TY - JOUR
T1 - Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis
AU - Shinohara, Moritsugu
AU - Thornalley, P. J.
AU - Giardino, Ida
AU - Beisswenger, Paul
AU - Thorpe, Suzanne R.
AU - Onorato, Joelle
AU - Brownlee, Michael
PY - 1998/3/1
Y1 - 1998/3/1
N2 - Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, 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 glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo- transfected control cells (21.80±0.1 vs. 0.76±0.02 μmol/min/mg protein, n = 3, P < 0.001). In neo-transfected 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/106 cells, n = 3, P < 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration 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/106 cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58±0.15 vs. 0.19±0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.
AB - Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, 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 glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo- transfected control cells (21.80±0.1 vs. 0.76±0.02 μmol/min/mg protein, n = 3, P < 0.001). In neo-transfected 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/106 cells, n = 3, P < 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration 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/106 cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58±0.15 vs. 0.19±0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.
KW - Advanced glycation endproducts
KW - Cell lines
KW - Endocytosis
KW - Glyoxalase-I
KW - Methylglyoxal
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U2 - 10.1172/JCI119885
DO - 10.1172/JCI119885
M3 - Article
C2 - 9486985
AN - SCOPUS:0032032578
SN - 0021-9738
VL - 101
SP - 1142
EP - 1147
JO - Journal of Clinical Investigation
JF - Journal of Clinical Investigation
IS - 5
ER -