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Adipose tissue inflammation is closely linked with the development of insulin resistance, heart disease, diabetes and cancer. The nuclear factor-κB (NF-κB) p65 pathway is one of the most inflammation signaling pathway, moreover, free fatty acid (FFA) is one of the most important inducements to NF-κB pathway in adipose tissue inflammation. It is well known that exposure of 3T3-L1 adipocytes to FFA is a strong inducement to NF-κB pathway. Activation of IκB kinase (IKK) is a critical step that lead to NF-κB activation. IKK phosphorylates the NF-κB regulatory protein IκB, resulting in the release of NF-κB for nuclear translocation. In addition, IKK directly phosphorylates p65 subunit of NF-κB Ser536, Ser276, and Ser468, enhancing the transcriptional activity of NF-κB. Activated X-box binding protein 1 (XBP1) is a key transcription factor which protects injured cells by modulate the function of unfolded protein response (UPR). This study is to investigate whether spliced XBP1 blocks FFA-induced NF-κB pathway in 3T3-L1 adipocytes.
Small RNA interference (siRNA) was used to achieve knock-down of XBP1. The knock-down efficiency was tested by measuring the protein level of spliced XBP1 using western blotting. Briefly, 3T3-L1 adipocytes were transfected with scramble siRNA or XBP1 siRNA using Lipofectamine 2000 for 6 h. Overexpression of spliced XBP1 was achieved by using adenovirus encoding mouse spliced XBP1 (Ad-XBP1s) in cultured 3T3-L1 adipocytes. The effect of overexpression by spliced XBP1 was examined by western blot analysis. Adenoviruses encoding green fluorescent protein (Ad-GFP) were used as negative control. 3T3-L1 adipocytes were infected with Ad-GFP or Ad-XBP1 for 48 h, and then were stimulated in the presence or absence of FFA (0.5 mM) for 4 h.
Phos-phorylation of IKK, phos-phorylation IκB at Ser32, and a phosphorylation of NF-κB p65 at Ser536 induced by FFA were determined at the protein levels by western blot analysis to measure the activation of NF-κB pathway.
Down-regulation of XBP1 by transfection with XBP1 siRNA increased phos-phorylation of IKK, phos-phorylation IκB at Ser32, and phosphorylation of NF-κB p65 subunit at Ser536 levels in 3T3-L1 adipocytes. In parallel, up-regulation of XBP1 by infection with Ad-XBP1 inhibited phosphorylation of IKK, phosphorylation IκB at Ser32, and phosphorylation of NF-κB p65 subunit at Ser536 levels in 3T3-L1 adipocytes.
Activated XBP1 suppresses FFA-induced NF-κB pathway in 3T3-L1 adipocytes.