title: Insulin-regulated protein palmitoylation impacts endothelial cell function.
abstract: OBJECTIVE
  Defects in insulin signaling are associated with abnormal endothelial cell function, which occurs commonly in cardiovascular disease. Targets of insulin signaling in endothelial cells are incompletely understood. Protein S-palmitoylation, the reversible modification of proteins by the lipid palmitate, is a post-translational process relevant to cell signaling, but little is known about the role of insulin in protein palmitoylation.
  
  
  RESULTS
  To test the hypothesis that insulin alters protein palmitoylation in endothelial cells, we combined acyl-biotin exchange chemistry with stable isotope labeling by amino acids in cell culture to perform quantitative proteomic profiling of human endothelial cells. We identified ≈380 putative palmitoylated proteins, of which >200 were not known to be palmitoylated; ≈10% of the putative palmitoylated proteins were induced or suppressed by insulin. Of those potentially affected by insulin, <10 have been implicated in vascular function. For one, platelet-activating factor acetylhydrolase IB subunit gamma (PAFAH1b3; not previously known to be palmitoylated), we confirmed that insulin stimulated palmitoylation without affecting PAFAH1b3 protein abundance. Chemical inhibition of palmitoylation prevented insulin-induced angiogenesis in vitro; knockdown of PAFAH1b3 had the same effect. PAFAH1b3 knockdown also disrupted cell migration. Mutagenesis of cysteines at residues 56 and 206 prevented palmitoylation of PAFAH1b3, abolished its capacity to stimulate cell migration, and inhibited its association with detergent-resistant membranes, which are implicated in cell signaling. Insulin promoted the association of wild-type PAFAH1b3 with detergent-resistant membranes.
  
  
  CONCLUSIONS
  These findings provide proof of principle for using proteomics to identify novel insulin-inducible palmitoylation targets relevant to endothelial function.
authors.affiliation: From the Division of Endocrinology, Metabolism, and Lipid Research (X.W., H.S., C.F.S.) and Department of Cell Biology and Physiology (C.F.S.), Washington University School of Medicine, St. Louis, MO.
authors.forename: Xiaochao
authors.initials: X
authors.lastname: Wei
authors.forename: Haowei
authors.initials: H
authors.lastname: Song
authors.forename: Clay F
authors.initials: CF
authors.lastname: Semenkovich
mesh_terms: D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002522:Chlorocebus aethiops; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D054878:Lipoylation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D018919:Neovascularization, Physiologic; D019308:Palmitic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D015398:Signal Transduction; D013311:Streptozocin; D014162:Transfection
chemical_list: D007328:Insulin; D019308:Palmitic Acid; D013311:Streptozocin; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; C568468:PAFAH1B3 protein, human
keywords: endothelial cells; insulin; lipoylation