The mechanisms underlying the pathogenesis of diabetic retinopathy are poorly understood, but studies have suggested that leukocytes may play a major role in its development. Previously, we reported that the activity of UDP-GlcNAc:Galβ1-3GalNAcαRβ-N-acetylglucosaminyltransferase (core 2 GlcNAc-T), a developmentally regulated enzyme of O-linked glycans biosynthesis pathway, is increased in polymorphonuclear leukocytes (PMN) in diabetic patients (Type 1 and Type 2), through PKCβ2-dependent phosphorylation (Chibber et al. 2000). The aim of this study was to establish an animal model to provide direct experimental evidence for the role of core 2 GlcNAc-T in increased leukocyte-endothelial cell adhesion in pathogenesis of diabetic retinopathy.
BB-rats and Wistar-rats where maintained by Taconic M & B, Denmark and euthanized according to Danish Animal regulations. Leukocytes suspensions were prepared by density gradient centrifugation. Briefly, 3 ml of blood was layered onto equal volume of Histopaque 1270, and centrifuged (400 g, 30 min), PMN-rich buffy coat was carefully removed, resuspended in phosphate-buffered saline (PBS), centrifuged (250 g, 15 min), and the pellet stored at -20°C until used for the measurement of core 2 GlcNAc-T as described (Chibber et al. 2000). Heart tissue was also isolated, rinsed in PBS and homogenized at 4°C in lysis buffer (0.9 % NaCl, 0.4 % Triton X-100, and 0.1 mM PMSF). The data were analysed using unpaired t test.
The activity of core 2 GlcNAc-T was significantly higher in PMN leukocytes of BB-rats compared to age-matched control Wistar-rats [2390 ± 439.3 pmol h-1 mg protein-1 (n = 6) vs. 244.6 ± 50.2 (n = 6), mean ± S.E.M., P = 0.0007, unpaired Student’s t test]. The activity of core 2 GlcNAc-T was also higher in the heart tissue of BB-rats compared to age-matched Wistar-rats [1622 ± 181 pmol h-1 mg protein-1 (n = 6) vs. 125.1 ± 36.04 (n = 4), P = 0.0008].
These results are consistent with our recent data (Chibber et al. 2000) and suggest that BB-rats would be useful as an animal model in Type 1 diabetes to further explore the role of core 2 GlcNAc-T in pathogenesis of capillary occlusion in diabetic retinopathy.
This work was supported by Guy’s & St Thomas’ Charitable Foundation