Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

中文翻译：

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NDPK-B通过己糖胺生物合成途径的激活和O-GlcNAcase活性的抑制参与葡萄糖代谢介导的内皮损伤。

我们以前的研究发现，高血糖症或核苷二磷酸激酶-B（NDPK-B）缺乏引起的视网膜内皮损伤与血管生成素2（Ang-2）的升高和己糖胺生物合成途径（HBP）的激活有关。在这里，我们调查了内皮细胞（ECs）的HBP中NDPK-B是如何参与的。通过体外测定来测量NDPK-B和O-GlcNAcase（OGA）的活性。通过UPLC-PDA（具有光电二极管阵列检测的超高效液相色谱法）和免疫印迹分别评估核苷酸的代谢和O-GlcNAcyylated蛋白。用重组腺病毒实现了NDPK-B的重新表达。我们的结果表明，与高葡萄糖（HG）处理类似，ECs中的NDPK-B耗竭升高了UDP-GlcNAc的水平并降低了NDPK活性。此外，谷氨酰胺：果糖-6-磷酸酰胺基转移酶（GFAT）的表达和磷酸化被诱导，而OGA活性被抑制。此外，在NDPK-B消耗的EC中，总蛋白O-GlcNAcylation以及O-GlcNAcylated Ang-2增加。使用Thiamet G（TMG）或OGA siRNA的蛋白O-GlcNAcylation的药理学升高可提高Ang-2水平。然而，NDPK-B的核苷三磷酸二磷酸（NTP / NDP）转磷酸化酶和组氨酸激酶活性对于蛋白O-GlcNAcylation而言是必不可少的。NDPK-B缺乏症因此导致HBP的激活和OGA活性的抑制，从而导致蛋白O-GlcNAcy的增加和Ang-2的进一步上调。数据表明NDPK-B通过调节HBP在内皮损伤中起关键作用。果糖6磷酸酰胺基转移酶（GFAT）被诱导，而OGA活性受到抑制。此外，在NDPK-B消耗的EC中，总蛋白O-GlcNAcylation以及O-GlcNAcylated Ang-2增加。使用Thiamet G（TMG）或OGA siRNA的蛋白O-GlcNAcylation的药理学升高可提高Ang-2水平。然而，NDPK-B的核苷三磷酸二磷酸（NTP / NDP）转磷酸化酶和组氨酸激酶活性对于蛋白O-GlcNAcylation而言是必不可少的。因此，NDPK-B缺乏导致HBP的激活和OGA活性的抑制，从而导致蛋白质O-GlcNAcy的增加和Ang-2的进一步上调。数据表明NDPK-B通过调节HBP在内皮损伤中起关键作用。果糖6磷酸酰胺基转移酶（GFAT）被诱导，而OGA活性受到抑制。此外，在NDPK-B消耗的EC中，总蛋白O-GlcNAcylation以及O-GlcNAcylated Ang-2增加。使用Thiamet G（TMG）或OGA siRNA的蛋白O-GlcNAcylation的药理学升高可提高Ang-2水平。然而，NDPK-B的核苷三磷酸二磷酸（NTP / NDP）转磷酸化酶和组氨酸激酶活性对于蛋白O-GlcNAcylation而言是必不可少的。因此，NDPK-B缺乏导致HBP的激活和OGA活性的抑制，从而导致蛋白质O-GlcNAcy的增加和Ang-2的进一步上调。数据表明NDPK-B通过调节HBP在内皮损伤中起关键作用。在NDPK-B缺失的EC中，总蛋白O-GlcNAcylation以及O-GlcNAcylated Ang-2增加。使用Thiamet G（TMG）或OGA siRNA的蛋白O-GlcNAcylation的药理学升高可提高Ang-2水平。然而，NDPK-B的核苷三磷酸二磷酸（NTP / NDP）转磷酸化酶和组氨酸激酶活性对于蛋白O-GlcNAcylation而言是必不可少的。因此，NDPK-B缺乏导致HBP的激活和OGA活性的抑制，从而导致蛋白质O-GlcNAcy的增加和Ang-2的进一步上调。数据表明NDPK-B通过调节HBP在内皮损伤中起关键作用。在NDPK-B缺失的EC中，总蛋白O-GlcNAcylation以及O-GlcNAcylated Ang-2增加。使用Thiamet G（TMG）或OGA siRNA的蛋白O-GlcNAcylation的药理学升高可提高Ang-2水平。然而，NDPK-B的核苷三磷酸二磷酸（NTP / NDP）转磷酸化酶和组氨酸激酶活性对于蛋白O-GlcNAcylation而言是必不可少的。NDPK-B缺乏症因此导致HBP的激活和OGA活性的抑制，从而导致蛋白O-GlcNAcy的增加和Ang-2的进一步上调。数据表明NDPK-B通过调节HBP在内皮损伤中起关键作用。NDPK-B的三磷酸核苷二磷酸核苷（NTP / NDP）转磷酸酶和组氨酸激酶活性对于蛋白O-GlcNAcylation是不可或缺的。因此，NDPK-B缺乏导致HBP的激活和OGA活性的抑制，从而导致蛋白质O-GlcNAcy的增加和Ang-2的进一步上调。数据表明NDPK-B通过调节HBP在内皮损伤中起关键作用。NDPK-B的三磷酸核苷二磷酸核苷（NTP / NDP）转磷酸酶和组氨酸激酶活性对于蛋白O-GlcNAcylation是不可或缺的。因此，NDPK-B缺乏导致HBP的激活和OGA活性的抑制，从而导致蛋白O-GlcNAcy的增加和Ang-2的进一步上调。数据表明NDPK-B通过调节HBP在内皮损伤中起关键作用。