Increased leukocyte-endothelial cell adhesion is a key early event in the development of retinopathy and atherogenesis in diabetic patients. We recently reported that raised activity of glycosylating enzyme β-1,6-acetylglucosaminyltransferase (core 2 GlcNAc-T) is responsible for increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. Here we demonstrate that elevated glucose and diabetic serum increase the activity of core 2 GlcNAc-T (2040 ± 445 (15 mM glucose, n = 5) vs. 182.7 ± 65.5 (5.8 mM glucose, n = 5); P = 0.0033; 2035 ± 411 (diabetic serum, n = 25) vs. 196 ± 35 (control serum, n = 19), P = 0.0002) and adhesion of human leukocytes to retinal capillary endothelial cells (8.76 ± 0.5 (15 mM glucose, n = 18) vs. 3.7 ± 0.35 (5.8mM glucose, n = 18), P = 0.0001; 3.7 ± 0.48 (diabetic serum, n = 21) vs. 0.62 ± 0.09 (control serum, n = 15), P = 0.0001) through diabetes activated serine/threonine protein kinase C b2 (PKCb2)-dependent phosphorylation. This regulatory mechanism, involving phosphorylation of core 2 GlcNAc-T, is also present in polymorphonuclear leukocytes (PMNs) isolated from Type 1 and Type 2 diabetic patients. Inhibition of PKCb2 activation with the specific inhibitor, LY379196, attenuated serine phosphorylation of core 2 GlcNAc-T and prevented increased leukocyte-endothelial cell adhesion. Raised activity of core 2 GlcNAc-T was associated with a 3-fold increase in O-linked glycosylation of P-selectin glycoprotein ligand-1 (PSGL-1) on the surface of leukocytes of diabetic patients compared to age-matched controls. PKCb2-dependent phosphorylation of core 2 GlcNAc-T may thus represent a novel regulatory mechanism for activation of this key enzyme in mediating increased leukocyte-endothelial cell adhesion and capillary occlusion in diabetic retinopathy.

We thank Dr Allassandro Datti (Genetics Institute, Canada) for supplying the core 2 GlcNAc-T antibody. We also thank Dr Kumar (Genetics Institute, Canada), Professor Alan Fields and Dr Nicole Murphy (Sealey Center for Cancer Research, Galvaston, Texas, USA) for the PKCβ2 cDNA plasmid for transfection experiments, and Mr John Schilling (Eye Unit, St Thomas’ Hospital) for blood samples from diabetic patients. This study was supported by the Juvenile Diabetes Foundation International (JDFI).