1.CASK,APBA1,and STXBP1 Collaborate During Insulin Secretion 2.miR-139-5p Mediates Palmitate-induced Insulin Secretion By Targeting NPTX1 In INS-1 Cells

Part 1 CASK,APBA1,and STXBP1 collaborate during insulin secretionBackground:Calcium/calmodulin-dependent serine protein kinase(CASK)belongs to membrane-associated guanylate kinase(MAGUK)protein family,which plays an important role in insulin secretion and contains several protein-protein interaction domains.Here,we explored the CASK-interactng proteins during insulin secretion.Methods:The endogenous CASK interactome in INS-1 cells during insulin secretion were measured by co-immunoprecipitation(Co-IP)and liquid chromatography-mass spectrometry(LC-MS/MS).GO analysis and Ingenuity Pathway Analysis(IPA)were carried out to explore the bioinformatic implication.CASK-specific small interfering RNA(siRNA),pEGFP-N2-Cask and pEGFP-C2-Apbal-?CI plasmids were transiently transfected in INS-1 cells using the Lipofectamine 2000 reagent.Mice with conditionally CASK deleted in islets were constructed by Cre-loxp recombination system.During insulin secretion,the interaction and localization of CASK and CASK-interacting proteins in INS-1 cells or in islet of conditional CASK knockout mice were examined by Co-IP,confocal microscopy and subcellular fractionation analysis.Besides,we established lipotoxicity damage cell model and high fatty-diet induced diabetic mice model to explore the correlation between high fatty and CASK as well as CASK-interacting proteins.Results:Using co-immunoprecipitation,liquid chromatography-mass spectrometry and bioinformatic analysis,we identified that CASK,Adapter protein X11 alpha(APBA1),and Syntaxin binding protein 1(STXBP1)formed tripartite complex during insulin secretion.CASK enhanced APBA1-STXBP1 interaction and mediated their traffic from cytoplasm to plasma membrane during insulin release.High fatty acid stimulation decreased insulin secretion along with CASK,APB A1,and STXBP1 expression;Cask overexpression enhanced CASK/APBA1/STXBP1 tripartite complex function,and may thereby rescue lipotoxicity-induced insulin-release defects.Conclusion:Collectively,our results illustrated the function of CASK in insulin granules exocytosis,which broadens the underlying mechanism of insulin secretion and highlights the clinical potential of CASK as a drug target of type 2 Diabetes Mellitus.Part2 miR-139-5p mediates palmitate-induced insulin secretion by targeting NPTX1 in INS-1 cellsBackground:High fatty acid reduces insulin secretion in pancreatic ? cells,but the precise mechanisms remain unclear.Previous study determined that miR-139-5p was increased in diabetic pancreatic tissues.However,to date,there is no study exploring whether or not miR-139-5p is involved in high fatty acid-induced insulin secretion.Methods:INS-1 cells were exposed to different concentration(0.1,0.2,0.4 mM)of palimitate in different time(12,24,48 h).The expression levels of miR-139-5p and NPTX1 were evaluated by real-time PCR or western blot.The regulation of NPTX1 by miR-139-5p were examined using luciferase assays.Cell transfection was conducted by Lipo8000 or Lipofectamine RNAiMAX.Potassium or glucose-stimulated insulin-secretion levels were used to verify the function of miR-139-5p or NPTX1 in insulin secretion.Insulin secretion levles were detected by radioimmunoassay kit.Results:miR-139-5p was increased in INS-1 cells stimulated with palmitate.Using bioinformatics analysis and luciferase assay,we demonstrated that miR-139-5p could bind to the 3' UTR region of NPTX1.Overexpression of miR-139-5p could decrease the level of NPTX1 suggeting that miR-139-5p was able to negatively regulate the expression of NPTX1.Besides,palmitate declined the expression of NPTX1 and NPTX1 was still negatively regulated by miR-139-5p in high fatty condition.In addition,overexpression of miR-139-5p or knockdown of Nptxl decreased insulin secretion.Moreover,overexpression of Nptxl could resuce palmitate-induced insulin release defects,while upregulation of Nptxl failed to rescue fatty-acid mediated insulin secretion defects,when miR-139-5p mimics were co-transfected with pEGFP-N2-Nptxl.Besides,Knockdown of miR-139-5p could reverse high fatty acid-induced insulin secretion defects.But when miR-139-5p inhibitors were co-transfected with NPTX1 siRNA,knockdown of miR-139-5p was not able to reverse palmitate-induced insulin secretion defects.Conclusion:Collectively,our results illustrated that the expression of miR-139-5p was elevated while NPTX1 was declined in high fatty condition.Besides,NPTX1 could negatively regualted by miR-139-5p.Moreover,the induction of beta cell insulin secretion defects by fatty acids is mediated,at least in part,by miR-139-5p via downregulation of Nptxl expression.