【作者】 宋君；

【导师】 陈丽； 沈胡英； 王兴利；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 背景代谢综合征(MS)是伴有糖尿病／糖耐量异常、高血压、高血脂、中心性肥胖、内皮细胞功能异常等代谢方面异常的一组疾病总称,其发生动脉粥样硬化等心血管并发症的风险明显增加,然而具体机制尚不清楚。研究表明巨噬细胞脂质沉积在代谢综合征患者心血管并发症(包括动脉粥样硬化,高血压和心肌梗塞等)的发生发展中起到了关键作用。巨噬细胞过量的脂质沉积可通过多条信号转导通路启动氧化应激和炎症反应,损伤细胞形态和功能,从而促进心血管并发症的发生发展。因此深入探讨巨噬细胞脂质沉积的分子机制对今后预防代谢综合征心血管并发症的发生具有重要作用。近年来高游离脂肪酸在代谢综合征心血管并发症中的作用正日益引起研究者的关注。临床试验表明在代谢综合征动脉粥样硬化,心肌梗塞等疾病中均存在高血清浓度的游离脂肪酸。尽管高游离脂肪酸所致心血管并发症的发生机制并不清楚,但高游离脂肪酸参与动脉粥样硬化等心血管并发症发生发展的多个环节,如单个核细胞贴壁,血小板聚集,内皮细胞凋亡,胰岛素抵抗,细胞内脂质沉积等,直接损伤心血管功能,促进动脉粥样硬化等心血管并发症的进程。然而高游离脂肪酸能否促进巨噬细胞内脂质沉积进而参与动脉粥样硬化等并发症的发生并不清楚。脂肪酸结合蛋白4(FABP4)作为脂肪酸结合蛋白家族中的一员,特异性表达于巨噬细胞和脂肪细胞中,直接参与脂肪酸的转运和代谢,促进细胞内脂质沉积。近来研究表明,FABP4在连接肥胖和动脉粥样硬化的发生中发挥了重要作用。FABP4表达缺失可通过降低炎症因子在巨噬细胞中的表达保护高血脂老鼠避免动脉粥样硬化的发生。同时在脂肪细胞脂质积聚的过程中FABP4表达增加,然而,FABP4是否参与了高游离脂肪酸所致的巨噬细胞脂质沉积尚不清楚,其中的分子机制也有待于进一步研究。叉头状转录因子01(forkhead transcription factor 01, FOXO1)作为Foxo蛋白家族中的一员,定位于13号染色体并编码655个氨基酸,与细胞周期调控,能量代谢及细胞凋亡有关,近年来其在脂代谢中的作用日益受到关注。FOXO1可被高游离脂肪酸激活,参与细胞内脂质沉积。同时参与脂蛋白脂肪酶(LPL)等多种脂代谢关键酶的转录调节。但是,FOXO1是否参与FABP4的转录调节尚不清楚,同时游离脂肪酸对FOXO1转录活性的调节机制有待进一步研究。因此,基于以上研究,本研究将验证以下假说：高浓度游离脂肪酸通过转录因子FOXO1促进FABP4的表达增加脂肪酸的转运,从而促进细胞内脂质沉积。高浓度游离脂肪酸-FOXO1-FABP4信号通路可能是高游离脂肪酸下诱导巨噬细胞内脂质沉积的新的分子机制,从而为代谢综合征心血管并发症的干预治疗提供潜在靶点。方法1.细胞培养：人单核细胞株THP-1生长于含10%胎牛血清,100U／ml青霉素,100ug/ml链霉素和5%L-谷氨酰胺的RPMI-1640培养液中。每48-72小时传代一次。取对数生长期细胞进行实验。实验前采用100nm佛波酯(phorbol 12-myristate 13-acetate, PMA)孵育THP-1细胞72小时,使其诱导分化为巨噬细胞。诱导后的细胞予不同浓度的游离脂肪酸处理或转染siRNAs, DNAs以构建细胞模型。2.游离脂肪酸配制：饱和脂肪酸PA,多不饱和脂肪酸LA及单不饱和脂肪酸0A用于本研究中。首先将游离脂肪酸溶解于100%异丙醇配成200mM FFA储存液。后以10%无游离脂肪酸,低内毒性的BSA使其终浓度变为0.25-2mM。调PH至7.5滤器滤过后存于-20℃冰箱备用。3.siRNA及质粒DNA转染：采用特异的siRNA进行基因沉默,包括FOXO1 siRNA。并采用质粒DNA转染进行过表达,包括野生型FOXO1 (WT),负显性型FOXO1 (DN)及持续激活型FOXO1 (CA).使用lipofectamineTM 2000脂质体包裹并转染THP-1巨噬细胞。被转染的巨噬细胞,再予以游离脂肪酸处理。4. Western Blot:处理后的细胞采用细胞裂解液提取总蛋白。取15μg蛋白与蛋白Marker一起上样。在10%SDS-聚丙烯酰胺凝胶上电泳后,转移至聚偏氟乙烯膜(PVDF)上。电转膜采用牛奶封闭,一抗孵育过夜,PBST清洗三遍后,使用HRP标记的二抗孵育。ECL曝光显色。蛋白半定量采用Quantity One软件进行,以目的蛋白与β-actin条带积分光密度的比值表示蛋白表达的相对值。5.实时定量PCR：采用Trizol提取细胞的总RNA。mRNA应用iScript cDNA合成试剂盒逆转录成cDNA。iCycler iQ荧光探针系统进行实时PCR。采用FABP4与β-actin循环阈值(threshold cycle, Ct)的比值表示mRNA的相对水平。6.细胞内脂质沉积的检测：应用油红0染色检测细胞内脂质沉积。处理后的细胞以福尔马林固定,然后在油红0工作液中孵育30分钟,测定490nm的吸收光值。7.免疫荧光染色：载玻片上处理的细胞使用含有1%BSA的一抗进行孵育,清洗后用得克萨斯红标记的二抗进行处理。DAPI染核,封片后采用Olympus Fluoview 300荧光显微镜采集图像。结果1.高浓度游离脂肪酸增加巨噬细胞内脂质沉积应用不同浓度的游离脂肪酸处理巨噬细胞以构建细胞模型。结果发现游离脂肪酸可显著增加细胞内脂质沉积,并呈剂量依赖性(P<0.05)。2.高游离脂肪酸通过调节脂肪代谢基因表达增加脂质沉积游离脂肪酸可促进FABP4和DGAT的表达,而抑制CPT-1的表达,提示游离脂肪酸可能通过增加脂肪酸转运,抑制脂肪酸氧化,增加甘油三脂合成而增加巨噬细胞内脂质沉积。3.高浓度游离脂肪酸在转录水平调节FABP4的表达采用实时定量PCR发现,PA能显著增加FABP4的mRNA水平并呈剂量依赖性,表明游离脂肪酸能在转录水平增加FABP4的表达。4.转录因子FOXO1参与了PA所致的FABP4的表达增加我们进一步研究FOXO1是否介导PA所致的FABP4表达增加。采用特异性siRNA沉默FOXO1基因(P<0.01), FOXO1 siRNA可逆转棕榈酸所致的FABP4的上调。同时过表达野生型和持续活性型FOXO1能显著增加FABP4的表达而负显性型FOXO1降低FABP4的表达,以上结果表明FOXO1正性调节FABP4的表达。5. FOXO1参与了PA所致的巨噬细胞内脂质沉积FOXO1siRNA不仅降低基础状态下巨噬细胞内脂质沉积,也可逆转游离脂肪酸诱导的细胞内脂质沉积的增加,表明FOXO1参与了PA所致的巨噬细胞内脂质沉积。6.PA可通过促进FOXO1核转位激活FOXO1介导的转录活性免疫荧光显示,棕榈酸能促进FOXO1的核转位,提示PA能通过促进FOXO1核转位激活其转录活性而增加FABP4的转录水平。结论1.高浓度游离脂肪酸通过增加FABP4和DGAT的表达,降低CPT-1的表达而增加巨噬细胞脂质沉积,并呈剂量依赖性。2.棕榈酸在转录水平调节FABP4的表达,转录因子FOXO1参与该过程的调节。3.棕榈酸通过促进FOXO1核移位增加其转录活性。4.转录因子FOXO1参与游离脂肪酸所致的巨噬细胞脂质沉积5. FFA-FOXO1-FABP4信号通路可能是高游离脂肪酸下诱导巨噬细胞内脂质沉积的新的分子机制,为代谢综合征心血管并发症的干预治疗提供了理论基础。背景游离脂肪酸介导的脂质沉积在代谢综合征心血管并发症的发生发展中起关键作用。巨噬细胞脂质沉积可加剧血管壁胆固醇和甘油三脂的沉积,或增加心血管氧化应激和炎症反应引起组织损伤和细胞功能异常。因此有效减少细胞内脂质沉积对预防代谢综合征心血管并发症的发生具有重要作用。AMPK可启动分解代谢途径从而增加ATP的产生,因此在能量代谢调控中发挥重要作用。目前研究证实AMPK信号通路的激活具有心血管保护效应。但机制不明。以往研究表明二甲双胍可激活AMPK通路。同时AMPK的激活可有效降低肝脏,骨骼肌,脂肪等细胞内的脂质沉积。但是,AMPK通路的激活能否降低棕榈酸(palmitic acid, PA)所致的巨噬细胞内脂质沉积尚不明确,其中的机制尚不清楚。脂肪酸结合蛋白4 (FABP4)作为脂肪酸结合蛋白家族中一员,参与了细胞内脂肪的转运和脂肪酸代谢,可促进细胞内脂质的沉积。许多研究表明,FABP4在肥胖和动脉粥样硬化中表达增强,FABP4表达缺失能保护高血脂老鼠避免动脉粥样硬化的发生发展,提示FABP4是连接肥胖和动脉粥样硬化的重要分子。然而,FABP4是否参与AMPK通路对细胞内脂质沉积的调节以及AMPK通路如何调节FABP4的表达,均需要进一步的验证。最近研究发现转录因子FOXO1是调节脂肪代谢的关键转录因子。FOXO1可促进细胞内的脂质沉积,同时参与多种脂肪代谢相关酶的转录调节,促进脂肪酸转运及甘油三脂合成。本研究前期研究发现FOXO1参与了FABP4的转录调节,但AMPK通路对FOXO1的转录活性以及FABP4表达的影响尚不明确。因此,本研究将验证如下假说：AMPK通路的激活能够通过降低游离脂肪酸所致的FABP4的表达而降低细胞内脂质沉积。FOXO1作为转录因子参与了该过程的调控。AMPK-FOXO1信号通路可能是减少巨噬细胞内脂质沉积的重要防御机制,从而为治疗代谢综合征心血管并发症提供潜在治疗靶点。方法1.细胞培养：原代人单个核细胞系THP-1 (human monocytic leukemia cell line THP-1),生长于含10%胎牛血清,100U／ml青霉素,100ug/ml链霉素和5%L-谷氨酰胺的RPMI-1640培养基中。取对数生长期细胞进行实验。实验前采用100nM佛波酯(phorbol 12-myristate 13-acetate, PMA)孵育THP-1细胞72小时,使其诱导分化为巨噬细胞后,细胞被转染siRNAs,质粒DNA或刺激以不同浓度的二甲双胍或者PA以构建细胞模型。2.游离脂肪酸的配制：将棕榈酸(PA)溶解于200mM的异丙醇中,同时以10%无游离脂肪酸,低内毒素的BSA使其终浓度在1-5 mM。所有溶液的PH值调为7.5,使用滤器过滤,-20℃储存。将不含有PA的BSA溶液作为对照。在处理细胞前将PA按1：10的比例使用1640培养基进行稀释。3.siRNA及质粒DNA转染：采用特异siRNA进行基因沉默,包括AMPK siRNA和FOXO1 siRNA。质粒DNA转染分别用0.5μg野生型FOXO1表达质粒(FOXO1 WT),负显性型FOXOl表达质粒(dominant-negative FOXO1, DN)和持续激活型FOXO1表达质粒(constitutively active-FOXO1, CA)处理THP-1巨噬细胞。采用lipofectamineTM 2000脂质体包裹,并转染巨噬细胞。被转染的THP-1巨噬细胞,再使用PA和met等处理24h。4.免疫荧光染色：将处理好的细胞使用含有一抗1%BSA进行孵育,并用得克萨斯红标记的二抗进行孵育。使用DAPI染核,采用Fluoview 300 Olympus荧光显微镜采集图像。5. Western Blot:处理后的细胞使用细胞裂解液提取细胞总蛋白。将含有15μg蛋白的样本和蛋白Marker一起上样。在10%SDS-聚丙烯酰胺凝胶上电泳,再用电转仪电转染到聚偏氟乙烯膜(PVDF)上。电转膜封闭后,一抗孵育过夜,清洗后,再使用HRP标记的二抗孵育。滴加ECL显色。蛋白强弱采用目的蛋白和(3-actin积分光密度的比值表示。7.实时定量PCR：处理后的细胞采用Trizol提取细胞总RNA。应用iScript cDNA合成酶将mRNA逆转录成cDNA。后采用iCycler iQ荧光探针系统进行实时PCR。采用FABP4与P-actin的循环阈值(threshold cycle, Ct)的比值并通过公式2ΔCt (ΔCt=β-actin Ct-target gene Ct)表示mRNA的相对水平。8.细胞内脂质沉积检测：采用油红0染色检测细胞内脂质沉积。4%福尔马林固定后在油红0工作液中染色30分钟,测定490nm下的吸收光密度值。结果1. AMPK降低PA所致的巨噬细胞内脂质沉积：PA能显著增加巨噬细胞内脂质沉积。二甲双胍所致的AMPK激活可降低PA所致的的巨噬细胞内脂质沉积,同时采用特异小分子干扰RNA (siRNA)来抑制AMPK表达不仅增加基础状态下巨噬细胞内脂质沉积,同时增加PA所致细胞内脂质沉积。二甲双胍诱导的脂质沉积的减少可由AMPK SiRNA所阻断。2. AMPK降低FABP4的表达：二甲双胍引起的AMPK激活无论是在PA存在还是不存在的情况下均可显著抑制FABP4的表达。用特异siRNA敲除AMPK可增加基础状态FABP4的表达并逆转二甲双胍引起的FABP4的表达降低。同时野生型AMPK质粒能显著降低FABP4的表达而负显性AMPK能增加FABP4的表达,表明AMPK通路激活可下调FABP4的表达。3. AMPK在转录水平调节FABP4的表达：通过实时定量PCR,发现PA显著增加FABP4mRNA。二甲双胍引起的AMPK激活能显著降低FABP4 mRNA表达并呈剂量依赖性。特异性siRNA敲除AMPK可增加基础FABP4表达。同时逆转二甲双胍所致的FABP4 mRNA水平下降,表明AMPK在转录水平调节FABP4的表达。4.FOXO1正向调节FABP4的表达：为进一步探究AMPK如何下调FABP4 mRNA,我们研究FABP4的转录调节机制,验证FOXO1是否调节FABP4的表达。结果显示野生型FOXO1和持续活性型FOXO1能增加FABP4的表达,而负显形型FOXO1显著降低FABP4的表达,无论是在PA存在还是不存在的情况下,提示FOXO1可以正向调节FABP4的表达。同时特异性siRNA敲除FOXO1降低基础状态下和PA诱导的FABP4的表达,表明FOXO1参与了PA诱导的FABP4的表达上调。6. AMPK通过促进FOXO1核外排降低FABP4的表达：我们进一步研究AMPK通路激活是否通过调节FOXO1下调FABP4的表达。二甲双胍能逆转野生型FOXO1和持续活性型FOXO1引起的FABP4的上调,表明AMPK通过抑制FOXO1下调FABP4的表达。免疫荧光显示PA显著增加FOXO1的核积聚同时AMPK激活不仅可使FOXO1保持在胞浆同时能有效抑制FOXO1的核积聚。AMPK siRNA可逆转PA诱导的FOXO1核积聚,降低二甲双胍诱导的FOXO1核外排。以上研究表明AMPK通过促进FOXO1核外排抑制其转录活性,进而降低FABP4的表达。结论1.AMPK激活可通过下调FABP4表达显著降低PA所致的细胞内脂质沉积2.转录因子FOXO1正向调节FABP4的表达。3. AMPK通过促进FOXO1核外排,降低其转录活性而抑制FABP4的转录。4. AMPK-FOXO1信号通路对高游离脂肪酸所致的细胞内脂质沉积有保护作用,是治疗代谢综合征心血管并发症的潜在治疗靶点。更多还原

【Abstract】 BackgroudMetabolic syndrome, a disease complex of obesity, dyslipidemia, diabetes, hypertension and proinflammatory state, is associated with a significant increased risk of atherosclerosis and cardiovascular disease. Although the increased atherosclerosis in metabolic syndrome is well described, its risk factors and underlying mechanisms are largely unknown.The atherosclerosis in metabolic syndrome has a complex pathogenesis. Metabolic syndrome is often characterized by high circulating concentration of FFAs. Clinical studies have shown that chronically elevated FFA levels are associated with atherosclerosis, myocardial infarction and other cardiovascular diseases. Although the underlying mechanisms are not fully understood, it has been suggested that excess free fatty acids maybe involved in different processes related to atherosclerosis, such as adhesion of monocytes to endothelial cells, platelet aggregation, endothelial apoptosis, endothelial dysfunction and vascular insulin resistance and therefore facilitate atherosclerosis pathology. However, it is unclear whether FFA can directly induce lipid accumulation in macrophage and promote atherosclerosis. In the present study, we examined the effects of FFA on lipid accumulation in macrophages, and investigated the molecular mechanisms involved. We observed that FFAs increased lipid accumulation in human THP-1 macrophages in a dose dependent manner. PA promoted the expression of fatty acid binding protein 4 (FABP4) and diacylglycerol acyltransferase (DGAT), while inhibiting Carnitine palmitoyltransferase I (CPT-1) expression. PA regulated FABP4 expression at transcription level and Forkhead transcription factor 1 (FOXO1) was involved in this process. Thus, fatty acids may play important roles in increased lipid accumulation and atherosclerosis development in metabolic syndrome. FOXO1 pathway may be an important mechanism involved in lipid accumulation induced by dyslipidemia, and thereby provides a rational for therapeutic intervention for the atherosclerosis in metabolic syndrome.Methods1. Cell culture:Primary human monocytic leukemia cell line THP-1 were cultured at 37℃in 5% CO2 in RPMI 1640 medium, supplemented with 10% FBS, 100U/ml penicillin,100ug/ml streptomycin and 5% L-glutamine. Differentiation of THP-1 cells were induced by 100nM PMA for 72h.After 72h of differentiation, the cells were used for the experiments The cells were transfected with siRNAs, treated with FFA at various concentrations for the time periods indicated in the text.2. siRNA-induced gene silencing:Silencing gene expression was achieved using specific siRNA including FABP4 siRNA, FOXO1 siRNA. Transfection of THP-1 macrophage with siRNAs/DNAs was carried out using lipofectamineTM 2000, according to the manufacturer’s instruction. Transfected cells were then treated with FFAs at the designated concentrations for the time periods indicated in the text.3. Western blot analysis:Cell extracts were prepared with lysis buffer.Protein samples (15μg per lane) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked, treated with primary antibody, washed, and then incubated with the secondary horseradish peroxidase-labeled antibody. Bands were visualized with Enhanced Chemiluminescence. The expression of target protein was demonstrated by the ratio of integral optical density (IOD) between target protein and P-actin.4. Real-time quantitative PCR:Total RNA from treated cells was extracted with Trizol, according to the manufacturer’s protocol. The mRNAs were reverse-transcribed into cDNAs using iScript cDNA synthesis kit. Real-time PCR was performed using iCycler iQ real-time PCR detection system. The mRNA levels were acquired from the value of the threshold cycle (Ct) of FABP4 normalized against the Ct of p-actin.5. Immunofluorescence staining THP-1 macrophages were plated onto coverslips and grown as usual. Cells were fixed with 4% formaldehyde for 40min at room temperature and permeabilized with 0.1% Triton-X 100. After washing cells were blocked with PBS containing 10% horse serum to avoid non-specific bindings. Cells were then incubated with anti-FOXOl antibody and followed by incubation with Texas red conjugated rabbit monoclonal antibody. Cell nuclei were stained using 2ng/ml DAPI.Slides were mount with antifading agent and images were captured digitally by Fluoview 300.6. Intracellular lipid accumulation detection THP-1 macrophages were treated with FFAs for 24 hours and washed with PBS. Treated cells were fixed with 4% formalin for 90min and incubated with oil red O working solution for 30min. Absorbance at 490nm was measured.Results1. High FFAs Increases the intracellular lipid accumulation in Human THP-1 Macrophage:THP-1 macrophages were incubated with increasing amounts of FFAs. We found that FFAs significantly increased intracellular levels of lipid accumulation in a dose-dependent manner (all P<0.05).2. FFAs increased lipid accumulation by modulating lipid metabolism gene expression:Palmitic acid promoted the expression of fatty acid binding protein 4 (FABP4) and diacylglycerol acyltransferase (DGAT), while inhibited Carnitine palmitoyltransferase I (CPT-1) expression.Similar effects were also observed with OA and LA treatment.3. PA regulates FABP expression at the mRNA level.Using quantitative RT-PCR, we found that PA significantly increased the expression of FABP4 mRNA in a dose-dependent manner, indicating that FFA may increase FABP4 expression at the mRNA level either by increasing transcription or by inhibiting mRNA degration. Together, these data suggest that FFA is capable of increasing FABP4 transcript in THP-1 macrophage.4. Forkhead transcription factor 1 (FOXO1) was involved in PA induced FABP4 ExpressionSilencing FOXO1 with siRNA significantly prevented PA induced FABP4 upregulation. Moreover, We observed that overexpression of wild type (FOXO1 WT) and constitutively active FOXO1 (FOXO1 CA) significantly increased FABP4 expression; while domain negative FOXO1 (FOXO1 DN) dramatically decreased FABP4 expression in both absence and presence of PA, indicating that FOXO1 is capable of positively regulating FABP4. These data support a critical role for FOXO1 transcription factor in PA-induced upregulation of FABP4 transcription.5. FOXO 1 was involved in PA-induced lipid accumulationFOXO1 siRNA not only decreased basal intracellular lipid accumulation, but also prevented the PA-induced increase in intracellular lipid accumulation indicating FOXO1 was involved in PA-induced lipid accumulation.6. PA activated FOXO1 by promoting its nuclear translocationFinally, immunostaining assays showed that PA increased FOXO1 nuclear translocation, indicating PA may increase FABP4 expression by promoting FOXO1 nuclear translocation and subsequently affecting its target transcription.Conclusions 1. FFAs significantly increased intracellular lipid accumulation by modulating lipid metabolism gene expression in human THP-1 macrophage.2. PA regulates FABP4 expression at the mRNA level.3. Forkhead transcription factor 1 (FOXO1) was involved in PA induced FABP4 expression.4. FOXO1 was involved in PA-induced lipid accumulation.5. The FFA-FOXO1-FABP4 pathway may play a central role in control production of intracellular lipid accumulation, and thereby provides a rationale for therapeutic intervention in cardiovascular complication of metabolic syndrome.. BackgroudLipid accumulation in macrophages, a hallmark of atherosclerotic lesion formation, not only contributes to cholesterol and triglyceride retention within the vascular wall, but also increases vascular oxidative stress and inflammation. Developing strategies to reduce macrophage lipid accumulation may have therapeutic potential in preventing and treating atherosclerosis and cardiovascular complications.Metformin acts partially through activation of the AMP-activated protein kinase (AMPK) pathway, which mediates metformin’s metabolic and cardiovascular protective effects. AMPK, an energy-sensing kinase, is a key factor in controlling intracellular lipid metabolism. Activation of AMPK pathway has been shown to reduce cholesterol fatty acid and triglyceride synthesis and storage, while increase lipolysis and fatty acid oxidation in skeletal muscle, liver and adipoctyte. It is conceivable that activation of this pathway may also reduce lipid accumulation in macrophage and thus prevent atherosclerosis formation.FABP4, a member of cytosolic 14-to 15-kDa fatty acid binding proteins family, is ubiquitously expressed in macrophage and adipocytes. FABP4 is involved in fatty acid metabolism and cellular lipid transport that promote increased intracellular lipid accumulation. Recent evidence has supported a critical role of FABP4 in linking obesity with atherosclerosis and inflammation. Lack of FABP4 expression protects hyperlipedimic mice from development of atherosclerotic lesions.It is unclear that whether FABP4 is involved in the AMPK pathway reduced intracellular lipid accumulation and how the AMPK pathway regulates the expression of FABP4.The role of FOXO in regulating lipid accumulation has been partially clarified. Recent findings found that FOXOs could promote intracellular lipid accumulation while inhibition of FOXO1 has been shown to reduce hepatic fatty accumulation. However, whether FOXO is the transcriptional factor of FABP4 and whether the AMPK pathway affects the activity of FOXO are still unclear.In the present study, we hypothesized that the activation of AMPK pathway by metformin may reduced the intracellular lipid accumulation by decreasing the expression of FABP4. FOXO1 may be the transcriptional factor of FABP4. AMPK inhibited lipid macrophage accumulation by inhibiting FOXO1 mediated FABP4 transcription. Thus, AMPK pathway may be an important mechanism against excessive lipid accumulation, and thereby provides a rational therapeutic intervention for atherosclerosis.Methods1. Cell culture:The human monocytic leukemia cell line THP-1 was cultured in RPMI 1640 medium supplemented with 10% FBS, 100U/ml penicillin, 100ug/ml streptomycin and 5% L-glutamine. Differentiation of THP-1 cells were induced by 100nM PMA in RPMI 1640 medium for 72h.After 72h of differentiation, the cells were used for the experiments.2. siRNA-induced gene silencing:Silencing gene expression was achieved using specific siRNA including, AMPK siRNA and FOXO1 siRNA. Transfection of THP-1 macrophage with siRNAs was performed with LipofectamineTM 2000. Transfected cells were then treated with FFAs, metformin at the designated concentrations for the time periods indicated in the text.3. In Vitro Transfection of Plasmid DNA:THP-1 macrophage were transfected with 0.5ug of FOXO1 plamid DNA including wide type (FOXO1 WT), constitutively active (FOXO1 CA) and dominant-negative (FOXO1 DN). The transfection was carried out using LipofectamineTM 2000 according to the manufacturer’s instructions. Transfected cells were then treated with PA and metformin at the designated concentrations for the indicated time periods.4. Immunofluorescent staining:THP-1 macrophages were plated onto coverslips and grown as usual. Cells were fixed with 4% formaldehyde for 10min at room temperature and permeabilized with 0.2% Triton-X 100. After washing, cells were blocked with 1% bovine serum albumin to avoid non-specific bindings. Cells were then incubated with anti-FOXO1 antibody and followed by incubation with Texas red conjugated rabbit monoclonal antibody. Cell nuclei were stained using 0.1ug/ml DAPI.Slides were mount with antifading agent,5. Western blot analysis:The cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked, incubated overnight with primary antibody, washed, and then incubated with the secondary horseradish peroxidase-labeled antibody. Antigen detection was performed with SuperSignal(?) West Femto Maximum Sensitivity Substrate according to the manufactures’protocol.6. Real-time quantitative PCR:The total cellular mRNAs were reverse-transcribed into cDNAs using iScript cDNA synthesis kit. Real-time PCR was performed using iCycler iQ real-time PCR detection system. Primers were designed with the use of Beacon Designer 2.0 software. The primers for human FABP4 were used:forward:5’-ATGATAAACTGGTGGTGGAAT-3’; reverse:5’-ATCAGCTTGGGAGAAAATTAC-3’; the mRNA levels were acquired from the value of the threshold cycle (Ct) of target gene normalized against the Ct of P-actin.7. Intracellular lipid accumulation detection:THP-1 macrophages were treated with FFAs in the presence or absence of metformin for 24 hours and washed with PBS. Treated cells were fixed with 4% formaldehyde for 90min and incubated with oil red O working solution for 30min. Absorbance at 490nm was measured.Results 1. AMPK reduced fatty acid-induced lipid accumulation in THP-1 Macrophages:PA significantly increased intracellular lipid accumulation. Importantly, the PA-induced increase of intracellular lipid accumulation was reduced by metformin in a dose-dependent manner. This result indicates that metformin is capable of reducing intracellular lipid accumulation in macrophages. Additionally, suppression of AMPK by specific siRNA not only increased basal intracellular lipid accumulation, but also augmented PA-induced increase of intracellular lipid accumulation. Furthermore, AMPK induced reduction of intracellualar lipid accumulation was abolished by AMPK siRNA.2. AMPK reduced macrophage lipid accumulation by downregulating FABP4:Activation of the AMPK pathway by metformin significantly down-regulated the expression of FABP4 in a dose-dependent manner in the absence and the presence of PA. Prolonged PA exposure increased CPT-1 expression. Importantly, knockdown of AMPK by its specific siRNA promoted the basal FABP4 expression and the downregulation of FABP4 by metformin, implicating the involvement of the AMPK pathway in FABP4 upregulation.3. Activation of AMPK pathway mediated the downregulation of FABP4 and reduction of macrophage lipid accumulationAMPK pathway could be activated by metformin in the presence or absence of PA. Silencing this pathway with AMPKαsiRNA increased basal FABP4 expression, amplified PA-induced increase in FABP4 expression and reduced metformin-induced inhibition on FABP4 expression indicating that AMPK is involved in the metformin-induced downregulation of FABP4 expression. Additionally, overexpression of wild type AMPK significantly decrease FABP4 expression, while domain negative AMPK dramatically increase FABP4 expression further suggesting that AMPK is capable of downregulating FABP4 expression.Finally, suppression of AMPK by specific siRNA not only increased basal intracellular lipid accumulation, but also augmented the PA-induced increase in intracellular lipid accumulation and prevented metformin-induced inhibition of intracellular lipid accumulation. Taken together, these results suggest that AMPK pathway mediate metformin induced inhibition of FABP4 expression and intracellular lipid accumulation.4. AMPK regulates FABP4 expression at the mRNA level:Using quantitative RT-PCR, we found that PA increased FABP4 mRNA level, which was significantly reduced by metformin treatment in a dose-dependent manner. Knockdown of AMPKa by its specific siRNA reversed metformin-induced downregulation of FABP4 mRNA, indicating that AMPK pathway mediates metformin induced inhibition of FABP4 expression at transcriptional level. Together, these data suggest that activation of AMPK pathway by metformin is capable of reducing FABP4 transcript in THP-1 macrophages.5. Forkhead transcription factor 1 (FOXO1) positively regulated FABP4 expression We observed that overexpression of wild type (FOXO1 WT) and constitutively active FOXO1 (FOXO1 CA) significantly increased FABP4 expression; while domain negative FOXO1 (FOXO1 DN) dramatically decreased FABP4 expression Additionally, inhibiting FOXO1 expression using FOXO1 siRNA (Fig 6C) significantly reduced the basal as well as PA-induced FABP4 expression (Fig 6D) suggesting that FOXO1 mediated PA-induced upregulation of FABP4 expression..6. AMPK reduced FABP4 expression by inhibiting FOXO1:Treat the cell with metformin significantly reversed the upregulation of FABP4 expression induced by overexpression of of wild type (FOXO1 WT) and constitutively active (FOXO1 CA) supporting that metformin may downregulate FABP4 expression by inhibiting FOXO1. Immunostaining assay showed that PA increased FOXO1 nuclear translocation. Metformin not only maintained FOXO1 in cytoplasm, but also prevented PA-induced FOXO1 nuclear translocation suggesting that metformin can effectively inhibit FOXO1 nuclear translocation. Importantly, AMPK siRNA promoted PA-induced FOXO1 nuclear translocation and reduced metformin induced FOXO1 exclusion suggesting that metformin inhibit Foxo1 nuclear translocation through AMPK pathway. Conclusions1. The ativation of the AMPK pathway significantly reduced PA-induced intracellular lipid accumulation by decreasing the expression of FABP4.2. Forkhead transcription factor 1 (FOXO1) was identified as a positive transcriptional factor in FABP4 expression regulation.3. AMPK inhibited FOXO1 by promoting its nuclear exclusion,subsequently inhibiting FABP4 transcription.4. AMPK-FOXO1 pathway has protective effects against intracellular lipid accumulation induced by free fatty acid and could be a therapeutic target in treating cardiovascular complications in metabolic syndrome.更多还原