Effect And Mechanism Of Lysosomal Membrane Protein Sidt2 On Insulin Secretion

Objective:Previous studies showed that lysosomal membrane protein?Sidt2?eviscerated mice showed disorder of glucose metabolism and impaired insulin secretion.On the basis of this study,the specific mechanism of Sidt2 affecting insulin secretion was further explored by the detection of insulin secretion related proteins.To deepen the understanding of the relationship between lysosomal membrane protein?Sidt2?and diabetes mellitus,and enrich the pathogenesis of diabetes.Methods:1?Through LoxP-Flox-LoxP system?Gene recombination and targeting technology to get Sidt2^-/-mouse model,Mating with wild-type B129 strain mice,Extract DNA from the rat tail and identify the genotype,choose heterozygous Sidt2+/-offspring mice,mating again,finally choose the Sidt2^-/-offspring mice,and Sidt2^-/-male mice were selected as experimental group,Sidt2^+/+wild male mice born in the same nest or in the same batch were used as controls.After DNA identification,the pancreatic islets were observed by optical microscope and dissecting microscope.2?Detect the glucose tolerance in Sidt2^+/+and Sidt2^-/-mice.3?Immunohistochemistry and immunofluorescene detection of insulin in pancreatic tissues of Sidt2^+/+and Sidt2^-/-mice.4?The expression of SNAP25,VAMP1,Syntaxin,Syntaxin1 were detected by Western blot,which are key proteins that involved in insulin secretion.5?Using small interfering RNA technique to knock down Sidt2 at the level of INS1cells,and detect the expression of key proteins involved in insulin secretion by Western blot.6?Detect the insulin secretion in INS1 cells stimulated by high glucose.7?Using overexpression vector to overexpression Sidt2 at the level of INS1 cells,and detect the expression of key proteins involved in insulin secretion by Western blot.8?Three pairs of primers of the promoter sequences of Sidt2 gene were designed.In98 cases of patients with type two diabetes and 67 cases of normal human serum DNA as template,PCR,and sequencing of the PCR product for single nucleotide mutation sites,and to find the single nucleotide mutation sites for statistical analysis.Results:1?It is proved that the Sidt2^-/-gene knockout mouse model is constructed from the DNA molecular level.2?Sidt2^-/-mice in the growth and development,the luster of the skin and the general response to stress and other general aspects than the wild type are backward.Sidt2elimination leads to disorder of glucose metabolism.Under the optical microscope and dissecting microscope,the pancreatic islets of the control group were larger in size,more regular in morphology,and more in number,while the islets of Sidt2^-/-mice were smaller in size,irregular in morphology and less in number.3?The expression of insulin in pancreatic tissue of Sidt2^-/-mice was lower than that of control group by Immunohistochemistry assay?P<0.05?.4?Western blot detection showed that the expression of SNAP25,VAMP1,Syntaxin,were significantly lower in the Sidt2^-/-mice than in the wild-type mice?P<0.05?.5?The expression of Sidt2 in the INS1 cell line of rat islet tumor cells was detected by western blot,and the expression of VAMP1 was decreased in Sidt2 knockdown INS1cells compared with the control group?P<0.05?.6?The expression of Sidt2 in the INS1 cell line of rat islet tumor cells was detected by western blot,and the expression of SNAP25,VAMP1,Syntaxin were corrected in Sidt2 overexpression INS1 cells compared with the Sidt2 knockdown group.7?Three polymorphic sites A1521G,G1816A,C502A were found.Among them,the SNP of C502A has statistical significance between the two groups..Conclusion:1?Sidt2^-/-can affect the morphology of islet cells in mice,mainly manifested in the small size of islet cells,irregular shape,reduced number of islets.2?Sidt2 is a multiple transmembrane lysosomal membrane protein that involved in the secretion of insulin granules.Sidt2 elimination inhibited the expression of SNAP25,VAMP1 and Syntaxin which are key proteins that involved in insulin secretion and thus hindered the normal exocytosis of insulin secretion granules.Lead to insulin secretion disorder of pancreatic cells;eventually enter the stage of diabetes.3?At the cellular level,the down-regulation of sidt2 expression on the expression of SNARE protein was further verified,and this down-regulation can be corrected by exogenous Sidt2.4?C502A of Sidt2 gene polymorphism is associated with type 2 diabetes mellitus in Chinese Han population,and CA genotype may increase the risk of type 2 diabetes mellitus in Chinese Han population.