Effect Of Pancreatic β Cells-specific Deletion Of SNX16 On Glucosestimulated Insulin Synthesis And Secretion In Mice

Objective:Insulin is released by pancreaticβcells when they were stimulated by endogenous or exogenous substances,such as glucose,lactose,glucagon etc.Insulin is the only protein hormone that lowers blood glucose in the body.Absolute or relative insufficient of insulin secretion will lead to hyperglycemia,even diabetes,in the body.Sorting Nexins（SNXs）family is characterized by containing the Phoxhomology domain（PX domain）in phagocytic oxidase.Studies have shown that SNXs,as membrane proteins,play important roles in endocytosis,protein sorting,cell signal transduction,membrane transport,membrane remodeling and organelle movement.Sorting Nexin 16（SNX16）is a member of the SNXs family,in which SNX16 is involved in regulating development,tumors and cardiovascular diseases,etc.The effect of SNX16 on blood glucose homeostasis has not been explored.This study was designed to investigate the effects of SNX16 on blood glucose homeostasis,insulin synthesis and secretion in pancreaticβcell-specific SNX16 knockout mice and pancreaticβcell lines.Methods:1.Preparation of pancreaticβcell-specific SNX16 knockout mice:Pancreaticβcell-specific SNX16 knockout mice were obtained by crossbreeding SNX16 Lox P mice with pancreaticβcell-specific expressing Cre mice and the genotypes were identified by PCR.Pancreaticβcell-specific SNX16 knockout mice contained both Lox P and Cre sequences as the experimental group which were referred as FF+.The mice that did not contain Cre sequences were named FF-mice,as the control group.Western-blot and Q-PCR were performed to verify the knockout efficiency of SNX16 in isolated islets from mice.2.Energy metabolism assay in mice:Metabolic parameters and physiological behaviors were monitored in FF-and FF+mice in TSE Phenomaster caging system.The food intake,water intake and other metabolic parameters were recorded and analyzed.3.Glucose tolerance test and insulin tolerance test:The mice were fasted for 16hours,then intraperitoneally injected with glucose or insulin,the blood glucose concentrations were measured at desired time point.4.Detection of insulin content in mouse serum:After fasting 6 hours,the mice were intraperitoneally injected with glucose,the serum was collected at 0 min,30 min and 60 min after the injection.The serum insulin concentration was determined by insulin Elisa kit.5.Immunohistochemistry:The mice were fasted for 6 hours,and intraperitoneally injected with glucose.After 1 hour,the mice were sacrificed by cervical dislocation,the pancreas was isolated,fixed,dehydrated,embedded,and sectioned.The insulin contents in pancreatic sections were detected by Immunohistochemistry and immunofluorescence,and the structure of the pancreas was examined with HE staining.6.Determining the expressions of genes by Western-Blot and Q-PCR.in SNX16deleted or overexpressing pancreatic beta cells.Preparations of primary pancreatic islets and Beta-TC-6 cells over-expressing SNX16:Primary pancreatic islets and Beta-TC-6 over-expressing SNX16 cells were stimulated with different concentrations of glucose.Total protein and RNA were extracted and the expressions of genes were determined by Western-Blot and Q-PCR.7.Measurement of the intracellular calcium concentrations:The Beta-TC-6 cells overexpressing SNX16 were stimulated with different concentrations of glucose and their corresponding intracellular free calcium were measured by Fura 3.Results:1.Pancreaticβcells-specific SNX16 knockout mice were obtained successfully,in which the genotypes were identified by Q-PCR and the expression levels of SNX16protein and m RNA in pancreatic islets were significantly decreased in F/F+mice compared with control group.2.Under ordinary drinking conditions,the food and drinking intake and energy metabolism were not significantly different between F/F-and F/F+groups.When supplying with 11%glucose in drinking water,there were abnormal energy metabolism and significant higher blood glucose level in F/F+mice.3.Glucose tolerance tests showed that the glucose tolerance of F/F+mice were impaired compared with F/F-mice.The insulin tolerance tests showed that there was no significant difference between two groups.4.The insulin concentration of F/F+group were significantly lower than control F/F-group under glucose stimulation,indicating that insulin secretion was remarkedly reduced in F/F+group.5.There were less insulin contents in F/F+pancreatic islets and HE staining revealed that the lack of SNX16 did not modify pancreatic development and tissue structures.6.The insulin synthesis and proinsulin gene expression were significantly decreased in F/F+group.On the contrary,overexpression of SNX16 will lead to an increase in insulin synthesis and an increase in proinsulin gene expression in Beta-TC-6 pancreatic cells.7.Overexpression of SNX16 enhanced[Ca²⁺]i in the Beta-TC-6 cells during glucose-stimulated insulin secretion.Conclusion:Specific knockout of SNX16 gene in pancreaticβ-cells significantly reduced the synthesis and secretion of insulin and impaired glucose tolerance in mice.Overexpression of SNX16 would increase insulin synthesis and secretion via increasing the influx of calcium ions in the Beta-TC-6 cells in the first and second phases of glucose stimulation.Our study demonstrated that SNX16 played an important role in maintaining glucose homeostasis through modulating the biosynthesis and secretion of insulin.