Involvement of NDPK-B in Glucose Metabolism-Mediated Endothelial Damage via Activation of the Hexosamine Biosynthesis Pathway and Suppression of O-GlcNAcase Activity.

Anupriya Chatterjee,Santosh Lomada,Hans-Peter Hammes,Thomas Wieland,Gernot Poschet,Kerstin Wilhelm,Mahmoud Halawa,Yuxi Feng,Martina Schmidt,Rachana Eshwaran

doi:10.3390/cells9102324

Abstract

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.

Highlights

Hyperglycemia increases glucose uptake and metabolism and increases the flux through the hexosamine biosynthesis pathway (HBP), leading to vascular damage
150 pmol of UDP-GlcNAc was detected per retina
The data showed that the depletion of nucleoside diphosphate kinase-B (NDPK-B) in endothelial cells (ECs): (1) is involved in the activation of the HBP without altering glycolysis; (2) does not alter nucleotide triphosphate levels, despite altering nucleotide metabolism; (3) increases the level of the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) in the HBP

Summary

Introduction

Hyperglycemia increases glucose uptake and metabolism and increases the flux through the hexosamine biosynthesis pathway (HBP), leading to vascular damage. The HBP is an offshoot of glycolysis in which the glycolysis intermediate fructose-6-phosphate is utilized to generate uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in a series of biochemical reactions that involve amino acid, fatty acid, and nucleotide metabolism [1,2,3]. Amongst the enzymes involved in the HBP, glutamine:fructose-6-phosphate amidotransferase (GFAT) is rate-limiting as it catalyzes the initial step of formation of glucosamine-6-phosphate and controls UDP-GlcNAc availability [4,5]. The counteracting enzyme, O-GlcNAcase (OGA), unselectively catalyzes the removal of O-GlcNAc moieties. Its regulation is still not well understood [6]. Both proteins are well known as the enzymes performing O-GlcNAc cycling in cells

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