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GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis

Zibrova, Darya; Vandermoere, Franck; Göransson, Olga LU ; Peggie, Mark; Mariño, Karina V.; Knierim, Anne; Spengler, Katrin; Weigert, Cora; Viollet, Benoit and Morrice, Nicholas A., et al. (2017) In Biochemical Journal 474(6). p.983-1001
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 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... (More)

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.

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Biochemical Journal
volume
474
issue
6
pages
19 pages
publisher
Portland Press Limited
external identifiers
  • scopus:85016157569
  • wos:000395819600009
ISSN
0264-6021
DOI
10.1042/BCJ20160980
language
English
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yes
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24d7cb43-edeb-454b-a183-6552fa5fbe8a
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2017-04-05 14:17:23
date last changed
2018-01-07 11:58:05
@article{24d7cb43-edeb-454b-a183-6552fa5fbe8a,
  abstract     = {<p>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.</p>},
  author       = {Zibrova, Darya and Vandermoere, Franck and Göransson, Olga and Peggie, Mark and Mariño, Karina V. and Knierim, Anne and Spengler, Katrin and Weigert, Cora and Viollet, Benoit and Morrice, Nicholas A. and Sakamoto, Kei and Heller, Regine},
  issn         = {0264-6021},
  language     = {eng},
  month        = {03},
  number       = {6},
  pages        = {983--1001},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis},
  url          = {http://dx.doi.org/10.1042/BCJ20160980},
  volume       = {474},
  year         = {2017},
}