title: Metformin prevents glucose-induced protein kinase C-beta2 activation in human umbilical vein endothelial cells through an antioxidant mechanism.
abstract: Hyperglycemia determines the vascular complications of diabetes through different mechanisms: one of these is excessive activation of the isoform beta2 of protein kinase C (PKC-beta2). Metformin, a widely used antidiabetic agent, is associated with decreased cardiovascular mortality in obese type 2 diabetic patients. Therefore, we assessed the role of metformin in glucose-induced activation of PKC-beta2 and determined the mechanism of its effect in human umbilical venous endothelial cells grown to either normo- (5 mmol/l) or hyperglycemia (10 mmol/l) and moderately and acutely exposed to 25 mmol/l glucose. We studied PKC-beta2 activation by developing adenovirally expressed chimeras encoding fusion protein between green fluorescent protein (GFP) and conventional beta2 isoform (PKC-beta2-GFP). Glucose (25 mmol/l) induced the translocation of PKC-beta2-GFP from the cytosol to the membrane in cells grown to hyperglycemia but not in those grown in normal glucose medium. Metformin (20 micromol/l) prevented hyperglycemia-induced PKC-beta2-GFP translocation. We also assessed oxidative stress under the same conditions with a 4-((9-acridine-carbonyl)amino)-2,2,6,6-tetramethylpiperidin-oxyl,free radical (TEMPO-9-AC) fluorescent probe. We observed significantly increased radical oxygen species production in cells grown in hyperglycemia medium, and this effect was abolished by metformin. We show that in endothelial cells, metformin inhibits hyperglycemia-induced PKC-beta2 translocation because of a direct antioxidant effect. Our data substantiate the findings of previous large intervention studies on the beneficial effect of this drug in type 2 diabetic patients.
authors.affiliation: Department of Clinical and Experimental Medicine, University of Padova, Padova, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Gallo
authors.forename: Giulio
authors.initials: G
authors.lastname: Ceolotto
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Iori
authors.forename: Ellen
authors.initials: E
authors.lastname: Murphy
authors.forename: Guy A
authors.initials: GA
authors.lastname: Rutter
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
authors.forename: Andrea
authors.initials: A
authors.lastname: Semplicini
authors.forename: Angelo
authors.initials: A
authors.lastname: Avogaro
mesh_terms: D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D005947:Glucose / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000201:enzymology*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008687:Metformin / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D017382:Reactive Oxygen Species; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects
chemical_list: D007004:Hypoglycemic Agents; D017382:Reactive Oxygen Species; D008687:Metformin; C579054:PRKCB protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D005947:Glucose