IL-6上调SCD1表达对HepG2细胞脂质合成功能的影响*

目的 探讨IL-6对HepG2细胞硬脂酰辅酶A去饱和酶1 （SCD1）基因的影响及其SCD1基因表达的改变对细胞脂质合成的影响。方法 应用rIL-6刺激HepG2细胞,检测细胞内脂质合成以及细胞SCD1基因水平变化。分别构建人SCD1真核表达质粒和小干扰（small interfering） RNA,转染HepG2细胞,观察改变SCD1水平后HepG2细胞脂代谢相关基因表达的变化以及细胞内脂质合成的变化。结果 rIL-6刺激HepG2细胞甘油三酯水平显著高于对照组（P<0.05）;与空质粒组比,转染真核表达质粒细胞SCD1 蛋白表达增加,细胞脂质合成相关基因SREBP1c和FASN相对水平增加1.35倍和1.27倍,脂质氧化代谢相关基因PPARα和ASCL3水平降低12.3%和13.5%;细胞甘油三酯水平显著升高（P<0.01）,而转染small interfering RNA,再用rIL-6刺激HepG2细胞SCD1蛋白表达显著降低,细胞甘油三酯水平也显著降低（P<0.05）,脂质合成相关基因SREBP1c和FASN水平显著下降了32.3%和51.9%,脂质分解相关基因PPARα相对水平也下降了80%（P<0.05）,而ASCL3水平无显著变化（P=0.832）。结论 rIL-6通过上调HepG2细胞SCD1基因表达,引起细胞内脂质合成增加,导致甘油三酯含量增多。     

RNA干扰Ref-1表达对肝癌顺铂敏感性的影响

目的建立表达氧化还原因子-1（Ref-1）小分子干扰RNA的重组逆转录病毒载体pmscv/siRef-1,观察人肝癌细胞株HepG2下调内源性Ref-1后对顺铂的敏感性变化。方法采用基因重组技术构建逆转录病毒载体pmscv/siRef-1。包装逆转录病毒,感染HepG2。RT-PCR及Western blot法检测感染后细胞内Ref-1 mRNA和蛋白表达,MTT法检测HepG2细胞下调Ref-1表达后对顺铂的敏感性变化。结果pmscv/siRef-1能有效被包装并感染靶细胞,感染pmscv/siRef-1病毒的HepG2细胞Ref-1蛋白与mRNA表达量明显降低,细胞对顺铂敏感性显著增加（P〈0.05）。结论成功构建以逆转录病毒为基础的抑制内源性Ref-1表达的RNA干扰载体;RNA干扰Ref-1基因可显著提高HepG2细胞对顺铂的敏感性。     

亚油酸对纤溶酶原激活物抑制剂-1表达的影响及其机制

目的探讨过氧化体增殖物激活型受体α（PPARα）的特异性激活物亚油酸对HepG2细胞1型纤溶酶原激活物抑制剂（PAI-1）mRNA表达及其活性的影响和在该基因转录调控中的作用机制.方法用不同浓度亚油酸为诱导因素刺激HepG2细胞,采用半定量RT-PCR法检测PAI-1mRNA水平,发色底物法检测PAI-1的活性变化.构建四个含PAI-1启动子序列从-804～＋17间不同长度片段驱动的荧光素酶报告基因质粒,体外瞬时转染HepG2细胞,检测荧光素酶的活性.结果与对照组相比,亚油酸组能使HepG2细胞PAI-1mRNA表达及蛋白活性显著升高（P＜0.05,P＜0.01）,且呈一定剂量依赖性;亚油酸诱导可使PAI-1转录活性显著升高（P＜0.01）;与转染质粒PAI-pGL3-A（-804/＋17）相比较,当转染质粒含有PAI-pGL3-B（-636/＋17）、PAI-pGL3-C（-449/＋17）时,荧光素酶活性显著降低（P＜0.01）;共转染PPARα表达质粒（PPARα-pSG5）的细胞在亚油酸诱导下PAI-1转录活性显著升高（P＜0.01）.结论亚油酸可以增加HepG2细胞PAI-1mRNA表达及其蛋白活性,调节PAI-1的基因转录,PPARα参与亚油酸对PAI-1基因的表达调控;在PAI-1启动子-804～-636、-449～-276区域内存在亚油酸作用的调控PAI-1基因表达的序列.     

高糖对THP-1巨噬细胞CD36表达及脂质蓄积的影响

目的 观察高糖对THP-1巨噬细胞清道夫受体CD36表达和脂质蓄积的影响。方法 用不同浓度的D-葡萄糖（分别为5.6、11、20、30及35 mmol/L）、50 mg/L氧化型低密度脂蛋白（ox-LDL）、50 mg/L ox-LDL＋20 mmol/L D-葡萄糖孵育THP-1巨噬细胞24 h,油红O染色观察细胞内脂质蓄积情况,高效液相色谱分析法检测细胞内总胆固醇水平,定量PCR与免疫印迹分析法分别检测THP-1巨噬细胞CD36 mRNA和蛋白的表达。结果 随着D-葡萄糖处理THP-1巨噬细胞浓度的增加,CD36 mRNA和蛋白的表达逐渐增加（P<0.05）;高糖可协同ox-LDL诱导THP-1巨噬细胞CD36 mRNA和蛋白的表达上调（P<0.05）,并增加细胞内总胆固醇水平（P<0.05）。结论 高糖可诱导THP-1巨噬细胞CD36的表达上调,并促进细胞内脂质蓄积。     

小檗碱靶向HDAC/H3K9ac/KLF4抑制棕榈酸诱导的HepG2脂肪变性体外实验研究*

目的 探讨小檗碱(BBR)调控HDAC/H3K9ac/KLF4对棕榈酸(PA)诱导的非酒精性脂肪性肝病(NAFLD)细胞模型的保护作用及其可能的机制。方法 应用PA诱导HepG2细胞构建NAFLD细胞模型,采用油红O染色法测定细胞脂肪变,采用Western blot法检测细胞HDAC1、H3K9ac、KLF4、SREBP-1c和PPARγ表达,使用染色质免疫沉淀技术(ChIP)测定KLF4启动子区H3K9ac水平,采用ELISA检测细胞培养上清细胞因子,采用SiRNA技术沉默KLF4基因验证BBR 靶向HDAC/H3K9ac/KLF4对PA诱导的HepG2细胞脂肪变的保护作用。结果 与模型组比,BBR处理使PA诱导的HepG2细胞脂质沉积显著改善,培养上清TNF-α、IL-1β和IL-6水平分别为(514.7±46.4)pg/ml、(241.5±37.7)pg/ml和(362.7±19.9)pg/ml, 均显著低于模型组【分别为(1162.0±110.8)pg/ml、(635.8±73.4)pg/ml和(1110.0±85.1)pg/ml,P＜0.001】;与模型组比,BBR干预组HDAC1蛋白表达降低了19.0%,而H3K9ac和KLF4蛋白表达增加了1.53倍和1.52倍,KLF4启动子区H3K9ac水平增加了3.97倍,脂质合成相关基因SREBP-1c蛋白表达降低了49.1%,脂质分解相关基因PPARγ蛋白表达增加了1.84倍;与空质粒转染组比,转染真核表达质粒细胞HDAC1蛋白表达水平增加了2.02倍,而H3K9ac蛋白表达水平降低了57.1%;与SiRNA-NC转染组比,转染SiRNA-KLF4的HepG2细胞KLF4蛋白表达水平显著下降(P＜0.01),而细胞脂质沉积显著增加,培养上清TNF-α、IL-1β和IL-6水平分别为(887.9±89.9)pg/ml、(471.9±38.4)pg/ml和(793.1±59.3)pg/ml, 均显著高于SiRNA-NC转染组【分别为(534.2±46.1)pg/ml、(260.3±26.9)pg/ml和(372.0±30.6)pg/ml,P＜0.01】,脂质合成相关基因SREBP-1c蛋白表达增加了1.77倍,而脂质分解相关基因PPARγ蛋白表达降低了41.6%。结论 本研究结果提示BBR可能通过调节PA诱导的HepG2细胞HDAC/ H3K9AC表达,激活了KLF4,抑制了细胞内炎症反应和脂质沉积,为临床干预NAFLD提供了新的思路。     

IL-6上调SCD1表达对HepG2细胞脂质合成功能的影响

目的探讨IL-6对HepG2细胞硬脂酰辅酶A去饱和酶1(SCD1)基因的影响及其SCD1基因表达的改变对细胞脂质合成的影响。方法应用rIL-6刺激HepG2细胞,检测细胞内脂质合成以及细胞SCD1基因水平变化。分别构建人SCD1真核表达质粒和小干扰(small interfering) RNA,转染HepG2细胞,观察改变SCD1水平后HepG2细胞脂代谢相关基因表达的变化以及细胞内脂质合成的变化。结果 rIL-6刺激HepG2细胞甘油三酯水平显著高于对照组(P0.05);与空质粒组比,转染真核表达质粒细胞SCD1蛋白表达增加,细胞脂质合成相关基因SREBP1c和FASN相对水平增加1.35倍和1.27倍,脂质氧化代谢相关基因PPARα和ASCL3水平降低12.3%和13.5%;细胞甘油三酯水平显著升高(P0.01),而转染small interfering RNA,再用rIL-6刺激HepG2细胞SCD1蛋白表达显著降低,细胞甘油三酯水平也显著降低(P0.05),脂质合成相关基因SREBP1c和FASN水平显著下降了32.3%和51.9%,脂质分解相关基因PPARα相对水平也下降了80%(P0.05),而ASCL3水平无显著变化(P=0.832)。结论 rIL-6通过上调HepG2细胞SCD1基因表达,引起细胞内脂质合成增加,导致甘油三酯含量增多。     

肿瘤坏死因子α通过NF-kB上调HepG2细胞中蛋白酪氨酸磷酸酶1B的表达

用不同浓度的肿瘤坏死因子α(TNF-α)诱导人肝癌细胞(HepG2细胞),结果表明TNF-α上调HepG2细胞蛋白酪氨磷酸酶1B(PTP-1B)的表达,激活NF-kB,并呈浓度依赖性,抑制NF-kB能阻断TNF-α对HepG2细胞PTP-1B的上调作用.提示TNF-α可能通过激活NF-kB从而诱导HepG2细胞PTP-1B表达的上调.     

莪术油对棕榈酸诱导的肾小管上皮细胞脂毒性反应的抑制作用观察

目的 观察莪术油对棕榈酸诱导的肾小管上皮细胞毒性反应的抑制作用,并探讨其可能分子机制。方法 体外培养肾小管上皮细胞HK-2细胞株分为对照组、棕榈酸组和莪术油组。对照组不作处理,棕榈酸组分别加入终浓度为0.2、0.8 mmol/L的棕榈酸,莪术油组加入终浓度为0.2、0.8 mmol/L的棕榈酸与20 mg/L的莪术油。采用油红染色法检测两组细胞内脂质沉积情况;MTT法检测细胞增殖情况;Real-time PCR法测定细胞内TNF-α、p53 mRNA表达;Western blot法检测p53上调凋亡调控因子（PUMA）、细胞死亡调节因子（Bim）蛋白表达。结果 对照组HK-2细胞无明显脂质沉积,棕榈酸组与莪术油组细胞内均有明显脂质沉积,但两组差异不大。棕榈酸组与莪术油组细胞增殖量低于对照组（P均〈0.05）;TNF-α、p53 mRNA、PUMA相对表达量高于对照组,且莪术油组低于棕榈酸组（P均〈0.05）;各组Bim蛋白表达无明显差异。结论 莪术油可抑制棕榈酸诱导的肾小管上皮细胞增殖,其机制可能与抑制TNF-α、p53 mRNA及PUMA蛋白表达有关。     

MiR-384对HFFA诱导的Hepa1-6细胞sirt3/FOXO1信号通路的影响*

目的 观察MiR-384对HFFA诱导的Hepa1-6细胞sirt3/FOXO1信号通路的影响。方法 取Hepa1-6细胞,加入OA和PA储存液,使两者终浓度分别为1 mmol/L和0.5 mmol/L,培养24 h,建立非酒精性脂肪性肝炎体外细胞模型。测定肝细胞内活性氧水平评估模型建立的情况。分别以miR-384模拟物、miR-ctrl、miR-384抑制剂、miR-384抑制剂-ctrl、沉默信息调节因子3（Sirt3）或si-ctrl转染细胞48 h。使用FCM测定各组细胞ROS水平,采用Western blot法检测各组细胞sirt3、FOXO1及抗氧化蛋白锰超氧化物歧化酶（MnSOD）和抗氧化蛋白过氧化氢酶（CAT）表达,采用专用试剂盒检测SOD和CAT活性。结果 与正常组（0.66±0.01）比,miR-384模拟物组和si-sirt3组细胞内活性氧（ROS）水平显著升高[分别为（37.3±1.13）和（10.4±0.36）,P<0.01];与HFFA组（29.4±0.98）比,HFFA/miR-384抑制剂组细胞 ROS水平显著降低[（12.8±0.41）,P<0.01];与正常组（0.75±0.04）Hepa1-6细胞sirt3蛋白表达水平比,HFFA组显著降低[（0.23±0.01）,P<0.01];与HFFA组比,HFFA/sirt3组显著增加[（0.83±0.03）,P<0.01];与HFFA/sirt3组比,HFFA/sirt3/miR-384组显著降低[（0.46±0.02）,P<0.01];与对照组比, miR-384模拟物组Hepa1-6细胞sirt3蛋白、Forkhead 转录因子O亚家族( FOXO)成员 FOXO1蛋白表达显著减少[分别为（0.2±0.01）和（0.3±0.01）,P<0.01],而CAT和MnSOD表达显著增加[分别为（2.3±0.05）和（2.4±0.06）,P<0.01];与HFFA组比,HFFA/ miR-384抑制剂组Hepa1-6细胞sirt3蛋白和FOXO1蛋白表达显著增加[分别为（0.5±0.02）和（0.7±0.01）,P <0.01],MnSOD和CAT表达水平显著降低[分别为（1.6±0.04）和（2.0±0.03）,P<0.01];与NAFLD组SOD（327±3.45）和CAT（386±4.03）活性比,正常组SOD和CAT[分别为（425±5.49）和（512±6.04）,P<0.01]和 miR-384抑制剂组SOD和CAT活性[分别为（406±4.79）和（447±5.38）,P<0.01]显著升高。结论 miR-384表达可导致HFFA诱导的Hepa1-6细胞氧化损伤加重,部分可能是通过抑制sirt3/FOXO1途径实现的。     

泽泻汤对巨噬细胞泡沫化脂质沉积及其LXRα和ABCA1表达的影响

目的 以大鼠腹腔巨噬细胞为研究对象,经ox-LDL（50 mg/L）诱导后建立巨噬细胞泡沫化模型,观察泽泻汤对细胞LXRα 和ABCA1表达的影响。 方法 分别采用ox-LDL（50 mg/L）和Dil-ox-LDL（10 mg/L）处理大鼠腹腔巨噬细胞24 h,同时20%泽泻汤血清干预后,油红O染色和荧光显微镜观察细胞内脂质沉积情况;蛋白免疫印迹检测细胞LXRα 和ABCA1的表达情况。结果 ox-LDL（50 mg/L）和Dil-ox-LDL（10 mg/L）处理巨噬细胞成功建立巨噬细胞泡沫化模型;与空白组的巨噬细胞比较,模型组巨噬细胞内脂质沉积增加,泽泻汤血清干预组巨噬细胞内脂质沉积明显减少。空白组巨噬细胞和模型组巨噬细胞LXRα呈低水平表达,泽泻汤血清干预组细胞LXRα表达明显增加;与空白组比较,模型组巨噬细胞ABCA1表达明显增加,泽泻汤血清干预组巨噬细胞中ABCA1亦呈高表达,但较模型组略低。结论 ox-LDL（50 mg/L）和Dil-ox-LDL（10 mg/L）处理巨噬细胞可以成功建立巨噬细胞泡沫化模型;泽泻汤能改善巨噬细胞泡沫化过程的脂质沉积,可能通过上调LXRα表达来实现的。     

Hepatocyte-specific hypoxia-inducible factor-1α is a determinant of lipid accumulation and liver injury in alcohol-induced steatosis in mice

Chronic alcohol causes hepatic steatosis and liver hypoxia. Hypoxia-regulated hypoxia-inducible factor 1-α, (HIF-1α) may regulate liporegulatory genes, but the relationship of HIF-1 to steatosis remains unknown. We investigated HIF-1α in alcohol-induced hepatic lipid accumulation. Alcohol administration resulted in steatosis, increased liver triglyceride levels, and increased serum alanine aminotransferase (ALT) levels, suggesting liver injury in wild-type (WT) mice. There was increased hepatic HIF-1α messenger RNA (mRNA), protein, and DNA-binding activity in alcohol-fed mice compared with controls. Mice engineered with hepatocyte-specific HIF-1 activation (HIF1dPA) had increased HIF-1α mRNA, protein, and DNA-binding activity, and alcohol feeding in HIF1dPA mice increased hepatomegaly and hepatic triglyceride compared with WT mice. In contrast, hepatocyte-specific deletion of HIF-1α [HIF-1α(Hep(-/-) )], protected mice from alcohol- and lipopolysaccharide (LPS)-induced liver damage, serum ALT elevation, hepatomegaly, and lipid accumulation. HIF-1α(Hep(-/-) ), WT, and HIF1dPA mice had equally suppressed levels of peroxisome proliferator-activated receptor α mRNA after chronic ethanol, whereas the HIF target, adipocyte differentiation-related protein, was up-regulated in WT mice but not HIF-1α(Hep(-/-) ) ethanol-fed/LPS-challenged mice. The chemokine monocyte chemoattractant protein-1 (MCP-1) was cooperatively induced by alcohol feeding and LPS in WT but not HIF-1α(Hep(-/-) ) mice. Using Huh7 hepatoma cells in vitro, we found that MCP-1 treatment induced lipid accumulation and increased HIF-1α protein expression as well as DNA-binding activity. Small interfering RNA inhibition of HIF-1α prevented MCP-1-induced lipid accumulation, suggesting a mechanistic role for HIF-1α in hepatocyte lipid accumulation. CONCLUSION: Alcohol feeding results in lipid accumulation in hepatocytes involving HIF-1α activation. The alcohol-induced chemokine MCP-1 triggers lipid accumulation in hepatocytes via HIF-1α activation, suggesting a mechanistic link between inflammation and hepatic steatosis in alcoholic liver disease.     

胰淀素对脂肪变性人L-02肝细胞甘油三酯代谢的影响

目的 研究胰淀素对脂肪变性人L-02肝细胞甘油三酯代谢的影响及其可能机制.方法 以人L-02肝细胞为实验对象,用含50％胎牛血清的高糖改良杜氏伊格尔培养基（DMEM）孵育肝细胞72 h,制备肝细胞脂肪变性模型.实验分为正常肝细胞组（A组）、脂肪变性肝细胞组（B组）、不同浓度胰淀素孵育脂肪变性肝细胞组：0.01 μmol/L（C组）、0.1μmol/L（D组）,1μmol/L（E组）和5μmol/L（F组）,胰淀素孵育时间为24 h.油红O染色观察每组肝细胞的形态和肝细胞内脂滴情况,甘油三酯定量检测试剂盒检测每组肝细胞内甘油三酯的含量,逆转录聚合酶链反应（RT-PCR）检测每组肝细胞内脂肪酸合成酶（FAS） mRNA和乙酰辅酶A羧化酶（ACC） mRNA的表达情况.进行正态性和方差齐性检验后,两组间比较采用t检验,多组间比较采用单因素方差分析.结果 用含50％胎牛血清的高糖DMEM培养基孵育肝细胞72 h后,肝细胞胞浆内充满大量红色脂滴,细胞内甘油三酯含量显著升高,说明肝细胞发生脂肪变性.A组甘油三酯含量为（415±52） mg/g蛋白,B组为（1129±96） mg/g蛋白（t=874,P＜0.05）；C、D、E、F组肝细胞内红色脂滴数量减少,甘油三酯含量分别为（898±77）、（740±83）、（515 ±30）、（628±48） mg/g蛋白（F =48.0,P＜0.05）.RT-PCR结果显示肝细胞脂肪变性后细胞内FAS mRNA和ACC mRNA表达增加,分别是A组的（1.35 ±0.03）、（1.51 ±0.04）倍,差异具有统计学意义（t=47.2、62.9,P ＜0.05）.不同浓度胰淀素处理后各组肝细胞内FAS mRNA和ACC mRNA表达有不同程度的下降.C、D、E、F组FAS mRNA的表达量分别是B组的（0.89 ±0.02）、（0.61 ±0.06）、（0.53 ±0.04）和（1.27±0.01）倍,与B组相比差异均具有统计学意义（t =16.2、58.7、70.4、41.2,均P＜0.05）.当胰淀素浓度为5μmol/L时,FASmRNA表达水平较B组升高.C、D、E、F组ACC mRNA的表达量分别是B组的（0.74±0.02）、（0.66 ±0.01）、（0.49 ±0.02）和（1.46±0.05）倍,与B组相比差异均具有统计学意义（t=30.6、40.6、61.1、57.3,均P＜0.05）.当胰淀素浓度为5μmol/L时,ACC mRNA的表达水平较B组升高.结论 胰淀素孵育脂肪变性肝细胞后肝细胞内甘油三酯含量下降,脂肪变性程度减轻,可能与肝细胞内FASmRNA和ACC mRNA表达水平变化有关.     

Hepatitis C Virus Core Protein Modulates Fatty Acid Metabolism and Thereby Causes Lipid Accumulation in the Liver

We studied the roles of hepatitis C virus (HCV) core protein in hepatic steatosis and changes in hepatic lipid metabolism. HCV core protein expression plasmid was transfected in HepG2 . Triacylglyceride (TG) and mRNA level associated with lipid metabolism were measured. Male C57BL/6 mice were infected with HCV core recombinant adenovirus and used for lipids and mRNA studies. In HCV core protein-expressing cells, peroxisome proliferator-activated receptor (PPAR)α, multidrug resistance protein (MDR) 3, and microsomal triglyceride transfer protein (MTP) were down-regulated 48 hr after transfection. In HCV core protein-expressing mice, hepatic TG content and hepatic thiobarbituric acid-reactive substances increased. PPARα, MDR2, acyl-CoA oxidase (AOX), and carnitine palmitoyl transferase-1 (CPT-1) were down-regulated. HCV core protein down-regulated lipid metabolism-associated gene expression, Mdr2, CPT, and AOX, accompanied by down-regulation of PPARα. There findings may contribute to the understanding of HCV-related steatosis, induction of reactive oxygen species, and carcinogenesis.     

Novel modulators of hepatosteatosis,inflammation and fibrogenesis

Alcoholic steatosis, instead of being innocuous, plays a critical role in liver inflammation and fibrogenesis. The severity of fatty liver is governed by the concerted balance between lipid transport, synthesis, and degradation. Whereas scavenger receptor class B, type I (SR-B1) is critical for reverse cholesterol uptake by the liver, peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator-1α and -β (PGC1α and PGC1β) are critical for lipid degradation and synthesis, respectively. Because betaine is a lipotropic agent, we have evaluated its effects on alcoholic steatosis. Betaine effectively prevented chronic alcohol-mediated (i) impaired SR-B1 glycosylation, plasma membrane localization, and consequent impaired cholesterol transport; and (ii) up regulation of PGC-1β, sterol regulatory element-binding protein 1c and downstream lipogenic genes with concomitant increased liver cholesterol, triglycerides and hepatic lipid score. Similarly, because of its anti-inflammatory and anti-fibrotic effects in other organs, we evaluated the protective effects of thymosin β4 (Tβ4) against carbon tetrachloride (CCl₄)-induced hepatotoxicity in rat. Tβ4 prevented CCl₄-induced (i) necrosis, inflammatory infiltration and up-regulation of α1(2)collagen, alpha-smooth muscle actin (α-SMA), platelet derived growth factor beta (PDGF-β) receptor and fibronectin mRNA expression; (ii) down-regulation of adipogenic gene, PPARγ and the up-regulation of epigenetic repressor gene, methyl CpG binding protein 2 (MeCP2) mRNA levels, suggesting that the anti-fibrogenic actions of Tβ4 involve the prevention of trans-differentiation of quiescent hepatic stellate cells into myo-fibroblasts largely by up-regulating PPARγ and by down-regulating MeCP2 genes. We therefore conclude that betaine and Tβ4 can effectively protect against alcoholic hepatosteatosis and hepatic fibrogenesis, respectively.     

SIRT3 reduces lipid accumulation via AMPK activation in human hepatic cells

OBJECTIVE: Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD)⁺-dependent protein deacetylase localized on mitochondria and regulates the adaptive thermogenesis in brown adipocytes. This study aims to investigate the role of SIRT3 in hepatic lipid accumulation, and whether the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) is required. METHODS: A retroviral system was used for overexpressing of SIRT3 in HepG2 cells, whereas a lentivirus-mediated vector encoding SIRT3 small interfering RNA (siRNA) was used to infect these cells for knocking down endogenous SIRT3 expression. The cells were treated with oleate to induce lipid accumulation and Nile red staining was used to assess the number of lipid droplets in HepG2 cells. The AMPK signaling pathway was facilitated with the administrating of isoproterenol and an immunoblot analysis was performed to assess the phosphorylation of AMPK and acetyl coenzyme A carboxylase (ACC). Compound C was adopted to inhibit AMPK activity. RESULTS: The number of lipid droplets in HepG2 cells overexpressing SIRT3 was significantly lower than that in the control cells (P < 0.05). SIRT3-infected cells exhibited significantly more phosphorylation of AMPK and ACC (P < 0.05), which was reversed by the treatment of compound C, an inhibitor of AMPK. Knocking down SIRT3 downregulated phosphorylation of AMPK and ACC by 60–80% (P < 0.05) and promoted lipid accumulation. The activation of AMPK by SIRT3 was dependent on SIRT3 deacetylase activity. CONCLUSION: SIRT3 reduces lipid accumulation via AMPK activation in human hepatic cells.     

Major role of apolipoprotein B in cycloheximide‐induced acute hepatic steatosis in mice

Aim: Hepatic steatosis accompanied by impaired protein synthesis is often observed in hepatic dysfunction. To assess whether protein synthesis inhibition directly induces hepatic steatosis, we investigated the molecular mechanisms of cycloheximide (CHX)‐induced fatty liver mice. Methods: C57/BL6CR mice were i.p. administrated CHX (20 mg/kg) three times every 4 h to induce hepatic steatosis. Hepatic lipid secretion, fatty acid oxidation, hepatic lipogenesis and hepatic lipid uptake were evaluated. Results: Twenty‐four hours after the first CHX injection, hepatic lipid levels increased in CHX‐treated mice to 1.8‐fold of that in controls but returned to normal within 48 h. The hepatic triglyceride (TG) secretion rate decreased significantly to 22% of controls, and the apolipoprotein B (apoB) protein level, but not microsomal TG transfer protein, decreased in CHX‐treated mice. The apob gene expression was not significantly different between controls and CHX‐treated mice. On the other hand, plasma free fatty acid and lipogenic protein levels did not increase and plasma β‐hydroxybutyrate level remained stable, suggesting that the coordinated balance between fatty acid oxidation, hepatic lipid uptake and lipogenesis was not disrupted in this model. Cellular lipid accumulation and decreased cellular and secreted apoB were also observed in CHX‐treated HepG2 cells. Knockdown of apoB in HepG2 cells also resulted in the cellular TG accumulation. Conclusion: We demonstrated that decreased hepatic lipid secretion due to acute apoB reduction is involved in the pathogenesis of CHX‐induced liver steatosis.