Furthermore, 8-Bromo cAMP, a membrane permeable cAMP analogue, also suppressed caspase-3 activation thus supporting the results obtained with MRS2211. to be involved in beta cell survival and insulin secretion. These findings provide further support for the concept that P2Y13 plays an important role in beta cell apoptosis and suggest that autocrine/paracrine mechanisms, related to ADP and P2Y13 receptors, contribute to glucolipotoxicity. test using GraphPad InStat, Version 5.0 (GraphPad Prism Software, San Diego, CA, USA). For immunocytochemical Relugolix Caspase-3 activity in mouse islets, statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. Statistically significant differences were considered at denote probability level of random difference, by Student’s test. *for 3?min. Supernatants were transferred to 96-well Relugolix plates and analysed for Caspase-3 activity using a commercial kit. Result of Caspase-3 was normalized to protein concentration and expressed as the percentage switch in specific activity. Data are given as the means??SEM (show Hoechst staining for the represented islet. a Control treatment, islet treated Relugolix with low glucose (5.6?mmol/l). b Glucose treatment, islet treated with high glucose (16.7?mmol/l). c Palmitate treatment, islet Relugolix treated with palmitate (150?mol/l). d Control?+?MRS2211 treatment, islets treated as a with MRS2211 (10?mol/l). e Glucose?+?MRS2211 treatment, islets treated as b with MRS2211 (10?mol/l). f Palmitate?+?MRS2211 treatment, islets treated as c with MRS2211 (10?mol/l). g Frequency analysis of Caspase-3 activated islets cells to total number of islet cells. are normalized to control treatment. Statistical analysis, one-way ANOVA (equals 20?m CREB, Bad and IRS-1 are activated upon blocking the P2Y13 receptor To determine if pathways, important for cellular survival, are activated by autocrine/paracrine activation of the P2Y13 receptor, we carried out western blot analysis using antibodies against Ser-133 phospho-CREB, Ser-612 phospho-IRS-1 and Ser-112 phospho-Bad. MIN6c4 cells incubated in high glucose (25?mmol/l) medium in the presence of 10?mol/l of the P2Y13 receptor antagonist, MRS2211, displayed an enhanced CREB activation (Fig.?7a, b). Blocking P2Y13 receptor by MRS2211, in the presence of palmitate (100?mol/l), also elevated the CREB activation (Fig.?7c, d). These results were paralleled by the results of the analysis of Bad, since P2Y13 inhibition also produced a strong phosphorylation of Bad (Fig.?7e, f). The effect around the IRS-1 phosphorylation state was significant although less pronounced (Fig.?7g, h). Open in a separate window Open in a separate window Fig. 7 Effects of high glucose and palmitate around the phosphorylation status of CREB, IRS-1 and Bad in MIN6c4 Rabbit Polyclonal to EHHADH cells. MIN6c4 cells were incubated for 30?min in medium containing control, high glucose (25?mmol/l) or palmitate (100?mol/l) with or without MRS2211 (10?mol/l). Cell lysates were prepared and then subjected to western blot analysis (20?g protein/lane), using antibodies against phosphorylated ser-133-CREB, ser-112-Bad and ser-612-IRS-1, respectively. a, b ser-133-CREB. c, d ser-112-Bad. e, f ser-612-IRS-1 . Membranes were re-probed with the anti-CREB, anti-Bad and anti-IRS-1 antibody. Each represents the fold increase of phospho-CREB, phospho-Bad or phospho-IRS-1 relative to control after normalizing against total non-phosphorylated CREB, Bad or IRS-1. Data for western blots (b, d, f) are given as the means??SEM in each group ( em n /em ?=?3). * em p /em ? ?0.05, ** em p /em ? ?0.01 Autocrine mechanisms involving the P2Y13 receptor Relugolix influence the viability and proliferation of MIN6c4 cells To investigate the functional consequences of autocrine P2Y13 activation, created by palmitate or by high glucose, the.