Effect Of HDL And ApoA-I Mimetic Peptide On Secretion In Adipocytes And The Possible Mechanisms Investigation

BackgroundAtherosclerosis diseases are the leading cause threatening humanhealth. Obesity is an independent risk factor for coronary heart disease(CHD). Since dysfunction of adipocytes plays a key role for obesity,studying function of adipocytes is of great significance for the preventionand treatment of obesity and CHD. Adipocyte functions have been furtherexplored and recognized recently. Dysfunctional adipocytes have beenthought to be in close relation with atherosclerosis.Adipose tissue is the largest energy reservoir in the body. It storesexcess energy in form of triglyceride. Moreover, adipose tissue showsactive endocrine function that secrets various adipokines, many of whichmight play significant roles in atherogenesis. Adipose tissue may be asignificant contributor to increase systemic chronic inflammation throughsecreting a number of pro-inflammatory adipokines. Adiponectin, anadipose-specific secreted and inflammation-related protein, is a protectivefactor for insulin-resistance, diabetes and atherosclerosis. The elevatedlevel of tumor necrosis factor-α(TNFα), an inflammatory cytokine, isclosely associated with the severity of atherosclerosis. The adipose tissueis a significant source of endogenous TNFαproduction. Monocytechemoattractant protein-1 (MCP-1) is a potent chemotactic factor formonocytes and may serve as a signal that triggers inflammation ofatherosclerosis. Adipocytes may be induced to produce MCP-1 withseveral types of stimulators.Lipopolysaccharide (LPS) is an acknowledged inducer ofinflammation. Low-density lipoproteins (LDLs) are thought to becomeatherogenic after undergoing oxidative modifications. In addition to theirrole in foam cell formation, oxidized low-density lipoproteins (oxLDL) may participate in local chronic inflammatory response. High densitylipoproteins (HDLs), in contrast to LDLs, are antiatherogenic andcardioprotective, but the detailed molecular mechanisms remain poorlyunderstood. The effects that HDL regulates the intracellular lipidsmetabolism in adipocytes have been confirmed. However, the effects ofHDL on regulating adipocytes inflammation have not been investigatedso far. Apolipoprotein A-I (apoA-I), the main protein of HDL and apoA-Imimetic peptide are potent anti-inflammatory agents that may havetherapeutic potential. But whether apoA-I mimetic peptide showsanti-inflammatory effects in adipocytes is not understood.Peroxisome proliferator-activated receptorγ(PPARγ) is a nuclearreceptor which mainly be expressed in adipose tissue. Nuclear factor-κB(NF-κB) is a crucial transcription factor, which controls the transcriptionof many genes with an established role in atherosclerosis.CCAAT/enhancer binding proteins (C/EBPs) is another important kind ofnuclear transcription factor. It was shown that PPARγ,NF-κB andC/EBPs all participate in the transcription control of some cytokines. Andthey may have actions on the regulation of transcription and secretion ofadipokines at least in part, which need to be further studied.ObjectiveThe aim of this study was to evaluate the effect of HDL and L-4F,an apoA-I mimetic peptide, on the secretion and expression ofadiponectin, TNFαand MCP-1 in fully differentiated 3T3-L1adipocytes with inflammatory status, and to elucidate the possiblemechanisms,MethodsFully differentiated 3T3-L1 adipocytes were divided into threegroups: (1)LPS stimulated group: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentrations of HDL(10-100μg/ml) or L-4F (1-50μg/ml) with 100μg/L LPS (n=3). Evaluatethe levels of adiponectin in supernatant, the mRNA expression ofadiponectin and PPARγ, and the activity of NF-κB. (2) OxLDLstimulated group (for 1h): Fully differentiated 3T3-L1 adipocytes wereincubated in the medium containing various concentrations of HDL (10-100μg/ml) or atorvastatin (0.1-10μM) with oxLDL (50μg/ml)stimulated for 1 hour, with/without H-89 (10μM) preincubated. TNFαand PPARy mRNA expression, NF-κB activity and IκB protein level wereevaluated. (3) OxLDL stimulated group (for 1h,6h,12h): Fullydifferentiated 3T3-L1 adipocytes were incubated in the mediumcontaining various concentrations of HDL (10-100μg/ml) or L-4F (10-100μg/ml) with oxLDL (50μg/ml) stimulated for 1h, 6h or 12h,with/without H-89 (10μM) preincubated. The concentrations of MCP-1in supernatant, the mRNA expression of MCP-1, the levels of C/EBPαand C/EBPβwere evaluated. The monocyte chemotaxis assay wasperformed by micropore filter method using a modified Boyden chamber.Results1. The secretion and mRNA expression of adiponectin decreased by44％and 60％after LPS stimulation in full differentiated 3T3-L1adipocytes (P＜0.05).2. HDL and L-4F dose-dependently increased adiponectin secretionand mRNA expression. Compared with LPS stimulation, treatment withHDL at 10, 50, 100μg/ml significantly enhanced adiponectin secretionabout 1, 1.9, 3.2-fold respectively (P＜0.05), and treatment with apoA-Imimetic peptide at 1, 10, 50μg/ml significantly enhanced adiponectinsecretion about 1.2, 2.9, 3.4-fold respectively (P＜0.05). Compared withLPS stimulated group (0.36±0.05), adiponectin mRNA expression increased to 0.72±0.13, 1.05±0.15 and 1.51±0.11 respectively by 10, 50and 100μg/ml HDL and to 0.79±0.12, 1.40±0.18 and 1.58±0.10respectively by 1, 10 and 50μg/ml L-4F (all P＜0.05).3. PPARγpresents high level expression in fully differentiated3T3-L1 adipocytes. Compared with LPS stimulated group, HDL andL-4F significant enhanced PPARγmRNA expression (2.85±0.69 vs0.38±0.19, 2.92±0.96 vs 0.38±0.19; P＜0.05) and the activity of NF-κBdecreased significantly after using HDL and L-4F (0.48±0.09 vs1.93±0.59, 0.43±0.08 vs 1.93±0.59; P＜0.05).4. OxLDL stimulation for 1 hour induced a significant enhancementof TNFαsecretion (2.98±1.10 pg/ml vs 8.93±3.26 pg/ml, P＜0.001),TNFαmRNA expression (0.218±0.078 vs 0.633±0.157, P＜0.001) andNF-κB activity (1.54±0.44 vs 4.08±0.87, P＜0.05) in 3T3-L1 adipocytes.5. Atorvastatin reduced TNFαsecretion and mRNA expression,inhibited NF-κB activation, and enhanced PPARγmRNA expression in adose-dependent manner. Treatment with atorvastatin at 10μMsignificantly suppressed TNFαmRNA expression by 56.5％, inhibitedNF-κB activation by 41.2％, and increased PPARy mRNA expression toabout 2.83-fold, as compared with oxLDL stimulated group. HDLdose-independently inhibited TNFαsecretion and mRNA expression,NF-κB activation and IκB degradation. Treatment with HDL at 100μg/mlsuppressed TNFαmRNA expression by 64.5％, inhibited NF-κBactivation by 49％, and stabilized IκB significantly, as compared withoxLDL stimulated group, but had no effect on PPARγmRNA expression.The anti-inflammatory effect of HDL could be partially abolished byPKA inhibitor H-89.6. OxLDL stimulation for 12 hours induced a significant increase ofMCP-1 secretion and expression to 2.13-fold in 3T3-L1 adipocytes. And the mean of migration distances of human peripheral blood monocyteinduced by oxLDL was significantly longer than that of the randommigration group (69.88±8.19μm vs 136.75±8.03μm, P＜0.001).7. L-4F and HDL decreased MCP-1 secretion and expression in adose-dependent manner as well as reduced the chemotactic activity ofmonocyte. 50μg/ml L-4F and 100μg/ml HDL decreased MCP-1secretion and expression by 91±6％and 79±7％respectively (P＜0.001).PKA inhibitor H-89 (10μM) significantly reduced oxLDL-inducedMCP-1 expression by 71±7％(P＜0.001), but no further decreasingoccurred when H-89 (10μM) adding with the existing of L-4F(50μg/ml)or HDL(100μg/ml) (P＞0.05).8. OxLDL (50μg/ml) stimulation had no marked influence onC/EBPαexpression, but increased C/EBPβlevel in a time-dependentmanner. H-89, HDL and L-4F all attenuated oxLDL-induced C/EBPβlevel in 3T3-L1 adipocytes.Conclusions1. Full differentiated 3T3-L1 adipocytes would be induced to be ininflammatory status after LPS or oxLDL stimulation, which showed thatadiponectin secretion and expression decreased and TNF a and MCP-1secretion and expression increased.2. HDL and L-4F could upregulate the secretion and mRNAexpression of adiponectin in 3T3-L1 adipocytes in inflammatory status.PPARγand NF-κB pathway may participate in the process.3. Atorvastatin inhibited TNFαsecretion and mRNA expression inoxLDL-stimulated 3T3-L1 adipocytes by NF-κB and PPAR7 signalingpathways.4. HDL could suppress TNFαsecretion and mRNA expression inoxLDL-stimulated 3T3-L1 adipocytes by PKA-IκBα-NF-κB signaling pathway, without any contact between HDL and oxLDL. The intensity ofanti-inflammatory effect of HDL was similar to that of atorvastatin.5. OxLDL induced C/EBPβexpression in a time-dependentmanner.6. L-4F and HDL dose-dependently counterbalanced the influence ofoxLDL on MCP-1. The cAMP/PICA-C/EBPβsignaling pathway mayparticipate in the process.