title: Methylglyoxal-bovine serum albumin stimulates tumor necrosis factor alpha secretion in RAW 264.7 cells through activation of mitogen-activating protein kinase, nuclear factor kappaB and intracellular reactive oxygen species formation.
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 (TNFalpha) are increased in both type 1 and type 2 diabetes. TNFalpha 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 TNFalpha secretion in macrophage-like RAW 264.7 cells. Treatment of cells with MGO-BSA induced TNFalpha 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 TNFalpha 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 TNFalpha 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 NFkappaB activation as shown by electrophoresis mobility shift assay. The MGO-BSA-induced NFkappaB activation was prevented in the presence of PD98059, NAC, and parthenolide, a selective inhibitor of NFkappaB. Furthermore, the NFkappaB inhibitor parthenolide suppressed MGO-BSA-induced TNFalpha 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 TNFalpha 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 TNFalpha in diabetes.
authors.affiliation: Institute of Pathology, Case Western Reserve University, Clevelard, OH 44106, USA.
authors.forename: X
authors.initials: X
authors.lastname: Fan
authors.forename: R
authors.initials: R
authors.lastname: Subramaniam
authors.forename: M F
authors.initials: MF
authors.lastname: Weiss
authors.forename: V M
authors.initials: VM
authors.lastname: Monnier
mesh_terms: D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D048748:MAP Kinase Kinase Kinase 3; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D011765:Pyruvaldehyde / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D016328:NF-kappa B; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D012710:Serum Albumin, Bovine; D011765:Pyruvaldehyde; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048748:MAP Kinase Kinase Kinase 3; D020930:MAP Kinase Kinase Kinases; C482626:Map3k3 protein, mouse