OVEREXPRESSION OF GLYOXALASE-I IN BOVINE ENDOTHELIAL-CELLS INHIBITS INTRACELLULAR ADVANCED GLYCATION ENDPRODUCT FORMATION AND PREVENTS HYPERGLYCEMIA-INDUCED INCREASES IN MACROMOLECULAR ENDOCYTOSIS

Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentatio n of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglut athione, which in turn is converted to D-lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human gly oxalase-I were generated, Glyoxalase-I activity in these cells was inc reased 28-fold compared to neo-transfected control cells (21.80 +/- 0. 1 vs. 0.76 +/- 0.02 mu mol/min/mg protein, n = 3, P < 0.001). In neo-t ransfected cells, 30 mM glucose incubation increased MG and D-lactate concentration approximately twofold above 5 mM (35.5 +/- 5.8 vs, 19.6 +/- 1.6, P < 0.02, n = 3, and 21.0 +/- 1.3 vs. 10.0 +/- 1.2 pmol/10(6) cells, n = 3, P < 0.001, respectively). in contrast, in glyoxalase-I- transfected cells, 30 mM glucose incubation did not increase MG concen tration at all, while increasing the enzymatic product D-lactate by > 10-fold (18.9 +/- 3.2 vs. 18.4 +/- 5.8, n = 3, P = NS, and 107.1 +/- 9 .0 vs. 9.4 +/- 0 pmol/10(6) cells, n = 3, P < 0.001, respectively), Af ter exposure to 30 mM glucose, intracellular AGE formation in neo cell s was increased 13.6-fold (2.58 +/- 0.15 vs. 0.19 +/- 0.03 total absor bance units, n = 3, P < 0.001), Concomitant with increased intracellul ar AGEs, macromolecular endocytosis by these cells was increased 2.2-f old. Overexpression of glyoxalase-I completely prevented both hypergly cemia-induced AGE formation and increased macromolecular endocytosis.