肿瘤坏死因子α、吡格列酮对3T3-L1脂肪细胞脂联素mRNA表达的影响

目的观察吡格列酮（PIO）和肿瘤坏死因子α（TNF-α）对3T3-L1脂肪细胞脂联素mRNA表达的影响。方法以不同浓度PIO和TNF-α于各时段处理3T3-L1细胞,用RT-PCR技术检测各条件下脂联素mRNA的表达。结果（1）3T3-L1前体脂肪细胞无脂联素mRNA表达。（2）TNF-α抑制分化及成熟的3T3-L1脂肪细胞脂联素mRNA表达。（3）PIO增强分化及成熟的3T3-L1脂肪细胞脂联素mRNA表达。（4）PIO能改善TNF-α对脂联素mRNA表达的抑制。结论在分化及成熟的脂肪细胞中,TNF-α抑制脂联素mRNA表达,而PIO增强其表达;PIO促进前体脂肪细胞分化及激活脂联素表达;PIO改善TNF-α对成熟脂肪细胞脂联素的抑制。     

TNF-α对3T3-L1脂肪细胞PPAR-γ2 mRNA表达及脂联素分泌的影响

用肿瘤坏死因子α（TNF-α）分别处理未分化、已分化的3T3-L1细胞，检测培养细胞过氧化物酶体增殖物激活受体γ2（PPAR-γ2）mRNA的表达和脂联素的分泌。结果表明TNF-α可明显抑制3T3-L1脂肪细胞的PPAR-γ2 mRNA表达和脂联素的分泌（P〈0.05或P〈0.01），提示TNF-α可能通过PPAR-γ影响脂联素的分泌。     

吡格列酮和肿瘤坏死因子α对3T3-L1脂肪细胞脂联素受体mRNA表达的影响

观察吡格列酮（PIO）和TNF-α对3T3-L1脂肪细胞中两种脂联素受体（AdipoR）mRNA表达的影响，发现AdipoR mRNA存3T3-L1细胞分化过程中的表达呈逐渐上调趋势；PIO能增加未分化和已分化3T3-L1细胞的AdipoR mRNA表达，且其促进效应与时间、剂量旱依赖关系；TNF—α对AdipoR mRNA的表达无影响     

肿瘤坏死因子α对人脂联素3T3-L1细胞PPARγ2 mRNA表达的影响

目的探讨肿瘤坏死因子α（TNF-α）对稳定表达人脂联素3T3-L1细胞过氧化物酶体增殖物激活受体（PPAR）γ2 mRNA表达的影响，为进一步研究脂联素功能提供了实验基础。方法重组脂联素真核表达质粒（pcDNA3．1^＋-hADPN）脂质体法稳定转染3T3-L1细胞。用TNF-α（100ng／m1）处理未转染、转染空载载体、转染pcDNA3．1^＋-hADPN的3T3-L1细胞，RT-PCR检测PPARγ2 mRNA的表达量。结果（1）稳定转染了pcDNA3．1^＋-hADPN的未分化和已分化3T3-L1细胞中，PPARγ2表达量较未转染组明显增加（P〈0.01）。（2）TNF-α可明显抑制PPARγ2 mRNA的表达（P〈0.05）。（3）稳定转染pcDNA3．1^＋-hADPN可改善TNF-α抑制作用（P〈0.05）。结论稳定转染pcDNA3．1^＋-hADPN可明显增加未分化和已分化的3T3-L1细胞PPARγ2 mRNA表达。TNF-α抑制PPAR-γ2 mRNA表达，而转染pcDNA3．1^＋-hADPN可改善TNF-α抑制作用。     

抵抗素结合多肽对3T3-L1脂肪细胞分化、脂代谢及葡萄糖转运体4基因表达的影响

目的观察抵抗素结合多肽（RBP）对3T3-L1脂肪细胞分化、脂代谢及葡萄糖转运体4（GLUT-4）基因表达的影响。方法构建大鼠抵抗素真核表达载体并转染3T3-L1前体脂肪细胞，获得稳定表达抵抗素基因细胞株；采用台盼蓝排斥试验，确定理想的RBP干预浓度，于诱导细胞分化第0天加入培养液；采用油红O染色，观察脂肪细胞分化及脂质积聚情况；采用RT-PCR技术检测脂肪细胞分化标志基因及GluT-4基因表达变化；采用全自动生化仪比色法，检测脂肪细胞内TG和游离脂肪酸FFAs含量的变化。结果（1）RBP浓度10^-12mol／L时，脂肪细胞活细胞数比例较高，且细胞形态无明显改变。（2）RBP对正常脂肪细胞分化进程无明显影响，RBP虽未影响抵抗素稳定表达脂肪细胞内脂滴的出现时间，但细胞内脂滴的数目明显减少。（3）RBP对正常脂肪细胞分化标志基因及抵抗素稳定表达细胞分化早期标志基因Pref-1的表达无明显影响，但明显下调抵抗素稳定表达细胞分化中晚期标志基因C/EBPα和FAS的表达水平。（4）RBP对正常脂肪细胞内TG、FFAs含量无影响，但可显著降低抵抗素稳定表达脂肪细胞内的TG、FFAs含量。（5）RBP干预对正常脂肪细胞及抵抗素稳定表达脂肪细胞中GluT-4基因的表达水平均无显著影响。结论RBP对正常3T3-L1脂肪细胞的分化、脂代谢、GluT-4基因表达均无明显影响，但能有效拮抗抵抗素基因，显著促进3T3-L1脂肪细胞分化及脂代谢。     

TNF-α和吡格列酮对3T3-L1脂肪细胞adiponutrin mRNA表达的影响

目的探讨肿瘤坏死因子α（TNF-α）和吡格列酮（PIO）对3T3-L1脂肪细胞中，脂肪滋养蛋白（adiponutrinADPN） mRNA表达的影响及时间效应。方法用100Fmol／L的PIO和100ng／ml的TNE-α处理不同阶段的3T3-L1脂肪细胞，RT-PCR检测ADPN的表达水平。结果在分化过程中和分化成熟的3T3-L1细胞中，TNF-α均增加ADPN的表达，PIO则可明显抑制其mRNA的表达。结论PIO和TNF-α可影响3T3-L1脂肪细胞的ADPN的表达。     

小檗碱对3T3-L1脂肪细胞内脂素表达的影响

目的观察小檗碱对3T3-L1脂肪细胞内脂素（visfatin）表达的影响和探讨小檗碱改善胰岛素抵抗的机制。方法采用RT—PCR法检测不同浓度小檗碱和不同作用时间后，3T3-L1脂肪细胞内脂素mRNA的表达，并以Western印迹方法检测其蛋白水平的表达。结果0～10μmol／L小檗碱剂量依赖性地增加内脂素mRNA的表达，其中10μmol／L时最明显，是空白对照组的4．96倍，其后随着小檗碱浓度的进一步增加，内脂素mRNA的表达反而降低；10μmol／L的小檗碱作用3h后内脂素mRNA的表达开始明显增强，至作用12h时表达增强最明显，是基础组的4．57倍，随着作用时间的延长内脂素mRNA的表达虽然有所下降，但到48h时内脂素mRNA的表达仍比空白对照组明显增高；5、10、20μmol／L的小檗碱均可使内脂素蛋白的表达增加1．13、2．46、2．34倍，与空白对照组相比差异有统计学意义（P〈0．05或P〈0．01）。结论在一定浓度范围内小檗碱可剂量和时间依赖性地促进离体脂肪细胞内脂素mRNA及蛋白表达。小檗碱对内脂素表达的调节作用可能是其改善机体胰岛素抵抗、降低血糖的机制之一。     

葡萄糖和胰岛素浓度对3T3-L1脂肪细胞脂联素mRNA表达的影响

大量研究表明，动物和人的脂肪细胞脂联素表达和其血浆浓度与空腹血糖、空腹胰岛素、胰岛素抵抗程度呈负相关；与胰岛素敏感性呈正相关。为探讨葡萄糖浓度和胰岛素浓度单独对体外脂肪细胞脂联素表达的影响，我们用不同浓度的葡萄糖和不同浓度的胰岛素分别干预体外培养的3T3-L1脂肪细胞，观察其脂联素mRNA表达的改变，进一步阐明脂联素表达的调控机制。     

葡萄糖和胰岛素对3T3-L1脂肪细胞内脏脂肪素mRNA表达的影响

目的研究不同浓度葡萄糖和胰岛素对3T3-L1脂肪细胞中内脏脂肪素（Visfatin）mRNA表达的影响。方法通过real—time RT-PCR方法检测不同浓度葡萄糖和胰岛素培养下3T3-L1脂肪细胞Visfatin mRNA的表达。结果葡萄糖增加了3T3-L1脂肪细胞Visfatin mRNA的表达;胰岛素降低其表达。结论葡萄糖和胰岛素对3T3-L1脂肪细胞中Visfatin mRNA的表达有凋控作用。     

氧自由基降低脂肪细胞脂联素表达的机制研究

目的 研究活性氧簇(ROS)对3T3-LI脂肪细胞脂联素表达的调节机制.方法 以实时定量PCR检测分化成熟的3T3-L1脂肪细胞脂联素mRNA表达水平,联合应用多重磷酸化蛋白分析系统与各种蛋白激酶抑制剂,筛查ROS下调脂联索表达的信号通路.结果 作为一种重要的ROS,H_2O_2激活了313-L1脂肪细胞细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基端激酶(JNK)、蛋白激酶B(Akt)、p70 S6 激酶(p70 S6K)及Janus激酶/信号转导和转录激活因子(JAK/STAT)等多种信号转导通路.其中Akt和JAK/STAT抑制剂完全逆转H_2_O2对脂联素表达的下调作用(均P<0.01).结论 ROS可能通过激活Akt、JAK/STAT信号途径下调脂肪细胞脂联素的表达,从而在肥胖及其相关疾病的发生、发展中发挥作用.     

Endothelin-1 regulates adiponectin gene expression and secretion in 3T3-L1 adipocytes via distinct signaling pathways

Adiponectin, which is specifically and highly expressed in adipose tissue, has pleiotropic insulin-sensitizing effects. Endothelin-1 (ET-1) is a potent vasoconstrictive peptide mainly produced by endothelial cells. We previously showed that ET-1 can induce insulin resistance in vitro and in vivo and proposed that it might regulate adiponectin expression and secretion, thus affecting the homeostasis of whole-body energy metabolism. In the present study, we explored the regulatory effects of ET-1 on adiponectin expression and secretion and the underlying mechanisms in 3T3-L1 adipocytes using Northern blotting and ELISA. ET-1 was found to cause a significant time- and dose-dependent decrease in adiponectin expression, and this effect was inhibited by the ET type A receptor (ETAR) antagonist BQ-610 but not by the ETBR antagonist BQ-788. To explore the underlying mechanism, we examined the involvement of the cAMP-dependent protein kinase A-, phospholipase A2-, protein kinase C-, and MAPK-mediated pathways using inhibitors and found that only PD98059 and U0126, inhibitors that blocked MAPK/ERK kinase's ability to activate the ERKs, prevented ET-1-induced down-regulation of adiponectin. Furthermore, acute ET-1 treatment significantly stimulated adiponectin secretion by 3T3-L1 adipocytes, and this effect was inhibited by the ETAR antagonist BQ-610, the inositol-1,4,5-triphosphate receptor blocker 2-APB, and phospholipase C inhibitor U73122, showing that the release of adiponectin stimulated by ET-1 was mediated through the ETAR and the inositol-1,4,5-triphosphate pathway. In conclusion, ET-1 regulates adiponectin expression and secretion by two different signaling pathways in 3T3-L1 adipocytes. These findings suggested that the cardiovascular system affects adipocyte physiology by regulating the expression of adipocytokines and, consequently, energy homeostasis via vasoactive factors, such as ET-1.     

脂联素球状结构域对3T3-L1脂肪细胞磷酸戊糖途径关键酶表达的影响

目的:通过探讨脂联素球状结构域(gAd)对3T3-L1脂肪细胞磷酸戊糖途径关键酶表达的影响,进而探讨gAd促进脂肪细胞摄取的葡萄糖是否经磷酸戊糖途径代谢。方法:用gAd干预分化成熟的3T3-L1脂肪细胞,干预结束后测定细胞残液的葡萄糖浓度,并以实时荧光定量PCR(RT-PCR)法检测各组细胞磷酸戊糖途径关键酶葡萄糖-6-磷酸酶(G6PD)转录水平的表达情况,进行统计学分析。结果:各实验组细胞残液中葡萄糖浓度均显著低于对照组(均P     

Reactive oxygen species up-regulates SOCS-3 in 3T3-L1 adipocytes

We investigated the effect of increased intracellular reactive oxygen species(ROS) on SOCS-3 expression in 3T3-L1 adipocytes. Increased intracellular ROS levels in 3T3-L1 adipocytes were achieved by two methods of exposure to H2O2 and the occurrence of oxidative stress in cells was assessed by flow cytometry . Expression of SOCS-3 mRNA and that of some adipokines were measured by real time PCR. The level of SOCS-3 protein was determined by western blot. The effect of the antioxidant alpha-lipoic acid was also investigated. Both the relatively mild increased intracellular ROS and the acute but transient increased ROS elevated the levels of SOCS-3 mRNA and protein in 3T3-L1 adipocytes, acompanied with elevated levels of TNF-α mRNA and resistin mRNA and the decreased levels of adiponectin mRNA and secretory adiponectin in culture medium. α-lipoic acid could attenuate the effects of ROS on 3T3-L1 adipocytes. We hypothesized that in mature 3T3-L1 adipocytes, SOCS-3 could be upregulated directly by the induction of increased intracellular ROS and to some extent which was up-regulated by adipokine modulation.

     

沉默脂肪细胞型脂肪酸结合蛋白基因对脂肪细胞合成甘油三酯及脂联素分泌的影响

目的应用RNA干扰(RNAi)技术,构建针对3T3-L1脂肪细胞型脂肪酸结合蛋白(A-FABP)的microRNA表达载体,研究沉默A-FABP基因的表达对脂肪细胞合成甘油三酯及脂联素分泌的影响。方法构建靶向A-FABP基因的microRNA表达载体转染3T3-L1脂肪细胞后,RT-PCR及Western blot法检测A FABP mRNA及蛋白表达。建立A-FABP基因沉默细胞模型,0.5 mmol/L游离脂肪酸和成熟脂肪细胞共孵育24 h,分为对照组、脂肪酸组、RNAi组、RNAi+脂肪酸组。ELISA法分别检测甘油三酯及脂联素水平,RT PCR法检测脂肪细胞A-FABP及脂联素mRNA的表达。结果 microRNA表达载体转染脂肪细胞后,能显著抑制A-FABP mRNA和蛋白表达。随着脂肪酸浓度的升高,脂肪细胞甘油三酯合成明显增加,脂联素分泌明显减少(P<0.05)。与对照组和脂肪酸组比较,RNAi组和RNAi+脂肪酸组甘油三酯水平明显降低,脂联素水平明显升高,脂联素mRNA表达明显上调(P<0.05)。结论阻断A-FABP基因有可能成为防治动脉粥样硬化及糖尿病的新靶点。     

Interleukin-18 enhances glucose uptake in 3T3-L1 adipocytes

In order to characterize the potential causative effects of interleukin-18 (IL-18) on insulin resistance, we measured glucose uptake in 3T3-L1 adipocytes treated with mouse recombinant IL-18. IL-18 surprisingly enhanced, rather than reduced insulin-mediated glucose uptake in adipocytes. Moreover IL-18 could counteract the glucose uptake suppression caused by tumor necrosis factor α in 3T3-L1 adipocytes. The mechanism dissection showed that the IL-18 upregulated phosphorylated Akt and downregulated phosphorylated P38 MAPK. These findings indicated that the elevated serum IL-18 levels in obesity and diabetes might be a compensatory response to insulin resistance.     

Regulation of adiponectin secretion by insulin and amino acids in 3T3-L1 adipocytes

Adiponectin is a fat cell-derived hormone with insulin-sensitizing properties. Low plasma adiponectin levels are associated with insulin resistance as found in obesity. One of the mechanisms for this finding is hampered insulin signaling via phosphatidylinositol 3-kinase (PI3K) with concomitant decreased adiponectin secretion. Because insulin can also stimulate signaling at the level of mammalian target of rapamycin (mTOR) by a mechanism that is dependent on the presence of amino acids, the role of mTOR signaling in adiponectin secretion was studied. In view of the vesicular nature of adiponectin secretion, the role of lysosomes was explored as well. In 3T3-L1 adipocytes, both insulin and amino acids stimulated adiponectin secretion. The stimulation by insulin was PI3K dependent but mTOR independent. The stimulation by amino acids was independent of both PI3K and mTOR. Whereas the effect of insulin via PI3K was mainly on adiponectin secretion from adipocytes, the effect of amino acids was predominantly due to their role as substrates for adiponectin synthesis. The acidotropic agents ammonia and methylamine, but not the lysosomal protease inhibitor leupeptin and the autophagy inhibitor 3-methyladenine, strongly inhibited adiponectin secretion and increased the intracellular adiponectin pool. In conclusion, adiponectin production is substrate driven. Phosphatidylinositol 3-kinase and an acidic lysosomal pH, but not amino acid-mediated mTOR signaling or lysosomal breakdown, are involved in adiponectin secretion.     

C-reactive protein inhibits adiponectin gene expression and secretion in 3T3-L1 adipocytes

C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. The aim of the present study is to investigate the effects of CRP on the production of adiponectin in 3T3-L1 adipocytes. Northern and western blot analysis revealed that CRP treatment inhibited adiponectin mRNA expression and secretion in a dose- and time-dependent manner. Co-incubation of adipocytes with rosiglitazone and CRP decreased induction of adiponectin gene expression by rosiglitazone. However, luciferase reporter assays did not show that CRP affected the activity of approximately 2.1 kb adiponectin gene promoter, which was increased by rosiglitazone alone. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by LY294002 partially reversed inhibition of adiponectin gene expression by CRP. These results collectively suggest that CRP suppresses adiponectin gene expression partially through the PI-3 kinase pathway, and that decreased production of adiponectin might represent a mechanism by which CRP regulates insulin sensitivity.     

氯化钴诱导低氧上调3T3-L1脂肪细胞单核细胞趋化蛋白1的表达

研究氯化钴体外模拟低氧对小鼠3T3-L1脂肪细胞单核细胞趋化蛋白1表达的影响.体外培养3T3-L1前脂肪细胞,并将其诱导分化为成熟脂肪细胞,油红O染色鉴定脂肪细胞分化程度和脂质积聚情况.氯化钴化学模拟脂肪细胞低氧环境,采用实时荧光定量PCR与蛋白免疫印迹法检测低氧诱导因子-1α的mRNA和蛋白水平;采用实时荧光定量PCR与酶联免疫吸附法检测单核细胞趋化蛋白1的mRNA和蛋白水平,并对两者的蛋白水平进行相关性分析.氯化钴处理分化成熟的3T3-L1脂肪细胞后,低氧诱导因子-1α在mRNA及蛋白水平均显著上调;同时发现单核细胞趋化蛋白1 mRNA的表达水平和培养液中单核细胞趋化蛋白1蛋白的分泌水平均升高,且低氧诱导因子-1α与单核细胞趋化蛋白1蛋白水平呈现线性相关( r=0.864,P＜0.01).氯化钴诱导低氧上调3T3-L1脂肪细胞单核细胞趋化蛋白1的表达,参与脂肪组织慢性低度炎症的发生.