A large body of evidence now implicates increased leukocyte-endothelial cell adhesion as a key early event in the development of diabetic retinopathy. We recently reported that raised activity of the glycosylating enzyme core 2 beta 1,6-N-acetylglucosaminyltransferase (GlcNAc-T) through protein kinase C (PKC)beta2-dependent phosphorylation plays a fundamental role in increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. In the present study, we demonstrate that following exposure to plasma from diabetic patients, the human promonocytic cell line U937 exhibits a significant elevation in core 2 GlcNAc-T activity and increased adherence to cultured retinal capillary endothelial cells. These effects of diabetic plasma on enzyme activity and cell adhesion, mediated by PKCbeta2-dependent phosphorylation of the core 2 GlcNAc-T protein, were found to be triggered by increased plasma levels of tumor necrosis factor (TNF)-alpha. Levels of enzyme activity in plasma-treated U937 cells were closely dependent on the severity of diabetic retinopathy, with the highest values observed upon treatment with plasma of patients affected by proliferative retinopathy. Furthermore, we noted much higher correlation, as compared with control subjects, between increased values of core 2 GlcNAc-T activity and cell adhesion properties. Based on the prominent role of TNF-alpha in the development of diabetic retinopathy, these observations further validate the significance of core 2 GlcNAc-T in the pathogenesis of capillary occlusion, thereby enhancing the therapeutic potential of specific enzyme inhibitors.

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 [beta]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 increases the activity of core 2 GlcNAc-T and adhesion of human leukocytes to retinal capillary endothelial cells, in a dose-dependent manner, through diabetes-activated serine/threonine protein kinase C beta2 (PKCbeta2)-dependent phosphorylation. This regulatory mechanism, involving phosphorylation of core 2 GlcNAc-T, is also present in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients. Inhibition of PKCbeta2 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 threefold increase in O-linked glycosylation of P-selectin glycoprotein ligand-1 on the surface of leukocytes of diabetic patients compared with age-matched control subjects. PKCbeta2-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.

The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyte-endothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Galbeta1-3GalNAcalphaRbeta-N-acetylglucoaminyltr ansferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with age-matched control subjects (1,638 +/- 91 [n = 42] vs. 249 +/- 35 pmol x h(-1) x mg(-1) protein [n = 24], P = 0.00013; 1,459 +/- 194 [n = 58] vs. 334 +/- 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA(1c) (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyte-endothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy. 

The activities of serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in 72 Libyan diabetic patients were determined. The respective mean values were 24 +/- 1.1 U/L and 23.03 +/- 0.87 U/L. Significant correlations were found between the activities of GOT or GPT and fasting blood glucose, age, body mass index, heart rate and blood pressure levels. The mean values of GOT and GPT in diabetic patients with secondary complications were significantly higher than those without complications. On the other hand, patients with a family history of diabetes have higher mean values of GOT and GPT when compared with those without diabetes in the family.