The Role And Mechanism Of TRB3 In AGEs-induced Oxidative Damage And Apoptosis In β-cells

In the pathogenesis of type 2 diabetes, many important factors such as high blood glucose, high free fatty acids (FFAs), inflammatory cytokines and amyloid deposits can lead to β-cell damage and apoptosis. Recent studies showed that advanced glycation end products (AGEs) could also promote β-cell damage through receptor for advanced glycation end products (RAGE) in the pathogenesis of type 2 diabetes. The production is accelerated when blood glucose is high, thereby increasing the accumulation of AGEs in the body, therefore, AGEs might be an important factor leading to pancreatic β-cell failure in diabetes. However, previous studies have not focused on the molecular mechanisms of AGEs on β-cells. Therefore, the roles of AGEs on β-cell apoptosis and their mechanisms warrant further study.Tribbles homolog 3 (TRB3), a protein that have no kinase activity, can act as a scaffolding-like protein bind to others signaling molecules in several signal pathways with multiple biological functions.TRB3 is widely expressed in insulin targeted tissues and and closely related to insulin resistance through the inhibition of insulin-signaling pathway. In addition, TRB3 can be induced by C/EBP homologous protein (CHOP) which is a mediator of endoplasmic reticulum (ER) stress-mediated apoptosis, and is involved in promoting apoptosis. However, the role of TRB3 in apoptosis remain controversial. Our previous studies showed that TRBs played an important role in β-cell apoptosis. High blood glucose, high fat, and ER stress upregulate the TRB3 that mediates β-cell apoptosis. whether TRB3 involved in AGEs-induced β cell damage deserves investigation.Objectives To examine the potential influence of AGEs and TRB3 on pancreatic islet β-cell damage in vivo. To identify the role of TRB3 in AGEs-induced β-cell damage and its mechanism.Methods Analysis of the relationship between TRB3 and AGEs levels and cell apoptosis:In vivo experiments:Serum levels of AGEs in diabetic rats were assayed by enzyme linked immunosorbent assay (ELISA); Caspase-3/7 activity in isolated rat islets was assayed by Caspase-Glo3/7 assay; expression levels of RAGE and TRB3 in isolated rat islets were assayed by Real-time PCR and Western blot.In vitro experiments:Cell viability was analyzed when INS-1 cells were cultured in conditions with different concentrations of AGEs. After treated with AGEs, apoptosis in INS-1 cells were assessed by caspase-Glo3/7 assay, TUNEL staining and flow cytometric assay; expression levels of RAGE, TRB3 and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) were measured; reactive oxygen species (ROS) levels were measured by a reactive oxygen species assay kit.Analysis of the mechanism of TRB3 in AGEs-mediated injury in INS-1 cells:Dox was used to induce TRB3 expression in TRB3 cells and lentiviral vector-mediated gene silencing was used to knock down expression of TRB3. The effect of TRB3 knockdown or overexpression on TRB3 or NOX4 expression, cell apoptosis and ROS generating were measured respectively.Analysis of the influence of TRB3 on PKC signaling pathway:After treated with AGEs, the effect of TRB3 knockdown on phosphorylation of AKT, nuclear translocation of protein kinase C 8 (PKC δ), expression and membrane translocation of PKC β2 in INS-1 cells. INS-1 cells were pretreated with LY333531 to block the activation of PKC 02 and then co-treated with AGEs, cell apoptosis, ROS generating and NOX4 expression were measured respectively.Results The levels of serum AGEs in diabetic GK rats were gradually increased during diabetes. Meanwhile, caspase 3/7 activity and expression of RAGE, TRB3 and CHOP in isolated islets of diabetic GK rats were increased.Results of CCK-8 showed that,200 ug/ml AGEs significantly reduced cell viability as 48 h treatment. After treated with AGEs (200 μg/ml) for 48 hours, apoptosis increased in INS-1 cells, expression levels of RAGE, TRB3 and NOX4 as well as ROS level were also increased.After overexpression of TRB3, AGEs-induced apoptosis, expression of NOX4 and producing of ROS were increased, conversely, TRB3 silencing significantly reduced AGEs-induced apoptosis, expression of NOX4 and producing of ROS.Results of Western blot showed that, PKC β2 expression and membrane translocation in INS-1 cells were both up-regulated after AGEs treatment. After TRB3 expression was inhibited, PKC β2 expression and membrane translocation were both down-regulated. Furthermore, after pretreated with a PKCβ2 specific inhibitor, the AGEs-induced INS-1 apoptosis, the activity of NOX4, and intracellular ROS levels were significantly reduced.Conclusion TRB3 mediated AGEs-induced oxidative injury and apoptosis in β-cells through the PKC β2 pathway.