Thus, we conclude that the ability of OEA to maintain -cell viability in the presence of cytotoxic stimuli, does not involve any of the putative receptors known to bind OEA
Thus, we conclude that the ability of OEA to maintain -cell viability in the presence of cytotoxic stimuli, does not involve any of the putative receptors known to bind OEA. In an attempt to identify the target for OEA’s cytoprotective actions, experiments were undertaken with AM404, a compound that acts as an inhibitor of the uptake of certain fatty acid ethanolamides into cells (Hillard and Jarrahian, 2000; 2003; Fowler and Jacobsson, 2002; Hogestatt et al., 2005). BRIN-BD11 and INS-1E cells and OEA was cytoprotective in these cells. However, cytoprotection was not reproduced by any of a range of selective, synthetic ligands of GPR119. The cytoprotective response to OEA was lost during exposure to inhibitors of fatty acid amide hydrolase (FAAH) suggesting that OEA is not the cytoprotective species but that release of free oleate is required. Similar data were obtained with anandamide, which was cytoprotective only under conditions favouring release of free arachidonate. CONCLUSIONS AND IMPLICATIONS Activation of GPR119 is not required to mediate the cytoprotective actions of OEA in BRIN-BD11 or INS-1E cells. Rather, OEA is internalised and subjected to hydrolysis by FAAH to release free oleate, which then mediates the cytoprotection. 2011) has attracted considerable attention as ligands at this receptor stimulate insulin secretion in a glucose-dependent manner (Overton test. Materials Glutamine, penicillin/streptomycin and RPMI-1640 medium were purchased from Invitrogen (Paisley, Scotland). Fetal calf serum was purchased from PAA laboratories (Yeovil, England). Fatty acid free BSA was purchased from MP Biomedicals (Thame, UK). Palmitate, oleate, OEA, AM404 and anandamide were from Sigma (Poole, England). URB597 and URB532 were purchased from Calbiochem (Darmstadt, Germany) and JNJ-1661010 from Tocris (Bristol, UK). The polyclonal anti-fatty acid amide hydrolase (FAAH) antibody, FAAH blocking peptide and the Idasanutlin (RG7388) FAAH positive control of recombinant rat FAAH were all purchased Idasanutlin (RG7388) from IDS-Ltd (Newcastle, England). Rat FAAH primers were designed in house and supplied by Invitrogen. The cAMP Direct Biotrak EIA came from GE Healthcare (Little Chalfont, UK). Results Effects of OEA on the viability of BRIN-BD11 and INS-1 cells Initial studies revealed that treatment of either of two rat -cell lines (BRIN-BD11 or INS-1) with OEA at concentrations up to 250 M did not Mouse monoclonal to MER lead to any loss of viability during culture periods of at least 24 h (not shown). By contrast, and as expected from previous studies (Newsholme < 0.001) cells and was dose- dependent over the concentration range 5C100 M OEA. Essentially, complete protection of cell viability was achieved with 60 M OEA in BRIN-BD11 cells. When the cytoprotective actions of OEA were compared with those of its parent free fatty acid, oleate, the ethanolamide derivative appeared to somewhat more potent than oleate under the conditions used, although formal EC50 values were not established due to uncertainties about the absolute binding affinity of each fatty acid to BSA. Open in a separate window Figure 1 Effects of the mono-unsaturated fatty acid oleate and its ethanolamide derivative, OEA, on the loss of viability caused by exposure of BRIN-BD11 cells to palmitate. Cells were treated with 250 M palmitate in the presence of increasing concentrations of oleate or OEA as shown. Cell viability was assessed after culture for 18 h. ***< 0.001 compared with the equivalent concentration of OEA. In addition to its Idasanutlin (RG7388) ability to attenuate the cytotoxic effects of palmitate, OEA also provided dose-dependent protection against the loss of viability arising from withdrawal of serum from the cell culture medium over a period of 30 h in BRIN-BD11 cells (Figure 2). Again, OEA appeared to be marginally more potent than oleate under these conditions (Figure 1). Open in a separate window Figure 2 Effects of oleate and OEA against the loss of viability of BRIN-BD11 cells caused by removal of serum from the culture medium. BRIN-BD11 cells were incubated with increasing concentrations of oleate or OEA in serum free medium for 30 h. Cell viability was assessed at the end of this culture period. Effects of GPR119 agonists on cell viability, cAMP generation and insulin secretion in -cells Because OEA has been suggested to act as an endogenous agonist of the lipid responsive receptor GPR119 in mammalian cells (Overton < 0.001 compared with control; **< 0.001 compared with palmitate alone. These results led us to re-evaluate the actions of this range of GPR119 agonists in rat -cells because the majority of previously published functional studies have been conducted in HIT-T15 cells, a hamster -cell line. Initially, RT-PCR was used to amplify GPR119 transcripts from BRIN-BD11 and INS-1 cells.