Zibrova Darya, Vandermoere Franck, Göransson Olga, Peggie Mark, Mariño Karina V, Knierim Anne, Spengler Katrin, Weigert Cora, Viollet Benoit, Morrice Nicholas A, Sakamoto Kei, Heller Regine
Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany.
Institut de Génomique Fonctionnelle, CNRS UMR5203, INSERM U1191, Université de Montpellier, Montpellier, France.
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 -linked β--acetylglucosamine (-GlcNAc). In the present study, we tested the hypothesis that AMPK controls -GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and 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 -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.
内皮细胞中AMP激活的蛋白激酶（AMPK）的激活可调节能量稳态、应激保护和血管生成，但其潜在机制尚未完全明确。通过无标记磷酸化蛋白质组学分析，我们鉴定出谷氨酰胺：果糖-6-磷酸酰胺转移酶1（GFAT1）为AMPK底物。GFAT1是己糖胺生物合成途径（HBP）中的限速酶，因此可控制蛋白质的O-连接β-N-乙酰葡糖胺（O-GlcNAc）修饰。在本研究中，我们使用生化、药理学、遗传学和血管生成方法，验证了AMPK通过GFAT1磷酸化控制内皮细胞O-GlcNAc水平和功能的假说。5-氨基咪唑-4-甲酰胺核苷（AICAR）或血管内皮生长因子（VEGF）激活原代人内皮细胞中的AMPK，可导致GFAT1丝氨酸243位点磷酸化。当通过小干扰RNA（siRNA）下调AMPK时，未观察到这种效应。AMPK激活后，含有野生型（WT）-GFAT1的内皮细胞中GFAT活性降低，O-GlcNAc水平降低，但在表达不可磷酸化的S243A-GFAT1的细胞中未观察到这种现象。GFAT1的药理学抑制或siRNA介导的下调增强了VEGF诱导的芽生，表明GFAT1作为血管生成的负调节因子发挥作用。在表达S243A-GFAT1的细胞中，VEGF诱导的芽生减少，提示VEGF通过AMPK介导的GFAT1磷酸化减轻了GFAT1/HBP对血管生成的抑制作用。高糖激活GFAT1/HBP会导致血管芽生受损，而GFAT1抑制即使在血糖水平较高时也能改善芽生。我们的研究结果为HBP在血管生成中的作用提供了新的机制性见解。它们表明，靶向内皮细胞中的AMPK可能有助于改善与代谢紊乱相关的高血糖诱导的血管功能障碍。
