Abstract
Purpose: :
Alpha–dicarbonyl compounds methylglyoxal (MGO) and glyoxal (GO) are highly reactive protein modifiers present in the human lens. Several products of their reaction are present in the lens. Such products accumulate in significant quantities in cataractous lenses. Glyoxalase system consisting of glyoxalase I (requires GSH) and glyoxalase II metabolizes α–dicarbonyl compounds to less reactive products. In this study we have investigated the effect of hyperglycemia on the activity of glyoxalase I and its role in MGO metabolism in lens epithelial cells and lenses of streptozotocin–diabetic mice.
Methods: :
Primary mouse lens epithelial cells were cultured for 7 days in the presence of 5 or 25 mM D–glucose or 25 mM L–glucose (osmotic control). Glyoxalase I activity was measured spectrophotometrically and its mRNA levels were measured by quantitative PCR. We also measured MGO and reduced GSH in cell lysates. Similar measurements were done in lenses of diabetic mice.
Results: :
Glyoxalase I activity in 25 mM D–glucose treated cells was significantly higher when compared cells treated with 5 mM D–glucose or 25 mM L–glucose (mean±SD: 5 mM Glc–120.4±9.7 nmoles S–lactoylglutathione formed per min per mg protein; 25 mM D–Glc–144.7±12.0; 25 mM L–Glc–124.8 ±13.9). Glyoxalase I mRNA content was also significantly higher (P<0.01) in 25 mM D–glucose treated cells (fold change over control– 25 mM Glc–1.72±0.7; 25 mM L–Glc–1.05±0.6). In spite of the higher glyoxalase activity, MGO levels were significantly higher (P=0.007) in cells grown in 25 mM D–glucose when compared cells grown in 5 mM D–glucose or 25 mM L–glucose. GSH levels were considerably reduced in cells cultured with 25 mM D–glucose (nmoles of GSH/mg protein, mean±SD: 5 mM Glc–10.28±1.39; 25 mM D–Glc–9.04±0.39; 25 mM L–Glc–9.53±1.05). In 9–week old diabetic mouse lenses, the glyoxalase I activity and mRNA content were significantly higher than in non–diabetic control animals (glyoxalase I: control–137.8±11.1; Diab–161.1±24.2, P=0.003; fold change in glyoxalase I mRNA in diabetic lenses–1.31±0.2).
Conclusions: :
Induction of glyoxalase I may constitute an initial protective response against damaging effects of hyperglycemia in diabetes. Reduction in the GSH content in diabetic lenses may compromise MGO metabolism by this protective enzyme.
Keywords: diabetes • enzymes/enzyme inhibitors • protective mechanisms