Effectofvaspinonthelevelofpalmiticacid-inducedautophagyinINS-1cells

Abstract:

Objective：Toinvestigatetheeffectoftheadipocytokinevaspinonthelevelofpalmiticacid（PA）-inducedautophagyinINS-1cells.Methods：INS-1cellsweredividedintogroupsandculturedasfollows：normalgroup：culturedinanordinarymediumfor24hours；PAgroup：culturedwith0.5mmol/LPAfor24hours；PA+vaspingroup：pre-culturedwith320ng/mlvaspinfor2hours，andthenco-culturedwith0.5mmol/LPAand320ng/mLvaspinfor24hours；PA+vaspin+rapamycingroup：pre-cul－turedwith50nmol/Lrapamycinfor2hours，andthenco-cul－turedwith0.5mmol/LPAand320ng/mLvaspinfor24hours；PA+vaspin+3-methyladenine（3-MA）group：pre-culturedwith1mmol/L3-MAfor2hours，andthenco-culturedwith0.5mmol/LPAand320ng/mLvaspinfor24hours.Theglucose-stimulatedinsulinsecretion（GSIS）test（withglucoselevelsof3.3and16.7mmol/L）wasperformedtodeterminetheinsulinlevelineachgroup；Westernblotwasusedtodeterminethelevelsofmammaliantargetofrapamycin（mTOR），p-mTOR，Beclin-1，LC3-I，LC3-II，andP62；mRFP-GFP-LC3andflowcytometrywereusedtodeterminetheautophagicflowandcellapoptosis，respectively.Results：Comparedwiththenormalgroup，thePAgrouphadsignificantlyreducedGSISlevelsintheINS-1cellsupernatant[（1.02±0.08）ng/mLvs.（0.54±0.06）ng/mL，P=0.000；（1.15±0.13）ng/mLvs.（0.71±0.05）ng/mL，P=0.000）]，whilethevaspin-treatedgrouphadsignificantlyincreasedGSISlevelsthanthePAgroup[（0.76±0.03）ng/mLvs.（0.54±0.06）ng/mL，P=0.001；（0.91±0.04）ng/mLvs.（0.71±0.05）ng/mL，P=0.011）].Comparedwiththenormalgroup，thePAgrouphadasignificantlyreducedp-mTOR-to-mTORratio（2.04±0.14vs.1.19±0.09，P=0.000），whilethevaspin-treatedgrouphadasignificantlyincreasedratiocomparedwiththePAgroup（1.51±0.05vs.1.19±0.09，P=0.005）.Comparedwiththenormalgroup，thePAgrouphadsignificantincreasesintheleveloftheautophagy-associatedproteinBeclin-1andtheratioofLC3-IItoLC3-I（0.65±0.04vs.0.81±0.07，P=0.005；1.16±0.11vs.2.35±0.25，P=0.000）andasignificantreductioninP62level（1.04±0.19vs.0.60±0.08，P=0.000）；thevaspin-treatedgrouphadsignificantdecreasesintheBeclin-1levelandtheLC3-Ⅱ-to-LC3-Ⅰratio（0.58±0.04vs.0.81±0.07，P=0.001；1.95±0.08vs.2.35±0.25，P=0.007）andasignificantincreaseinP62level（0.82±0.03vs.0.60±0.08，P=0.032）comparedwiththePAgroup.ThePAgrouphadasignificantlyhighernumberofautolysosomesthanthenormalgroup（8.33±1.53vs.0.33±0.58，P=0.000），whilethevaspin-treatedgrouphadasignificantlyreducednumberofautolysosomescom－paredwiththePAgroup（3.67±1.53vs.8.33±1.53，P=0.001）.Aftertreatmentfor24hours，thePAgrouphadasignificantlyincreasedapoptosisrateofINS-1cellscomparedwiththenormalgroup（22.15±2.05vs.7.91±0.50，P=0.000），whilethevaspin-treatedgrouphadasignificantlyreducedapoptosisratecomparedwiththePAgroup（13.23±1.97vs.22.15±2.05，P=0.000）.Conclusion：VaspincaninhibitPA-inducedexcessiveautophagyandapoptosisofINS-1cells，thusimprovingthesecretoryfunctionofisletβcells.