Protection of Polyphenols against Glyco-Oxidative Stress: Involvement of Glyoxalase Pathway

Abstract

Chronic high glucose (HG) exposure increases methylglyoxal (MGO)-derived advanced glycation end-products (AGEs) and is involved in the onset of pathological conditions, such as diabetes, atherosclerosis and chronic-degenerative diseases. Under physiologic conditions the harmful effects of MGO are contrasted by glyoxalase system that is implicated in the detoxification of Reactive Carbonyl Species (RCS) and maintain the homeostasis of the redox environment of the cell. Polyphenols are the most abundant antioxidants in the diet and present various health benefits. Aims of the study were to investigate the effects of HG-chronic exposure on glyco-oxidation and glyoxalase system in intestinal cells, using CaCo-2 cells. Moreover, we studied the effect of apple polyphenols on glyco-oxidative stress. Our data demonstrated that HG-treatment triggers glyco-oxidation stress with a significant increase in intracellular Reactive Oxygen Species (ROS), lipid peroxidation, AGEs, and increase of Glyoxalase I (GlxI) activity. On the contrary, Glyoxalase II (GlxII) activity was lower in HG-treated cells. We demonstrate that apple polyphenols exert a protective effect against oxidative stress and dicarbonyl stress. The increase of total antioxidant capacity and glutathione (GSH) levels in HG-treated cells in the presence of apple polyphenols was associated with a decrease of GlxI activity.

Keywords: AGEs (advanced glycation end-products); glutathione; glyoxalase system; hyperglycemia; methylglyoxal.

Figure 1

Methylglyoxal (MGO) formation from intermediates of glucose, protein and fat metabolism, and its degradation by the Glyoxalase System. Glyoxalase I (GlxI) converts hemithioacetal formed from glutathione (GSH) and methylglyoxal (MGO) into S-D-Lactoylglutathione (SLG) which is hydrolyzed by Glyoxalase II (GlxII) to D-lactic acid and GSH. Abbreviations: AGEs, advanced glycation end products; DHAP, dihydroxacetone phosphate; ROS, reactive oxygen species; GSH, reduced glutathione; GR, glutathione reductase.

Figure 2

AGEs formation and GA-modified protein in HG-treated CaCo-2 cells in absence and in the presence of apple polyphenolic extract. (A) Levels of total fluorescent AGEs and (B) Representative western blot of GA-modified proteins and relative densitometric analysis, in control cells (25 mM glucose, Ctrl) or high glucose treated cells (50mM glucose) incubated in the absence (HG) or in the presence of apple polyphenolic extract (0.4 and 0.8 mmol GAE/L). Densitometric data are normalized on β-actin. Results are presented as mean ± SD of 5 determinations carried out in triplicate. Different letters a–c indicate significant statistic differences between samples (p < 0.05).

Figure 3

The glyoxalase system in HG-treated CaCo-2 cells incubated in the absence or in the presence of apple polyphenolic extract. (A) Glx I and (B) Glx II activity in intestinal CaCo-2 cells treated for one week with normal (25 mM) (Ctrl), or with high glucose (50 mM) concentrations in the absence (HG) or in the presence of polyphenolic extract (0.4 and 0.8 mmol GAE/L). Results are presented as mean ± SD of 5 determinations carried out in triplicate. Different letters a,b indicate significant statistic differences between samples (p < 0.05).

Figure 4

Glutathione level in HG-treated CaCo-2 cells in the absence or in the presence of apple polyphenolic extract Glutathione (GSH) level in control cells (25 mM glucose, Ctrl) or high glucose treated cells (50 mM glucose) incubated in the absence (HG) or in the presence of apple polyphenolic extract (0.4 and 0.8 mmol GAE/L). Results are represented as mean ± SD of 5 determinations carried out in triplicate. Different letters a,b indicate significant differences between samples (p < 0.05).