 and 14:0, respectively) enhanced glucose uptake by 3T3-L1 adipocytes, stearoyl, oleoyl, and arachidonoyl LPC (18:0, 18:1, and 20:4, respectively) were ineffective [2]. At a molecular level LPC 16:0, 18:0 and 18:1 were identified as novel ligands of G protein coupled receptor GPR119, which is one of the crucial targets responsible for regulation of insulin secretion from β cells of the islets of Langerhans [1]. GPR119 is preferentially expressed in pancreatic cells but its expression was also documented in enteroendocrine cells of the gut, where its activation was related to stimulation of secretion of glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) [3], [4], [5]. Homology clustering analysis indicated that the closest relatives of GPR119 are the cannabinoid receptors and a number of cannabinoid ligands and fatty-acid ethanolamides were recognized as endogenous agonists. Potencies of ethanolamides for GPR119 were assessed with maximum response observed with oleoylethanolamide (OEA) followed by and palmitoylethanolamide (PEA), stearoylethanolamide (SEA), and arachidonoylethanolamide (anandamide, AEA) [3]. Agonist binding to GPR119, which is coupled predominantly to Gαs results in an increase in intracellular cyclic adenosine monophosphate (cAMP) [6]. Indeed, LPC 16:0, 18:0 and 18:1 were shown to produce elevated cAMP levels in RH777 rat hepatoma cells stably expressing GPR119 and in NIT-1 insulinoma cells [1]. OEA was also demonstrated to utilize the adenylate cyclase pathway in stably GPR119-transfected HEK 293-derived cell line [3], [7] and in endogenous GPR119-expressing murine MIN6c4 insulinoma cells [8]. However, in addition to Gαs coupling, there is evidence for GPR119-mediated activation of ATP-sensitive K+ and voltage-dependent Ca channels [8]. Very recently it has been demonstrated that GPR119 can signal through not only Gαs but also Gαq, Gαi in addition to β-arrestin recruitment [9]. Taking into account the massive interest in GPR119 as a pharmaceutical target for the treatment of diabetes and obesity, its activation might therefore result in a beneficial dual response. However, the challenge to the medicinal chemists is to enhance the stability of normally unstable native ligands. Many of the known lysophospholipids are unstable when administered in vivo due to a labile phosphate moiety and this phenomenon limits their potential usefulness. Another factor limiting application of unmodified LPC may be intramolecular 1→2 migration of an acyl group [10]. Or aim was to protect LPC against the action of lysophospholipid phosphatases [11], [12] along with conservation of the 2-LPC configuration maintaining an acyl chain in the sn-1 position that is the most abundant LPL in nature [13]. All synthesized 2-OMe-LPC analogues (Fig. 1B) where an oxygen atom in position 2 of glycerol was protected by methylation 1.1(a–d) along with commercially available unmodified LPC (14:0, 16:0, 18:0, and 18:1; Fig. 1A) were subjected to biological characterization in GPR119-expressing pancreatic βTC-3 insulinoma cells (Fig. A.1). As far as GPR119 activation in β cells is associated with regulation of insulin secretion, primary biological assay performed among this study was dedicated to glucose-stimulated insulin secretion (GSIS). It was suggested that native LPC's activity is multidirectional and may proceed both directly and indirectly. Therefore we applied a concerted approach based on determination Tango β-arrestin translocation assay in engineered U2OS-GPR119-bla cell line, intracellular calcium mobilization, and cAMP levels. We also evaluated cytotoxic activity to measure proliferation, viability and lactate dehydrogenase leakage. Among the biological experiments dedicated to potential activation of GPR119 we included known GPR119 agonists, namely OEA being an endogenous ligand and synthetic oxadizone analogue PSN375963 (4-[5-(4-butylcyclohexyl)-1,2,4-oxadiazol-3-yl]-pyridine) [3], [8]. Finally, the possible binding mode of 2-OMe-LPC to the GPR119 receptor in the context of differences in chemical structure between various native LPCs and their 2-methoxy counterparts was determined. We created theoretical models of chosen LPC-GPR119 interactions to calculate differences in interaction energies, to predict the most important ligand interacting amino acid residues, and to correlate these data with the obtained biological results. We hope that presented multidirectional approach to characterization of native and modified LPCs' properties will help to understand the complexity of molecular mechanisms involved in signaling in pancreatic β cell lines and delineate potential role of various LPC types in modulation of β cell function.