Methylglyoxal–bovine serum albumin stimulates tumor necrosis factor alpha secretion in RAW 264.7 cells through activation of mitogen-activating protein kinase, nuclear factor κB and intracellular reactive oxygen species formation

Accumulating evidence suggests that the pathophysiology of diabetes is analogous to chronic inflammatory states. Circulating levels of inflammatory cytokines such as IL-6 and tumor necrosis factor alpha (TNFα) are increased in both type 1 and type 2 diabetes. TNFα plays an important role in the pathogenesis of insulin resistance in type 2 diabetes. However, the reason for this increase remains unclear. Levels of the dicarbonyl methylglyoxal (MGO) are elevated in diabetic plasma and MGO-modified bovine serum albumin (MGO-BSA) can trigger cellular uptake of TNF. Therefore we tested the hypothesis that MGO-modified proteins may cause TNFα secretion in macrophage-like RAW 264.7 cells. Treatment of cells with MGO-BSA induced TNFα release in a dose-dependent manner. MGO-modified ribonuclease A and chicken egg ovalbumin had similar effects. Cotreatment of cells with antioxidant reagent N-acetylcysteine (NAC) inhibited MGO-BSA-induced TNFα secretion. MGO-BSA stimulated the simultaneous activation of p44/42 and p38 mitogen-activated protein kinase. PD98059, a selective MEK inhibitor, inhibited MGO-BSA-induced TNFα release as well as ERK phosphorylation. Pretreatment of cells with NAC also resulted in inhibition of MGO-BSA-induced ERK phosphorylation. MGO-BSA induced dose-dependent NFκB activation as shown by electrophoresis mobility shift assay. The MGO-BSA-induced NFκB activation was prevented in the presence of PD98059, NAC, and parthenolide, a selective inhibitor of NFκB. Furthermore, the NFκB inhibitor parthenolide suppressed MGO-BSA-induced TNFα secretion. Confocal microscopy using dichlorofluorescein to demonstrate intracellular reactive oxygen species (ROS) showed that MGO-BSA produced more ROS compared with native BSA. MGO-BSA could also stimulate protein kinase C (PKC) translocation to the cell membrane, considered a key signaling pathway in diabetes. However, there was no evidence that PKC was involved in TNFα release based on inhibition by calphostin C and staurosporine. Our findings suggest that the presence of chronically elevated levels of MGO-modified bovine serum albumin may contribute to elevated levels of TNFα in diabetes.