title: GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.
abstract: Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins byO-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controlsO-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic andin vitroangiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reducedO-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany.
authors.forename: Darya
authors.initials: D
authors.lastname: Zibrova
authors.affiliation: Institut de Génomique Fonctionnelle, CNRS UMR5203, INSERM U1191, Université de Montpellier, Montpellier, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Vandermoere
authors.affiliation: Department of Experimental Medical Sciences, Lund University, 221 84 Lund, Sweden.
authors.forename: Olga
authors.initials: O
authors.lastname: Göransson
authors.affiliation: Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
authors.forename: Mark
authors.initials: M
authors.lastname: Peggie
authors.affiliation: Laboratorio de Glicómica Funcional y Molecular, Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (IBYME-CONICET), C1428 Buenos Aires, Argentina.
authors.forename: Karina V
authors.initials: KV
authors.lastname: Mariño
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Knierim
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Spengler
authors.affiliation: Division of Pathobiochemistry and Clinical Chemistry, University of Tübingen, 72076 Tübingen, Germany.|Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Zentrum München at the University of Tübingen, 72076 Tübingen, Germany.|German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany.
authors.forename: Cora
authors.initials: C
authors.lastname: Weigert
authors.affiliation: INSERM U1016, Institut Cochin, Paris, France.|CNRS UMR 8104, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Viollet
authors.affiliation: AB-Sciex, Phoenix House, Centre Park, Warrington WA1 1RX, U.K.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Morrice
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Kei
authors.initials: K
authors.lastname: Sakamoto
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany regine.heller@med.uni-jena.de.
authors.forename: Regine
authors.initials: R
authors.lastname: Heller
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000117:Acetylglucosamine / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006595:Hexosamines / Q000096:biosynthesis; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051379:Mice; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D061251:Primary Cell Culture; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
chemical_list: D006595:Hexosamines; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D012265:Ribonucleotides; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000620:Aminoimidazole Carboxamide; D012694:Serine; C497511:GFPT1 protein, human; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide; D005947:Glucose; D000409:Alanine; D000117:Acetylglucosamine
keywords: AMPK; GFAT1; O-GlcNAcylation; VEGF; angiogenesis; phosphoproteomics