Abstract
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.
Original language | English (US) |
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Pages (from-to) | 274-286 |
Number of pages | 13 |
Journal | Archives of Biochemistry and Biophysics |
Volume | 409 |
Issue number | 2 |
DOIs | |
State | Published - Jan 15 2003 |
Externally published | Yes |
Keywords
- Bovine serum album
- Methylglyoxal
- RAW cell
- Reactive oxygen species
- TNFα
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology