Abstract
The present study was designed to determine whether N-acetylcysteine (NAC), a potent antioxidant, modulates nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) in adipocytes. Stimulation by the combination of 5μg/ml of LPS and 100ng/ml of TNF-α (LT) significantly enhanced NO production in 3T3-L1 adipocytes. Preincubation of the cells with NAC (5-20mM) for 24h suppressed the increased NO production in a dose-dependent manner. The production of NO was decreased by 49% at the concentration of 20mM of NAC. The decrease in NO production by NAC was accompanied by a decrease in inducible nitric oxide synthase (iNOS) protein, detected by immunoblot analysis, and iNOS mRNA, determined by real-time reverse-transcriptase coupled polymerase chain reaction analysis. Nuclear factor-κB (NF-κB) was significantly activated by LT-treatment, while the pretreatment with 20mM of NAC prevented the activity by 42%. Pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, also inhibited the LT-mediated NO production dose-dependently. One hundred μM of PDTC inhibited the NO production by 46%. We also investigated the effect of NAC and PDTC on the production of interleukein-6 (IL-6), which is regulated transcriptionally by NF-κB in 3T3-L1 adipocytes. IL-6 production was markedly increased by LT stimulus, and the enhanced secretion of IL-6 was suppressed in a dose-dependent manner by pretreatment with NAC or PDTC. These results suggest that NAC regulates iNOS expression and NO production in adipocytes through the modulating activation of NF-κB.
