Carboxybutyrylated glucosamine inhibits LPS‐induced iNOS expression in RAW 264.7 macrophages via increased protein O‐GlcNAc levels

Abstract

We have shown that increasing levels of O‐linked‐N‐acetylglucosamine (O‐GlcNAc) on nucleocytoplasmic proteins, with glucosamine (GlcN) attenuated the inflammatory response of cells treated with lipopolysaccharides (LPS). Others reported that carboxybutyrated glucosamine (CGlcN) attenuated LPS induced inflammatory response in macrophage cell line (RAW264.7). We hypothesized, therefore, that the anti‐inflammatory effect of CGlcN was mediated via an increase in O‐GlcNAc levels. We found that CGlcN increased O‐GlcNAc levels in RAW cells in a dose dependent manner and that at 24 hrs the effect of CGlcN was greater than GlcN. Treatment with 0.1μg/ml LPS significantly decreased RAW O‐GlcNAc levels, which was prevented by both GlcN and CGlcN. LPS treatment significantly increased iNOS expression compared to untreated cells and this was attenuated by both GlcN and CGlcN; however CGlcN appeared to have a greater effect on attenuating iNOS expression that GlcN. We also tested other compounds based on variants of CGlcN; however, none of these compounds had any effect on RAW O‐GlcNAc levels or the response to LPS treatment. These data suggest that CGlcN inhibits LPS induced iNOS expression via increased O‐GlcNAcylation and that this effect appears to be more pronounced than GlcN; however, the mechanism by which this occurs remains to be determined. Grants: NIH HL067464/HL079364.