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  • br Materials and methods br Results br Discussion and

    2021-10-13


    Materials and methods
    Results
    Discussion and conclusions Our data demonstrate that native and 2-OMe-LPC analogues are GPR119 ligands and activate insulin secretion from βTC-3 pancreatic cells. LPC 18:1 was the first proposed endogenous ligand for GPR119 [1], however natural LPC has limited therapeutic value due to poor stability and relatively short half-life (minutes) [50]. Blocking a metabolically labile functional group at the sn-2 position of the LPC by methylation greatly enhances stability and protects against intramolecular 1→2 migration of an acyl group [12], [16]. In this study 2-OMe-LPC 14:0 appeared to be the most potent stimulator of insulin secretion, hence, its influence is clear even at low dosage affected by binding to BSA. Weaker or no influence on secretory activity of the cell model was observed in the case of the native and modified LPCs with longer acyl residues. We suspect that this atypical observation may be related to secretory properties of the βTC-3 cell line, which is less efficient compared with other insulinoma (i.e. NIT-1 or MIN6) [1], [51], [52]. Thus, βTC-3 could potentially serve as a β cell model with diminished ability to produce insulin, which accentuates therapeutical properties of the short-chain LPCs. As oppose to LPC, OEA was shown to stimulate insulin secretion partially independently of glucose concentration. It is consistent with previous observations in BRIN-BD11 rat β cell line [48] which makes it a potential hypoglycemic factor. WIN 18446 A synthetic GPR119 agonists, namely PSN375963, did not affect insulin secretion in βTC-3 cells. PSN375963 was previously reported not to stimulate insulin secretion in MIN6c4 and BRIN-BD11 up to 10-μM working concentration [8], [48]. Only 100-μM concentration was found to stimulate insulin secretion in BRIN-BD11 at high glucose conditions by 20% with respect to control whereas even 10nM concentration of OEA leads to enhanced GSIS in the same cell line [48]. The RT-qPCR analysis confirmed GPR119 mRNA expression in βTC-3 WIN 18446 hence we assumed that 2-OMe-LPC may act as ligands of this receptor. Indeed, all the tested compounds led to activation of GPR119 in Tango™ GPR119-bla U2OS cell line and triggered β-arrestin translocation signals. The observation that not all LPC species enhanced GSIS in the GPR119-expressing βTC-3 cells supports the theory about complexity of biological activity of the test compounds. We could suspect even ligand-dependent biased signaling [9], especially due to the observed differences between activities of the two control agonists of GPR119. Synthetic PSN375963 is clearly more potent activator of GPR119 in Tango™ GPR119-bla U2OS engineered cell line compared to OEA, it does not stimulate insulin secretion in βTC-3. Hence, we decided to study the matter of intracellular signaling triggered by the test compounds in β-TC3 more thoroughly. LPC 18:1 was previously shown to activate GPR119-Gαs pathways in RH7777 rat hepatoma cells stably expressing human GPR119 [1]. Unlike OEA, which appeared to stimulate cAMP accumulation also in many other GPR119-expressing cell lines (e. g. GPR119-EGFP-expressing HEK293 [4], mGLUTag [47], BRIN-BD11 [48]), natural and modified LPC species 1–1.1(a–d) led to lowering of intracellular cAMP concentration in βTC-3 cells. However, decrease in cAMP accumulation does not exclude possibility of GPR119 activation as it was found for one of its specific synthetic agonists – PSN375963, which affected cAMP synthesis differently dependent on the studied cell model [3], [8]. Due to this observation we assume that decrease in cAMP content in βTC-3 cells after stimulation with 1–1.1(a–d) is related to specific characteristics of the cell line and is a resulting effect of interactions with GPR119 and/or other lipid receptors expressed by the cell line. What is more, different natural LPCs (7:0; 10:0; 16:0; 18:1) as opposed to OEA were found to stimulate or arrest cAMP synthesis in COS-7 cells transiently expressing GPR119 dependently on their concentration [53]. Thus, the observed decrease in intracellular cAMP caused by stimulation of βTC-3 with the LPC-based test compounds may be related to biased signaling recently discovered for GPR119 [9]. One can suspect that 10-μM dose of LPCs and their analogues triggered signaling pathway switch from Gαs to Gαi at GPR119 expressed in βTC-3. LPC dose-dependent cAMP formation cannot be excluded as well.