Effect Of Endogenous Apolipoprotein O On Cytokines Secretion In Adipocytes And The Possible Mechanisms Investigation

BackgroundApolipoprotein O (apoO) is a very recently discovered apolipoprotein in2006. The biologic role of apoO remains incompletely unknown. Under physiological conditions, its plasma concentrations range from1.05to5.47μg/ml, with a mean value in normal subjects of2.21±0.83μg/ml. The secreted apoO mainly exists in high density lipoprotein (HDL). However, high expression of apoO resulting in a substantial increase in plasma apoO level as well as HDL apoO content exerts effects neither on HDL-cholesterol concentration nor HDL functionality. In line with this, there are no correlations between the secreted apoO and lipid components in plasma, suggesting that the function of the secreted apoO is very limited.Besides functions as a secreted protein, apoO also plays an improtant role within cells. Recently, we found that apoO levels were increased in acute coronary syndrome patients and positively associated with the inflammatory marker high-sensitive C-reactive protein (hs-CRP), suggesting a potential role as an inflammatory predictor. Consistent with this, the expression of several inflammation-related genes were significant changed in apoO-silencing cells. However, the role of apoO in regulating celluar inflammatory response and the possible mechanisms are still unknown. In view of the fact that apoO is highly expressed in adipocytes and inappropriate secretion of pro-inflammatory adipokines plays an important role in the development of obesity and atherosclerosis, it is necessary to explore the function of endogenous apoO in inflammation in adipocytes.ObjectiveThe aims of this study were to investigate whether the endogenous apoO can regulate the inflammatory response in adipocytes and the possible mechanisms.Methods1. Effects of apoO on the secretory function of adipocytesHuman adipose-derived mesenchymal stem cells (ADSCs) were derived from subcutaneous adipose tissue of patients undergoing abdominal surgery. Then ADSCs were cultured and induced into mature adipocytes and identified by flow cytometry and oil red O staining. An efficient lentiviral siRNA vector targeting the human apoO gene was designed and constructed. Differentiated adipocytes and the cells transfected with lentiviral vector were respectively incubated with low concentration of oxidized low-density lipoprotein (ox-LDL) for indicated time. Then the experiments were performed as follows:(1) mRNA expression of apoO, interleukin-6(IL-6), monocyte chemoattractant protein-1(MCP-1) and tumor necrosis factor-a (TNF-a) were assessed by real-time PCR;(2) western blot analysis were performed to test the protein level of apoO;(3) ELISA was used to determine the concentration of IL-6, MCP-1and TNF-a in medium.2. Possible mechanisms involving the inhibitory effect of apoO on inflammationSome ADSCs were induced for4days and transfected with lentiviral vector to silence apoO expression. These cells continue to differentiate into mature adipocytes for another10days in the presence of insulin, IBMX, dexamethasone and rosiglitazone. Both the normal adipocytes and apoO-silencing adipocytes were intervened by low concentration of ox-LDL for indicated time. Then the experiments were performed as follows:(1) mRNA expression of Toll-like receptor4(TLR4), marrow-like differentiation factor88(MyD88), interleukin-1receptor-associated kinase4(IARK4) and tumor necrosis factor receptor-associated factor6(TRAF6) were assessed by real-time PCR;(2) western blot analysis were performed to test the protein level of TLR4, nuclear factor κB inhibitor protein a (IκBα) and phosphorylated IκBα (phospho-IκBα).3. Modes of regulation of the TLR4/NF-κB signaling pathway by apoODifferentiated adipocytes were incubated with low concentration of ox-LDL for indicated time. Immunofluorescence was used to visualize the subcellular localization of apoO and caveolin-1. Images were captured with a confocal microscope. Isolation of caveolae was conducted using sucrose density gradient centrifugation and the existence of apoO in caveolae was detected. The interaction between apoO and caveolin-1was examined using co-immunoprecipitation assay. We finally investigate the cellular cholesterol contents in apoO-silencing adipocytes.Results1. The impact of apoO on secretory function of adipocytes1) We have successfully isolated ADSCs harvested from subcutaneous adipose tissue and the ADSCs can differentiate into mature adipocytes in vivo.2) We have successfully constructed the apoO-siRNA lentiviral vector which can effectively silence the expression of apoO in human adipocytes (P<0.005)3) Compared with the control group, the expression of apoO was markedly increased in adipocytes stimulated with low concentration of ox-LDL. The secretion of pro-inflammatory cytokines such as IL-6, MCP-1, TNF-α were dramaticly decreased in this group (P<0.01).4) The inhibitory effect of low concentration of ox-LDL on pro-inflammatory cytokines production was abolished after apoO silencing.2. Possible mechanisms involving the inhibitory effect of apoO on inflammation1) Compared with the control group, the stimulation of low concentration of ox-LDL did not affact the expression of TLR4in human adipocytes (P>0.05).2) Low concentration of ox-LDL can significantly inhibit the activation of TLR4/NF-κB signaling pathway (P<0.05).3) The inhibitory effect of low concentration of ox-LDL on TLR4/NF-κB signaling pathway was abolished after apoO silencing.3. Modes of regulation of the TLR4/NF-κB signaling pathway by apoO1) Confocal microscopy analysis clearly showed that apoO was distributed in a ring around the lipid droplets, and co-localized with perilipin on the surface of lipid droplets in adipocytes.Treatment of adipocytes with low concentration of ox-LDL induced apoO to translocate into caveolae and co-localize with caveolin-1.2) Co-immunoprecipitation indentified the interaction between apoO and caveolin-1.3) There is no alteration of cellular cholesterol contents in apoO-silencing adipocytes (P>0.05). Conclusions1. Endogenous apoO mediated the inhibitory effect of low concentration of ox-LDL on the production of the pro-inflammatory cytokines such as IL-6, MCP-1and TNF-a in human adipocytes.2. The inhibitory effect of apoO on inflammatory response probably mediated by its influence on the activation of TLR4/NF-κB signaling pathway.3. ApoO could impair the activation of TLR4/NF-κB signaling pathway by its interation with caveolin-1which could subsequently regulate the secretion of pro-inflammatory cytokines in adipocytes. This anti-inflammatory effect of apoO did not rely on the alteration of cellular cholesterol contents.