GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis

Biochem J. 2017 Mar 7;474(6):983-1001. doi: 10.1042/BCJ20160980.


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 by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis 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 reduced O-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.

Keywords: AMPK; GFAT1; O-GlcNAcylation; VEGF; angiogenesis; phosphoproteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / antagonists & inhibitors
  • AMP-Activated Protein Kinases / genetics
  • AMP-Activated Protein Kinases / metabolism*
  • Acetylglucosamine / metabolism*
  • Alanine / chemistry
  • Alanine / metabolism
  • Amino Acid Substitution
  • Aminoimidazole Carboxamide / analogs & derivatives
  • Aminoimidazole Carboxamide / pharmacology
  • Animals
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Glucose / pharmacology
  • Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / antagonists & inhibitors
  • Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / genetics
  • Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / metabolism*
  • Hexosamines / biosynthesis
  • Human Umbilical Vein Endothelial Cells / cytology
  • Human Umbilical Vein Endothelial Cells / drug effects
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Humans
  • Mice
  • Neovascularization, Physiologic / drug effects*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorylation / drug effects
  • Primary Cell Culture
  • Protein Processing, Post-Translational*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Ribonucleotides / pharmacology
  • Serine / chemistry
  • Serine / metabolism
  • Vascular Endothelial Growth Factor A / pharmacology*


  • Hexosamines
  • Phosphoproteins
  • RNA, Small Interfering
  • Ribonucleotides
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Aminoimidazole Carboxamide
  • Serine
  • GFPT1 protein, human
  • Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
  • AMP-Activated Protein Kinases
  • AICA ribonucleotide
  • Glucose
  • Alanine
  • Acetylglucosamine