Abstract
We investigated the regulatory effect of glucosamine (GlcN) for the production of nitric oxide (NO) and expression of inducible NO synthase (iNOS) under various glucose conditions in macrophage cells. At normal glucose concentrations, GlcN dose dependently increased LPS-stimulated production of NO/iNOS. However, GlcN suppressed NO/iNOS production under high glucose culture conditions. Moreover, GlcN suppressed LPS-induced up-regulation of COX-2, IL-6, and TNF-α mRNAs under 25 mm glucose conditions yet did not inhibit up-regulation under 5 mm glucose conditions. Glucose itself dose dependently increased LPS-induced iNOS expression. LPS-induced MAPK and IκB-α phosphorylation did not significantly differ at normal and high glucose conditions. The activity of LPS-induced nuclear factor-κB (NF-κB) and DNA binding of c-Rel to the iNOS promoter were inhibited under high glucose conditions in comparison with no significant changes under normal glucose conditions. In addition, we found that the LPS-induced increase in O-GlcNAcylation as well as DNA binding of c-Rel to the iNOS promoter were further increased by GlcN under normal glucose conditions. However, both O-GlcNAcylation and DNA binding of c-Rel decreased under high glucose conditions. The NF-κB inhibitor, pyrrolidine dithiocarbamate, inhibited LPS-induced iNOS expression under high glucose conditions but it did not influence iNOS induction under normal glucose conditions. In addition, pyrrolidine dithiocarbamate inhibited NF-κB DNA binding and c-Rel O-GlcNAcylation only under high glucose conditions. By blocking transcription with actinomycin D, we found that stability of LPS-induced iNOS mRNA was increased by GlcN under normal glucose conditions. These results suggest that GlcN regulates inflammation by sensing energy states of normal and fuel excess.
Highlights
We investigated the regulatory effect of glucosamine (GlcN) for the production of nitric oxide (NO) and expression of inducible NO synthase under various glucose conditions in macrophage cells
GlcN increased LPS-mediated NO generation at normal glucose conditions but GlcN strongly inhibited LPS-mediated NO generation at concentrations higher than 25 mM glucose (Fig. 1A)
LPS-mediated NO/inducible NO synthase (iNOS) Increases Were Inhibited by PDTC at 25 mM Glucose but Not at 5 mM Glucose Concentrations—We investigated the effects of the nuclear factor-B (NF-B) inhibitor, pyrrolidine dithiocarbamate (PDTC), on activation of NF-B as well as the induction of NO/iNOS in response to LPS and/or GlcN under ambient (5 and 25 mM) glucose conditions
Summary
GlcN, 2-amino-2-deoxy-D-glucose; HBP, hexosamine biosynthetic pathway; ActD, actinomycin D; TTP, tristetraprolin; ER, endoplasmic reticulum; MTT, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; WGA, wheat germ agglutinin. GlcN by regulating c-Rel O-GlcNAcylation and subsequent NF-B activity in both microglia and macrophage cells [16]. Vertebrate NF-B transcription complexes can be any of a variety of homo- and heterodimers formed by the subunits RelA (p65), RelB, c-Rel, p50 (a product of p105), and p52 (a product of p100) [22, 23] These complexes bind to DNA regulatory sites and usually activate specific target gene expression. We investigated the potential of both pro- and anti-inflammatory effects of GlcN to regulate NO/iNOS induction as well as O-GlcNAcylation in macrophage cells under normal and high glucose culture conditions. GlcN demonstrated an inverse effect on LPS-mediated iNOS induction and O-GlcNAcylation and DNA binding of c-Rel between normal and high glucose conditions. GlcN or the HBP metabolic pathway likely regulates inflammation and the related signaling pathway depends on nutrient availability
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