Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis

Moritsugu Shinohara, P. J. Thornalley, Ida Giardino, Paul Beisswenger, Suzanne R. Thorpe, Joelle Onorato, Michael Brownlee

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383 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1142-1147
Number of pages6
JournalJournal of Clinical Investigation
Volume101
Issue number5
DOIs
StatePublished - Mar 1 1998

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Keywords

  • Advanced glycation endproducts
  • Cell lines
  • Endocytosis
  • Glyoxalase-I
  • Methylglyoxal

ASJC Scopus subject areas

  • Medicine(all)

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