Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells

Abstract

Methylglyoxal is an ␣-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 M methylglyoxal was detected, 100-to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 M, 0.02% of cellular amino acid residues could be modified. As few as one or two ''hits'' with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture

“…The steady-state concentration of free MG was 0.035 lmol L-packedcell À1 ; the steady state concentration of reversibly bound MG was 3 lmol L-packed-cell À1 ; the steady state concentration of irreversibly bound MG was 1.3 lmol L-packed-cell À1 . These levels of reversibly bound MG were 1-2 orders of magnitude lower than found in CHO cells in culture (Table 1; Chaplen et al 1998).…”

“…Intracellular free MG exists as a complex function of cell metabolism and interactions with cellular structures, in particular, proteins (Chaplen et al, 1998). MG:protein interactions form the central element of Model 2 (Figure 2b), where they are treated similarly to Model 1, but with the arginine, lysine and cysteine residue concentrations adjusted to reflect the protein content and composition of mammalian cells.…”

“…Free MG in solution was measured at various times by derivatizing with o-PD followed by HPLC analysis (Chaplen et al 1998). The first-and second-order rate constants for disappearance of MG were determined by parameter estimation.…”

Methylglyoxal Impairs the Insulin Signaling Pathways Independently of the Formation of Intracellular Reactive Oxygen Species

“…Indeed, the methylglyoxal level in rat aortic tissue has been reported to be about sixfold higher than in plasma (37). The intracellular concentration of methylglyoxal (free and bound) in Chinese hamster ovary cells (CHO cells) was found to be ϳ0.3 mmol/l (38). Considering that only a few percent (ϳ3%) of the exogenously added methylglyoxal was taken up by our cultured L6 cells (Fig.…”

Buthionine Sulfoximine Promotes Methylglyoxal-Induced Apoptotic Cell Death and Oxidative Stress in Endothelial Cells

“…16,17) It is reported that plasma MG concentration is significantly increased in diabetic patients. 23,24) Intracellular MG concentration, which is higher than 300 mM, 25) would be considerably higher than plasma MG concentration because MG is formed during glycolysis. It is reported that only a small percentage of exogenous MG is incorporated into cultured cells.…”