Metformin prevents glucose-induced PKC beta2 activation in human umbilical endothelial cells

Hyperglycaemia determines the vascular complications of diabetes through different mechanisms: one of these is the excessive activation of the isoform ß2 of the protein kinase C (PKC ß2). Metformin, a widely used antidiabetic agent, is associated with a decreased cardiovascular mortality in obese type 2 diabetic patients. Therefore, we assessed the role of metformin in glucose-induced activation of PKC ß2 and determined the mechanism of its effect in human umbilical venous endothelial cells grown to either normal- (5 mmol/l) or hyper-glycaemia (10 mmol/l ) medium and acutely exposed to 25 mmol/l glucose. We studied PKC ß2 activation by developing adenovirally expressed chimeras encoding fusion protein between green fluorescent protein (GFP) and conventional ß2 isoform (PKC ß2-GFP). Glucose (25 mmol/l) induced the translocation of PKC ß2-GFP from the cytosol to the membrane in cells grown to hyperglycaemia but not in normal glucose medium. Metformin (20 µmol/l) prevented hyperglycemia-induced PKC ß2-GFP translocation. We also assessed oxidative stress in the same conditions with Tempo-9-AC fluorescent probe. We observed significantly increased radical oxygen species production in cells grown in hyperglycaemia medium and this effect was abolished by metformin. We show that, in endothelial cells, metformin inhibits hyperglycemia-induced PKC ß2 translocation because of a direct anti-oxidant effect. Our data substantiate the findings of previous large intervention studies on the beneficial effect of this drug in Type 2 diabetic patients.