SNX16 Is Involved In Insulin Synthesis And Secretion In Pancreatic Islet β Cells Via IR/PI3K/AKT Signaling Pathway

Objective: Type 2 diabetes mellitus(T2D)has long been referred to as non-insulin-dependent diabetes,or adult-onset diabetes characterized by insulin resistance.The interaction between insulin resistance and islet β cell dysfunction is the main factor promoting the development and progression of type 2 diabetes.Insulin is the only hormone secreted by the β cells that lowers blood sugar in the body.Insulin signaling first binds to insulin receptors(IRs)on the cell surface and triggers activation of the signaling cascade to regulate metabolism and cell growth.Tyrosine phosphorylated IR recruits and phosphorylates IR substrates,which subsequently activate PI3 K.PI3K/AKT signaling affects islet β cell metabolism and apoptosis by regulating insulin-related signaling pathways,thereby affecting insulin secretion and blood glucose homeostasis.SNX16 is a unique member of the SNX family,consisting of a PX domain and a curly coil(CC)domain.The PX structure locates SNX16 in endosomes,and recruits the cargo proteins to a variety of endomembrane systems by binding to phosphoinositol of the endosomal membrane,thereby regulating endocytosis and intracellular sorting of the cargo protein.Previous studies in our laboratory have suggested that pancreatic SNX16 deletion had altered metabolic function,and the synthesis and secretion of insulin in pancreatic islet β cells were significantly reduced.This study aims to explore the effect of SNX16 on the synthesis and secretion of insulin with pancreaticβ cell-specific knockout of SNX16 mice and its mechanism.Methods: 1.Construction of islet β cell-specific knockout SNX16 mice: SNX16flox/flox mice were used to mate with RIP CRE mice to obtain pancreatic β cell-specific knockout SNX16(F/F+)mice with Cre and(F/F-)mice without CRE 2.Construction and identification of SNX16 overexpression stable cell line: 293 T cells were used to prepare lentivirus containing SNX16 c DNA which were used to transfect the target cells 1.1B4,and then the SNX16-1.1B4 cells were screened and selected by puromycin,and the transfected efficiency was judged by fluorescence density;Western-blot and RT-q PCR were used to analyze the expression of SNX16 in 1.1B4 stable cell lines.3.The effect of SNX16 on insulin synthesis and secretion: the experimental group and control group mice were injected intraperitoneally with normal saline or glucose solution,pancreatic tissues were extracted,and the expression of insulin was detected by immunohistochemistry;Mouse islets were extracted,and stimulated with different concentrations of glucose,and the secretion of insulin was detected by Elisa kit in the supernatants.4.Detection of cellular proliferation via CCK8 method.1.1B4 stable cell lines were inoculated in 96-well plates,and high or low concentration of glucose were given for 16 hrs,then CCK8 tests were performed.5.Effect of SNX16 on IR/PI3K/AKT signaling pathway.mouse islets and,SNX16 1.1B4 cell lines were first starved with sugar-free KRBH solution for 2 hours,different concentrations of glucose or insulin were added.The proteins were extracted,and the expressions of IR/PI3K/AKT were detected by Western-blot.6.Effect of SNX16 on the expression of glucose transporter 2(Glut2).SNX16-1.1B4 cells were cultured,then starved with glucose-free KRBH solution for 2 hours,and stimulated with different concentrations of glucose.The cellular proteins and m RNA were extracted,and the expression of Glut2 was analyzed by Western-blot and RT-q PCR.7.Effect of IR inhibitors on insulin synthesis and secretion.IR inhibitors were applied into cells.The expressions of IR/PI3K/AKT were detected by Western-blot.Cellular RNA was extracted and the expression of Glut2 and Ins1 m RNA was analyzed by RT-q PCR.Cultured supernatants were collected and the contents of insulin were detected by the Elisa kit.8.Effect of PI3 K inhibitors on insulin synthesis and secretion.PI3 K inhibitors inhibitors were applied into cells.The expressions of PI3K/AKT were detected by Western-blot.Cellular RNA was extracted and the expression of Glut2 and Ins1 m RNA was analyzed by RT-q PCR.Cultured supernatants were collected and the contents of insulin were detected by the Elisa kit.Results: 1.SNX16-1.1B4 were successfully constructed and confirmed by flurescence of EGFP and the expressions of SNX16-EGFP fusion protein.2.The results of immunohistochemistry and insulin Elisa kit showed that the synthesis and secretion of insulin were significantly reduced in pancreatic islets with β cell specific deletion of SNX16.3.Under the stimulation of high concentration of glucose,the proliferation capacity of cells in the SNX16-1.1B4 was significantly higher than that of the control group.The results showed that SNX16 overexpression improved glucose-induced proliferation of islet β cells.4.Knocking out SNX16,the phosphorylations of IR and AKT were significantly reduced in pancreatic islets under the stimulation of high concentration of glucose.Conversely,when SNX16 was overexpressed,the phosphorylation levels of IR,PI3 K and AKT were increased significantly under high glucose or shortterm insulin induction.5.SNX16 overexpression significantly increased the expressions of Glut2 under high concentration of glucose.6.IR inhibitors and PI3 K inhibitors significantly inhibited the IR/PI3K/AKT pathway,significantly reducing the increase in Glut2 expression and insulin synthesis and secretion which were initiated by SNX16 overexpression.Conclusion: SNX16 is essential for insulin synthesis and secretion,and ipancreatic β cellspecific knockout of SNX16 significantly inhibited IR/PI3K/AKT signaling pathway and reduced glucose-induced insulin synthesis and secretion.Conversely,the overexpression of SNX16 in the 1.1B4 cells enhanced the IR/PI3K/AKT signaling pathway,increased the expression of Glut2,and promoted the activity and proliferation of cells.Our results show that SNX16 plays a positive regulatory role in insulin synthesis and secretion,and SNX16 regulates insulin synthesis and secretion in pancreatic β cells through the IR/PI3K/AKT signaling pathway,thereby participating in the regulation of blood glucose homeostasis.