The Role And Mechanism Of TRB3in Free Fatty Acid-induced Apoptosis In Pancreatic β Cells

Backgrounds:Diabetes, as one of the most common noncommunicable chronic diseases, has become the leading cause of disease and mortality burden worldwide. In2010, the estimated prevalence of diabetes Chinese adults was11.6%. The prevalence of diabetes has increased significantly in the recent decade and is now becoming epidemic proportions in China. Therefore, the pathogenesis, prevention and treatment of diabetes has become the research hotspot. More than90%of patients with diabetes is type2diabetes. Type2diabetes is characterized by insulin resistance and pancreatic β-cell failure,while the latter is the central link of type2diabetes. Therefore, how to protect and prevent islet β-cell dysfunction and apoptosis is critical prevention of type2diabetes.Epidemiological studies have shown that, about80to90percent patients with type2diabetes are obese or overweight. Accumulating evidence indicate that FFA are chronically elevated in obesity-linked T2DM. Numerous in vivo and in vitro studies have demonstrated the destructive effects of chronic exposure of FFA on β cell survival resulting in defective glucose-induced insulin secretion and β cell dysfunction and loss through apoptosis.The mechanisms underlying FFAs-induced β cell apoptosis and dysfunction may be associated with oxidative stress, endoplasmic reticulumstress, ceramide pathway, peroxisome proliferator-activated receptor pathway, hydrogen peroxide produced by peroxisomes, G protein-coupled receptors and so on. Recently, researchers discovered a new series of molecules, which could play an important role in pancreatic β-cell apoptosis induced by FFA. These molecules could serve as targets for therapeutic intervention, which may deter or delay pancreatic β-cell lipoapoptosis. Although the overall picture is known, mechanisms of lipoapoptosis in β cells are still very fragmentary and the details remain to be further examined.TRB3, the best studied member of the mammalian Tribbles family, coordinates crucial cellular processes, including adipocyte differentiation, lipid metabolism, regulation of collagen expression, and modulation of tumorigenesis. Several studies have described that TRB3promotes apoptosis, while others have revealed TRB3to possess an anti-apoptotic role. In diabetes mellitus, TRB3was involved in impairing insulin action in peripheral tissues by binding and inhibiting AKT/PKB phosphorylation. The potential significance of the regulation of TRB3function to FFAs-induced β cell apoptosis deserves further investigation.Aims1. Identify whether TRB3could be upregulated by the free fatty acids in vivo and in vitro; investigated the potential mechanism involved in the process.2. Identify the importance of TRB3in lipotoxicity induced β cell apoptosis and investigate the relevant mechanisms underlying TRB3’s activity in β cells.MethordsPart I:TRB3expression is upregulated by free fatty acid1. C57BL/6mice were injected intraperitoneally with or without palmitate for7days. Mice serum was separated the FFA concentrations were assayed following a modified version of the manufacturer’s protocol by the nonesterified fatty acid assay kit. Then pancreatic islets were isolated. Apoptosis of islets was determined by Caspase3/7activity assays.2. Quantitative real-time PCR and Western blot were utilized to test the mRNA and protein levels of TRB3、CHOP and ATF4in mice islets.3. To investigate the time-course of apoptosis induced by palmitate, INS-1cells were cultured in the presence of0.2mM palmitate for0,12,24,48h. To study the trend of apoptosis with changing concentrations of palmitate, cells were cultured with0,0.2,0.4,0.8mM palmitate for24h. Apoptosis was assessed by TUNEL staining.4. Quantitative real-time PCR and Western blot were utilized to test the mRNA and protein levels of TRB3、CHOP and ATF4.5. The siRNA of CHOP or negative control were introduced into INS-1cells by Lipofectamine2000, then TUNEL staining was utilized to test the apoptotic effect. The effects of CHOP silencing onTRB3expression was detected by Western blot.Part Ⅱ:TRB3is involved in free fatty acid-induced β cell apoptosis1. Establishment of INS-1derived cell lines, allowing inducible expression of TRB3by Tet-on system. The inducible TRB3cells were cultured in RPMI1640medium with G418, hygromycin, and10%FCS. TRB3cells were induced with500ng/ml Dox for48h. The induction of TRB3expression in the beta cell line was confirmed by Quantitative real-time PCR and Western blot and immunofluorescence staining.2. Caspase-3activity、TUNEL staining and Annexin V/PI staining were utilized to analysis the cell apoptosis in the normal and palmitate conditions with or without TRB3overexpression.3. The siRNAs of TRB3or negative control were introduced into INS-1cells or TRB3cells by Lipofectamine2000, then TUNEL staining and Caspase-3activity were utilized to test the apoptotic effect. The effects of on TRB3knockdown was detected by Western blot.4. TRB3cells were treated with or without500ng/ml Dox for48h to induce TRB3expression, and then incubated with or without0.2mM palmitate. The nuclear accumulation and activation of PKCδ was determined by western blot. TRB3cells were pretreated with U73122or rottlerin to block the activation or nuclear accumulation and then incubated with Dox followed by palmitate, cell apoptosis was assessed by TUNEL staining.5. Establish subrenal capsular transplantation animal model:Firstly, nu/nu mice were irreversibly induced to be diabetic by intraperitoneal injection with a relatively high single dose of STZ. At day6, TRB3cells (5×106) were transplanted into the left kidney of STZ-treated diabetic mice to establish the subrenal capsular animal model. The fasting blood glucose began to decline significantly18days post-transplantation, and from then on, we injected STZ intraperitoneally once daily with or without palmitate and Dox for8days. All mice were sacrificed at day32. Sections of the kidneys were then subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry. The different effect on apoptosis between groups was tested by TUNEL staining.6. Using the animal model described above, TUNEL staining was utilized assessed whether blocking PKC8activation by rottlerin could inhibit TRB3-mediated β cell apoptosis under high plasma FFAs conditions in vivo.ResultsPartⅠ1. High level serum free fatty acid resulted in an increased cell apoptosis in mice islets.We injected palmitate into mice intraperitoneally once daily for7days, and serum free fatty acid increased markedly. In addition, caspase-3/7activity in the isolated islets was increased in palmitate-injected mice (P<0.01).2. Free fatty acid up regulated the expression of TRB3、CHOP、ATF4in islets.Using quantitative real-time PCR, we found that TRB3、CHOP、ATF4mRNA in palmitate-injected mice were significantly increased. In addition, Western blot analysis revealed that TRB3and CHOP were upregulated in palmitate-injected mice.3. FFA induced apoptosis in INS-1cells in duration-and dose-dependent manner.TUNEL assay was used to investigate the time-course and dose-couse of apoptosis induced by palmitate, we found that palmitate induced apoptosis in INS-1cells in a duration-and dose-dependent manner. 4. TRB3, CHOP, ATF4were upregulated in INS-1cell by free fatty acid.Results of real-time PCR showed that, mRNA levels of TRB3, CHOP and ATF4were increased as the duration and concentrations increased. Meanwhile, Western blot analysis conformed that TRB3and CHOP was upregulated by palmitate in INS-1cells in duration-and dose-dependent manner.5. CHOP knockdown reduced the upregulation of TRB3and apoptotic effect induced by palmitateAccording to the result of western blot, CHOP knockdown reduced the upregulation of TRB3and CHOP induced by palmitate. In addition, TUNEL assay showed that CHOP silencing offset the apoptotic effect of palmitate in INS-1cells.Part II1. The inducible TRB3cell line was established by Tet-on system.In order to investigate the exact role of TRB3in palmitate-induced β cell apoptosis, we established a stable cell line capable of an inducible expression of TRB3by tet-on system, termed TRB3cells. According to the results of real-time PCR、Western blot and immunofluorescence analysis, induced with500ng/ml of doxycycline (Dox) for48h, TRB3expression was upregulated in TRB3cells.2. Overexpression of TRB3induced apoptosis and exacerbated lipoapoptosis.According to the results of Caspase-3activity analysis、TUNEL staining and AnnexinV/PI analysis, cell apoptosis was induced by TRB3overexpression and exacerbated by the addition of palmitate.3. TRB3knockdown significantly inhibited palmitate-induced βcell apoptosis.TUNEL staining showed that TRB3silencing largely offset the apoptotic effect of palmitate in TRB3cells and INS-1cells.4. TRB3mediated FFAs-induced apoptosis via PKCδ pathway. Accompanied with TRB3upregulation, PKC8was activated and accumulated in the nuclei, and these events were enhanced by the addition of palmitate. In contrast, TRB3silencing largely decreased the activity and nuclear accumulation. The inhibitor or preventing nuclear accumulation of PKC8reduced the apoptosis restricted to TRB3overexpression significantly, without altering the expression of TRB3. These results indicated that PKCδ is a novel and key downstream mediator of TRB3effects in lipoapoptosis.5. TRB3mediated FFAs-induced INS-1-derived cell apoptosis in vivo.Subrenal capsular transplantation animal model was used to validate our in vitro results. About2weeks post-transplantation, the fasting blood glucose of the mice began to decline gradually. Then, the mice were intraperitoneally injected once daily with Dox to induce TRB3overexpression and with palmitate to increase the plasma FFAs. After8days, the groups treated with Dox or palmitate exhibited increased blood glucose levels, which were further enhanced in the group treated with both Dox and palmitate. We assessed the apoptosis in TRB3cell xenografts by TUNEL staining for each treatment group. TRB3overexpression exacerbated FFAs-induced apoptosis.6.Blocking PKCS activation prevented TRB3-mediated β cell apoptosis under high plasma FFA conditions in vivo.We further assessed whether blocking PKCδ activation by rottlerin could inhibit TRB3-mediated β cell apoptosis under high plasma FFAs conditions in vivo. Injections of rottlerin inhibited PKCδ phosphorylation, which significantly prevented TRB3-mediated β cell apoptosis under high plasma FFA conditions. These results indicate that TRB3could activate PKCδ and mediate FFAs-induced INS-1-derived cell apoptosis in vivo.SignificanceThese studies provide novel insights into the central role of TRB3in lipoapoptosis in pancreatic β cells. TRB3expression is upregulated by free fatty acid via CHOP/ATF4 pathway in pancreatic β cells. We demonstrated that TRB3expression may explain FFAs-induced (3cell apoptosis and implicated the PKCδ pathway in this process for the first time. Inhibition of the TRB3/PKCδ axis could be relevant for preservation of β cell mass and function in conditions associated with increased serum FFA levels. These findings can be incorporated into a new potential therapeutic approach for the treatment of T2DM.