Abstract 635: Apolipoprotein A-I Inhibits Lipopolysaccharide-Induced Inflammation and Activation of Major Stress Signaling Kinases in 3T3-L1 Adipocytes in an ABCA1-Dependent Manner

Abstract

Obesity is characterised by adipose tissue inflammation and increased secretion of pro-inflammatory adipokines, such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6). A recently suggested mechanism linking obesity with its co-morbidities is an increase in stress signalling pathways, including p38 mitogen-activated protein kinase (p38MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK). Apolipoprotein A-I (apoA-I), the main lipoprotein of high density lipoproteins, has potent anti-inflammatory properties. It is also the primary acceptor of cholesterol that is exported from cells expressing the ATP-binding cholesterol transporter A1 (ABCA1). The role of apoA-I and ABCA1 in the inhibition of adipocyte inflammation is unknown. Aim: To determine if apoA-I inhibits inflammation and activation of stress signalling pathways in lipopolysaccharide (LPS)-stimulated 3T3-L1 adipocytes. Methods: Mature 3T3-L1 adipocytes were pre-incubated for 16 h with or without apoA-I (1 mg/mL), then stimulated for 6 h with LPS (100 ng/mL). MCP-1 and IL-6 secretion and mRNA levels were quantified by ELISA and qPCR, respectively. 3T3-L1 adipocytes were also pre-incubated with apoA-I, then stimulated with LPS for 10, 20, 30 and 60 min prior to quantification of ERK1/2, p38MAPK and JNK1/2 phosphorylation by western blotting. ABCA1 expression was silenced by transfection with ABCA1siRNA (50 nM) for 24 h. Results: Pre-incubation of 3T3-L1 cells with apoA-I reduced the LPS-mediated secretion of MCP-1 from 2666±216 to 983±65 pg/mg cell protein, while IL-6 secretion decreased from 120±22 to 55±11 pg/mg cell protein (p<0.05 for both). MCP-1 and IL-6 mRNA levels decreased by 43- and 5-fold, respectively, (p<0.05 for both). These effects of apoA-I were attenuated in 3T3-L1 cells in which ABCA1 expression was silenced. LPS induced phosphorylation of ERK1/2, p38MAPK and JNK1/2 in 3T3-L1 cells. Pre-treatment of the LPS-stimulated cells with apoA-I inhibited phosphorylation of ERK1/2, p38MAPK and JNK1/2 by 66.2±4%, 58.8±10.7% and 33.2±14% respectively (p<0.05 for all). Conclusion: ApoA-I exerts anti-inflammatory effects and inhibits activation of MAP kinases in 3T3-L1 adipocytes in an ABCA1-dependent manner.