The Interaction Between TERT And Par-4 Is Involved In The Mechanism Of Islet β Cells Apoptosis In Type 2 Diabetes Mellitus

Background and ObjectiveIn the previous study,we found Par-4 had an interaction with hTERT by yeast two-hybrid technology.It?s clear that Par-4 can promote the apoptosis of tumor cells,and there were specific functional areas of par-4 that play key roles in promoting cell apoptosis.Is the specific region of hTERT binding to Par-4 that promotes apoptosis of cells? We first use molecular biofilm interferometry to examine the direct interaction region of Par-4 and hTERT.Overexpression of TERT can lead to anti-apoptosis effect in islet βcells.However,the mechanism of TERT against apoptosis in islet β cells is still unclear.Par-4 is associated with a variety of age-related diseases,and Par-4 can induce apoptosis through the mitochondrial pathway.We first studied the interaction between Par-4 and TERT and its functional mechanism involved in apoptosis of islet β cells in vitro.Further studies are needed to determine an in vivo role of the interaction between TERT and par-4 in β cells apoptosis in type 2 diabetic mice.Methods1.hTERT was divided into four fragments according to its functional domains to construct the four deletion mutants.Then the full-length of Par-4 gene was constructed to interact with each deletion mutant to determine the specific binding region of hTERT.After the interaction region of hTERT was determined,three deletion mutants of Par-4 were constructed according to its functional regions to further verify the interaction between hTERT and par-4.Gene construction,synthesis and quality inspection were completed by Detai Biotechnology Company.Proteins expression and purification of hTERT deletion mutants,full length Par-4 and Par-4 deletion mutants were completed in E.coli system.Protein identification was completed by Western blot.Protein-protein interaction was detected by BLI.Full-length Par-4 protein was used to interact with each deletion mutant of hTERT,and only hTERT-2 can bind to Par-4.Then we further detected the interaction between hTERT-2 protein and each deletion mutant of Par-4.2.The NIT-1 cells were first divided into group C(normal group),group H12(high-glucose/fatty acid DMEM culture for 12 hours),group H24(high-glucose/fatty acid DMEM culture for 24 hours)and group H48(high-glucose/fatty acid DMEM culture for 48 hours).Then the cells were divided into Group C(control group),group H(high-glucose/fatty acid group),group C-Par-4(Par-4 inhibition group),and group H-Par-4(high-glucose/fatty acid + Par-4 inhibition group),CS group(SH5 inhibition group),HS group(high-glucose/fatty acid + SH5 inhibition group),CS-Par-4 group(SH5 inhibition+ Par-4 inhibition group),HS-Par-4 group(high-glucose/fatty acid + SH5 inhibition + Par-4 inhibition group).Cell survival rates were determined by MTT assays.Cell apoptosis was detected via TUNEL staining.Glucose-stimulated insulin secretion in each group was detected by ELISA.The protein expression of Par-4,TERT,Akt and p-Akt were measured by Western blot and immunocytochemistry.Immunofluorescence and immunoprecipitation were performed to detect the expression of Par-4 and TERT.3.The experimental animals were divided into N group(C57BL/6J mice),D group(C57BL/6J mice with type 2 diabetes),N-Par-4 group(normal diet Par-4-knockout C57BL/6J mice),and D-Par-4 group(Par-4-knockout C57BL/6J mice with type 2 diabetes).The body weight and tail vein blood glucose level of each animal were measured daily.At the end of the experiment,the pancreases were removed and stored at-80℃.The expression levels of Par-4 and insulin in serum were measured by ELISA.Protein expression of Par-4,TERT,Akt and p-Akt were tested by Western blot and Immunohistochemistry.The secretory ability of islet cells was evaluated using the HOMA-β index,and the apoptosis rate of islet β cells was determined by TUNEL staining.Results1.The proteins expression,purification and identification of the four deletion mutants of hTERT,the full length of Par-4 and its three deletion mutants were completed in the E.coli system.No direct interaction was observed between Par-4 and hTERT-1(1-183aa),hTERT-3(523-924aa)and hTERT-4(915-1132aa).However,hTERT-2(170-546aa)was detected to bind directly with Par-4 with five different concentrations.Further study showed hTERT-2 could bind directly to Par-4(1-299aa),Par-4(1-160aa)and Par-4(161-340aa)respectively.2.As the treatment time increased,Par-4 expression and the apoptosis rate increased significantly in cells treated with high glucose/fatty acid 48 hours compared with that in control cells and those treated for shorter periods of time(P < 0 05).Furthermore,TERT expression,cell survival rate and insulin secretion were significantly decreased in cells treated with high glucose/fatty acid 48 hours compared with those in control cells and those treated for shorter periods of time(P < 0.05).Inhibition of Par-4 in normal cells did not affect the TERT expression,the survival rate,apoptosis rate and insulin secretion(P>0.05).High-glucose/fatty acid treatment can increase cytoplasmic Par-4 levels and apoptosis rate while decrease cytoplasmic TERT levels,cell survival,and insulin secretion ability.Inhibition of Par-4 can increase cytoplasmic TERT levels,cell survival,and the insulin secretion ability under high-glucose/fatty acid conditions(P <0.05).The results of immunofluorescence and immunoprecipitation suggested that Par-4 and TERT interact mainly in the cytoplasm and Par-4 can inhibit the expression of TERT.High-glucose/fatty acid treatment induced Par-4 expression,inhibited TERT expression,and increased cell apoptosis.In contrast,reduced Par-4 expression in high-glucose/fatty acid condition could increase TERT expression and reduce cell apoptosis.Inhibiting Akt in the control group did not affect the apoptosis rate.High-glucose/fatty acid treatment can cause increases in Par-4 expression,and decreases in Akt and p-Akt expression,survival rates and eventrally lead to reduced insulin secretion,and increased NIT-1 cell apoptosis.Inhibition of Par-4 can alleviate these effects,whereas inhibiting p-Akt aggravates them.Thus,the Par-4-dependent induction of NIT-1 cell apoptosis mainly occurs via the Akt/p-Akt signalling pathway.3.There were no differences in apoptosis rate and insulin secretion between group N and group N-Par-4,suggesting that the knockout of Par-4 in normal mice did not affect apoptosis rate and insulin secretion.The apoptosis rate was significantly increased while the insulin levels and HOMA-β index were significantly decreased in type 2 diabetes group compared with normal group.Furthermore,the animals in D group had higher cell apoptosis rates and lower insulin levels and HOMA-β index than D-par-4 group,which suggested that Par-4 can promote the apoptosis of islet β cells in diabetic mice.The results of Immunocytochemistry and Western blot indicated that type 2 diabetes can induce the expression and secretion of Par-4 and reduce the expression of TERT,Akt,and p-Akt,leading to apoptosis and decreased insulin secretion.Inhibiting Par-4 expression can increase the levels of TERT,Akt,and p-Akt;reduce apoptosis;and improve the dysfunction of insulin secretion.Conclusion1.The full length and three deletion mutants of Par-4 can directly bind to hTERT-2(170-546aa),and we speculated that at least two sites of Par-4 can directly bind to hTERT-2(170-546aa).Therefore,the binding sites of Par-4 and hTERT are NLS and SAC(except NLS2)or LZ regions.2.Par-4 expression is positively associated with high-glucose/fatty acid treatment and the apoptosis rate,and negatively associated with TERT expression,the survival rate,and insulin secretion,and these effects are time dependent.Therefore,48 h was selected as the optimal treatment time.High-glucose/fatty acid treatment induces apoptosis of islet βcells via up-regulation of Par-4 expression and down-regulation of TERT expression.Par-4 interacts with TERT,and inhibition of TERT in the cytoplasm,as observed in NIT-1 cells with high-glucose/fatty acid treated,triggers Par-4 translocation to the nucleus.Diabetes activates Par-4 and inhibits p-Akt expression to induce islet β cell apoptosis.3.Par-4 can promote the apoptosis of islet β cells in diabetic mice.Type 2 diabetes can induce the expression and secretion of Par-4 and reduce the expression of TERT,Akt,and p-Akt,leading to apoptosis and decreased insulin secretion.Inhibiting Par-4 expression can increase the levels of TERT,Akt,and p-Akt;reduce β cells apoptosis;and improve the dysfunction of insulin secretion.