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

X. Fan, R. Subramaniam, M. F. Weiss, V. M. Monnier

Research output: Contribution to journalArticle

68 Scopus citations

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 languageEnglish (US)
Pages (from-to)274-286
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume409
Issue number2
DOIs
StatePublished - Jan 15 2003
Externally publishedYes

Keywords

  • Bovine serum album
  • Methylglyoxal
  • RAW cell
  • Reactive oxygen species
  • TNFα

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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