Study On The Mechanism Of Sphingomyelin Synthase 2 (SMS2) Gene Knockout Leading To Impaired Pancreatic Islet Function In Mice

Part Ⅰ Effects of SMS2 gene knockout on pancreatic islet function in miceObjective:To investigate the effect of SMS2 deficiency on the islet function in miceMethods:Spingomyelin synthase 2-knockout(SMS2-KO)mice was used as the research model.To observe the effects of SMS2 deficiency on glucose stimulated insulin secretion(glucose-stimulated insulin secreting)in mouse islet,we isolated islets from WT and SMS2-KO mice for GSIS and KSIS(potassium-stimulated insulin secreting).TEM was applied to distinct the ultrastructure and the number,size and density of insulin secretory granules of β cells in two groups of mice.Insulin and glucagon IHC staining(Immunohistochemisty)were also performed to investigate the proliferation of a cells and β cells in two groups of mouse.Last,Real-time PCR were also done to compare the expression of Pdx1,Ki67,Insl and Ins2.Results:(1)Compared with WT mice,GSIS(16.7mM glucose)of SMS2-KO mice islet was decreased,but the difference didn’t reach statistically significant While KSIS(50mM KCl)of SMS2-KO mice islet was significantly decreased.(2)Under the electron microscope,β cell organelles including Golgi complex,mitochondria and endoplasmic reticulum showed no obvious abnormalities.Compared with WT mice,area of insulin secretory granules in SMS2-KO β cell was increased,the density decreased and the gray of dense-nuclear decreased,which all had statistically significance.(3)IHC staining of insulin and glucagon showed:compared with WT mice,the ratio ofa cells and β cells in SMS2-KO mice islet had no significantly chang Also there were no significant changes in the expression of Pdxl,Ki67 and Ins1 and Ins2 genes.Conclusion:SMS2 deficiency leads to reduced GSIS and KSIS,which may be related to the changes of insulin secretory granules in beta cells.Part Ⅱ Primarily study on the mechanism of SMS2 knockout on the β cell dysfunctionObjective:To explore the mechanism of β cell dysfunction in SMS2-ko mice.Methods:Spingomyelin synthase 2-knockout(SMS2-KO)mice and INS-1 cell line in which SMS2 is overexpressed(INS-1 SGSMS2)or knockdown(INS-1 shRNA-SMS2)were used as the research models.Cell immunofluorescence technique was used to examine the expression of GLUT2(glucose transporter 2)and SNAP25 protein(synaptosomal-associated protein of 25 kDa)in islet beta cell of WT and SMS2-KO mice.Glucose analogue(2-NBDG)was used for glucose uptake experiments to compare the ability of glucose uptake between islets of WT and SMS2-KO mouse.Whole-cell patch clamp was applied to detect the changes of membrane capacitance(Cm)induced by current stimulation in β-cell of WT and SMS2-KO mouse and constructed INS-1 cells,which can indirectly reflect the secretion ability of insulin secretory granules in β-cell and constructed INS-1 cells.Results:(1)Glucose(2-NBDG)uptake during 60s,120s,300s,600s and 1200s were lower in islets of SMS2-KO mice than those in WT mice,which all have statistical significance.(2)Compared with WT mice,expression of GLUT2 was reduced inβ-cell of SMS2-KO mouse,however,there was no significant change in the expression of SNAP25.(3)Compared with WT mice.Cm1 and Cm2(Cm=IRP+RRP)were significantly reduced in pancreatic β-cell of SMS2-KO mouse(p<0.05).Similarly,compared with the control group,Cml and Cm2 in INS-1 shRNA-SMS2 cells also decreased significantly(p<0.05),however,there was no significant change in Cms in INS-1 SGMS2.Conclusion:SMS2 difficiency may reduce β-cell insulin secretion through change of membrane GLUT2 expression which decreased glucose uptake,thus affecting the secretion of insulin.Imbalance of membrane SM(sphingomyelin)dynamic caused by SMS2 knockout could impact the SM-enriched lipid rafts function.So,SMS2 knockdown may reduce the secretion of insulin by affecting the transport and secretion of intracellular insulin secretory granules.