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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Cover Image
Cover Image
The interfacial active site of the type IB GTP cyclohydrolase from Neisseria gonorrhoeae (grey and green ribbons), in complex with zinc (magenta ball) and the reaction intermediate analog and potent inhibitor 8-oxo-GTP (stick model). The structure sheds light on the complex and unique catalytic strategy of this potential antibacterial target, and offers a starting point for the design of specific inhibitors against the enzyme. For more information, please see study by Paranagama et al. in this issue, pages 1017–1039. Image provided by Manal Swairjo.
GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis
Darya Zibrova, Franck Vandermoere, Olga Göransson, Mark Peggie, Karina V. Mariño, Anne Knierim, Katrin Spengler, Cora Weigert, Benoit Viollet, Nicholas A. Morrice, Kei Sakamoto, Regine Heller; GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis. Biochem J 15 March 2017; 474 (6): 983–1001. doi: https://doi.org/10.1042/BCJ20160980
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