Tibbles Homolog 3 Participating In High Glucose Induced Beta Cell Apoptosis

The beta cell dysfunction is an important character of the late stage of type 2 diabetes.High glucose inducing beta cell apoptosis is a crucial cycle in the process of beta cell dysfunction.It is important for understanding the pathogenesis of type 2 diabetes to investigate the molecular mechanism of high glucose induced beta cell apoptosis.Tibbles homolog 3(TRB3) was found holding lots of cellular biological functions.TRB3 can inhibit the insulin signal pathway by binding AKT and associate with the onset of insulin resistant.In addition,TRB3 regulates ER stress through the ATF4/CHOP pathway.There was still no report about the relationship between TRB3 and high glucose induced beta cell apoptosis.Objectives:To explore the expression of TRB3 in pancreatic beta cells and the effect of TRB3 on high glucose induced beta cell apoptosis.Methods:The expression of TRB3 gene was detected by the real-time PCR in diabetic GK rat islets and INS-1 cells in high glucose condition.The DOX inducible TRB3 stable cell line was established in INS-1 cells by tetracycline Tet-on system.The induction of TRB3 expression in the beta cell line was confirmed by real-time PCR,immunofluorescence staining and Weston Blot,respectively.In high glucose condition,the effect of TRB3 over expression on cell proliferation and insulin secretion in beta cell line was respectively detected by MTT and ELISA.The beta cell apoptosis was respectively analyzed by DNA fragmentation,TUNEL staining and flow cytometry in the normal and high glucose conditions before and after TRB3 over expression.Furthermore,the related mechanisms of TRB3 participating in beta cell apoptosis were analyzed:the morphology of mitochondria and distribution were checked by cofocus microscope;the caspase-3 was detected by ELISA.The△ψm(membrane potential) of mitochondria and ROS(reactive oxygen species) production were determined by flow cytometry.Results:The mRNA expression of TRB3 was higher in diabetic GK rat islets than that in normal GK rat islets.The mRNA expression of TRB3 in INS-1 cells was significantly up-regulated by glucose.The DOX inducible TRB3 cell line was established and TRB3 expression was induced by DOX as dose and time dependent manner.In high glucose condition,TRB3 over expression in the inducible cell line suppressed cell proliferation and insulin secretion.By detection with DNA fragrmentation,TUNEL staining and flow cytometric analysis,more cell apoptosis was found in TRB3 over expressed cells than that in TRB3 normal expressed cells in high glucose condition.However,the number of cell apoptosis was significantly reduced when TRB3 expression was inhibited by siRNA technology.Furthermore,we found that the morphology of mitochondria was changed,the release of cytochrome C was increased,the△ψm of mitochondria was decreased,caspase-3 activity was up-regulated and ROS content was increased in TRB3 over expressed beta cells in high glucose condition,suggesting these factors might be related to the mechanisms of TRB3 participating in high glucose induced cell apoptosis.Conelusions:TRB3 participates in high glucose induced beta cell apoptosis.