Regulation of synthesis and activity of protein kinase C beta(1) in platelets of diabetic patients after in vitro incubation

Hyperglycemia is increasing the risk of macrovascular disease in diabetic patients. An important role in development of diabetic complications plays signal transduction enzyme protein kinase C (PKC). We investigated wheter activity and synthesis of protein kinase C is regulated in vitro in diabetic patients compared with healthy subjects by determing membrane and cytosol level of PKC beta(1) by Western blot technique. We incubated human platelets of 26 patients with type 2 diabetes (M) and 9 healthy controls for a different time. There was a significant increase of total PKC beta(1) in diabetic patients after incubation for 2, 4. 6, 8 and 10 hours and in controls after 2-, 6-, 8- and 10- hour incubation (p<0.05). The total PKC beta(1) concentration was significantly higher in diabetic patients than in the controls after 4, 6 and 8 hours (p<0.05). A significant increase of cytosolic PKC beta(1) was observed both in diabetic patients and in controls after 2, 4, 6 and 8 hours, being significantly higher in the diabetic patients compared to the controls after 4-, 6-, 8-, and 10- hour incubation (p<0.05). No significant increase after incubation and difference between both groups were found for the membrane fraction. No changes were observed in membrane fraction of PKC beta(1). Next we tested whether increase of activity of PKC beta(1) is regulated in vitro by hyperglycemia (16 mm) and hyperinsulinemia (25 mU/ml) for 15 min. We found in vitro no increase of activity of PKC P, by glucose and insulin in healthy volunteers (n=16) although 100 nmol/l 12-O-tetradecanoylphorbol 13-acetate caused maximal activation. In marked contrast we observed significant increase of activity of PKC beta(1) in patients with T2DM (n=10, p<0.05). The results showed increased PKC beta(1) synthesis and activity in platelets of diabetic patients compared to the controls.