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。     

3T3-L1前脂肪细胞分化过程中chemerin基因表达水平的变化

目的 探讨体外培养3T3-L1前脂肪细胞诱导分化过程中chemerin基因表达水平的变化与脂肪细胞分化、脂质积聚之间的关系.方法 应用3-异丁基-1-甲基黄嘌呤、胰岛紊、地塞米松联合方案诱导其分化为成熟的脂肪细胞,采用油红0染色观察脂肪细胞分化及脂质聚集情况,并应用RT-PCR和Western印迹技术检测chemerin基因表达的变化.结果 3T3 -L1脂肪细胞分化过程中,chemerin mRNA表达水平逐渐升高,分化至第6天达到较高水平且逐渐趋于稳定.利用Western印迹可观察到,随着脂肪细胞分化成熟.chemerin基因的蛋白表达水平逐渐增高.结论 chemerin mRNA及蛋白质在脂肪细胞分化成熟过程中表达水平升高,提示其很有可能参与了脂肪细胞分化和脂质聚集.     

胰岛素上调3T3-L1细胞apelin基因表达的初步观察

Northern印迹法证实apelin在分离的小鼠脂肪细胞上有表达，其表达量随3T3-L1细胞分化而渐增。胰岛素上调3T3-L1脂肪细胞apelin的表达，提示脂肪细胞apelin的表达可能与肥胖、胰岛素抵抗、高血压相关。     

罗格列酮对3T3-L1脂肪细胞chemerin表达的影响

目的观察罗格列酮对3T3-L1脂肪细胞chemerin基因表达的影响。方法用实时荧光定量PCR方法检测罗格列酮对chemerin基因表达的影响。结果 3T3-L1前脂肪细胞在诱导分化过程中第24、、6、81、0天chemerin基因的表达水平表现出逐渐上调的趋势,除第0天与第2天差异无显著性外,其余各时间点之间均有显著性差异（P〈0.05）。与对照组相比1,0μmol/L罗格列酮促进第2、46、、81、0天chemerin基因的表达（P〈0.01）。在诱导分化成熟的3T3-L1脂肪细胞中0,.11、1、0μmol/L的罗格列酮作用24 h使chemerin基因表达分别增加142%、230%、293%（P〈0.01）,表现出剂量依赖趋势。结论罗格列酮促进3T3-L1前脂肪细胞及诱导分化成熟的3T3-L1脂肪细胞中chemerin基因的表达,提示chemerin可能参与了脂肪细胞的分化,并可能与肥胖、代谢综合征等疾病的发生相关。     

脂肪酸受体GPR120影响胰岛素受体底物-1的表达

目的 研究在3T3-L1脂肪细胞中G蛋白耦联受体120(GPR120)与胰岛素受体底物-1(IRS-1)的关系.方法 利用经典“鸡尾酒法”诱导3T3-L1前脂肪细胞分化,以未诱导组作为阴性对照,通过油红O染色法检测细胞内脂滴的形成情况,从而确定前脂肪细胞分化为脂肪细胞,再利用实时PCR方法检测GPR120 mRNA的表达水平.采用siRNA技术下调3T3-L1前脂肪细胞中GPR120的表达,以无关干扰组为阴性对照,干扰24 h后诱导细胞分化,诱导后于3T3-L1脂肪细胞培养液中加入软脂酸孵育24 h,分别用实时PCR和Western印迹方法检测3T3-L1脂肪细胞中IRS-1的表达水平.结果 诱导分化的3T3-L1脂肪细胞中,GPR120 mRNA表达量较未诱导组明显升高(P＜0.05);干扰GPR120表达后3T3-L1脂肪细胞中脂滴体积和数量明显减小.另外,GPR120表达的量降低后,IRS-1 mRNA和蛋白表达水平均显著降低(P＜0.05).结论 GPR120参与胰岛素信号通路中IRS-1的表达调控.     

甘精胰岛素、地特胰岛素、人胰岛素对3T3-L1脂肪细胞PPARγ2mRNA表达的影响

目的 探讨人胰岛素、甘精胰岛素、地特胰岛素对体外培养3T3-L1脂肪细胞中过氧化物酶体增殖物激活受体-γ2(PPARγ2)基因表达的影响及可能机制.方法 体外培养3T3-L1前脂肪细胞,在分化培养液分别加入100 nmol/L、500 nmol/L、1 000 nmol/L人、甘精、地特胰岛素培养10 d,用RT-PCR检测脂肪细胞中PPARγ2 mRNA表达水平的变化.结果 在细胞分化的过程中,胰岛素浓度的增高促进PPARγ2的表达;相同浓度下,人胰岛素促进PPARγ2 mRNA表达最强,甘精胰岛素次之,地特胰岛素最弱.结论 胰岛素通过上调PPARγ2基因表达促进前脂肪细胞的分化,不同胰岛素促分化能力不同.     

肿瘤坏死因子α和罗格列酮对30T3-L1细胞分化过程中蛋白酪氨酸磷酸酶1B表达的影响

目的 观察3T3-L1前脂肪细胞分化过程中蛋白酪氨酸磷酸酶1B（PTP1B）蛋白水平的变化以及肿瘤坏死因子α（TNF—α）和罗格列酮在前脂肪细胞分化过程中对PTP1B表达的影响，探讨PTP1B在脂肪细胞分化中的作用。方法 体外培养3T3-L1前脂肪细胞，分别采用3组诱导剂（完全诱导剂：3-异丁基-1-甲基黄嘌呤＋地塞米松＋胰岛素，C组），完全诱导剂加20μg/LTNF—α（CT组），完全诱导剂加10^-5mol／L罗格列酮（CR组）诱导脂肪细胞分化，以Western印迹方法检测各组脂肪细胞分化过程中PTP1B蛋白表达变化。结果 3组中PTP1B蛋白均表现为在前脂肪细胞中（第1天）表达最高，随着脂肪细胞分化成熟表达逐步降低，至第10天降至最低；与C组相比，CT组中脂肪细胞分化较为迟缓，其分化后期（第7～10天）PTP1B蛋白表达水平较C组显著增高；而CR组则表现为脂肪细胞分化活跃，其分化后期（第7～10天）PTP1B蛋白表达水平较C组显著降低。结论 在脂肪细胞分化成熟过程中PTP1B蛋白表达呈降低趋势：TNF—α及罗格列酮影响脂肪细胞胰岛素敏感性的作用可能与其调控PTP1B的表达有关。     

S100A16基因促进3T3-L1前脂肪细胞分化

目的 研究S100A16基因在3T3-L1前脂肪细胞分化过程中的作用及机制.方法 构建过表达S100A16的慢病毒载体(PLJMI-S100A16-GFP),转染3T3-L1细胞.以Western印迹法检测S100A16正常3T3-L1细胞分化过程中S100A16的表达;采用油红O观察脂滴堆积情况;采用Western印迹和实时定量PCR方法检测前体脂肪细胞分化过程中相关基因的表达变化.免疫共沉淀方法检测S100A16是否与p53相互作用.结果 成功构建S100A16过表达3T3-L1细胞株;随着3T3-L1前脂肪细胞的分化,S100A16蛋白表达水平逐渐升高;高表达S100A16能够促进3T3-L1前脂肪细胞分化,促进甘油三酯在脂肪细胞内聚集(P＜0.01),同时上调脂肪细胞分化标志基因PPARy、CCAAT增强子结合蛋白α(C/EBP-α)、脂蛋白脂酶、脂肪细胞脂肪酸结合蛋白(aP2)及脂肪酸合成酶的表达(P＜0.05或P＜0.01);免疫共沉淀结果提示,S100A16蛋白与p53相互作用.结论 S100A16通过抑制p53活性进而促进3T3-L1前脂肪细胞的分化.     

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。     

糖基化终产物对3T3-L1脂肪细胞胰岛素敏感性及SAA3基因表达的影响

目的探讨糖基化终产物（AGE）对3T3-L1脂肪细胞胰岛素敏感性及SAA3基因表达的影响。方法以2-DG摄入法观察葡萄糖的摄取率,用RT-PCR检测脂肪因子SAA3mRNA的表达。结果AGE显著减少3T3-L1脂肪细胞在胰岛素刺激下的葡萄糖摄取,呈剂量和时间依赖效应;AGE显著增加脂肪细胞SAA3mRNA的表达;呈剂量依赖方式。结论AGE能降低3T3-L1脂肪细胞对葡萄糖的摄取,增加3T3-L1脂肪细胞对淀粉样蛋白的表达。     

Differential expression of adrenomedullin and resistin in 3T3-L1 adipocytes treated with tumor necrosis factor-alpha

DESIGN: It has recently been shown that deficiency of adrenomedullin (AM), a potent vasodilator peptide, leads to insulin resistance. We studied expression of AM in NIH 3T3-L1 adipocytes and compared it with expression of resistin, an adipocyte-derived peptide hormone that is proposed to cause insulin resistance. Moreover, we studied the effects of tumor necrosis factor-alpha (TNF-alpha), a known mediator of insulin resistance, on the expression of AM and resistin in 3T3-L1 adipocytes. METHODS: 3T3-L1 cells were induced to differentiate to adipocytes by insulin, dexamethasone and 3-isobutyl-1-methylxanthine. Expression of AM mRNA and resistin mRNA was examined by Northern blot analysis. Immunoreactive AM in the medium was measured by RIA. RESULTS: AM mRNA was expressed in preadipocytes, but barely detectable in adipocytes. Immunoreactive AM was detected in the medium of both preadipocytes and adipocytes, with about 2.5 times higher levels found in preadipocytes. In contrast, resistin mRNA was expressed in adipocytes, whereas it was not detected in preadipocytes. Treatment with TNF-alpha increased AM expression in both adipocytes and preadipocytes, whereas it decreased resistin mRNA levels in adipocytes. CONCLUSIONS: The present study has shown that AM expression was down-regulated and resistin expression was up-regulated during adipocyte differentiation of 3T3-L1 cells. TNF-alpha acted as a potent negative regulator of resistin expression and a potent positive regulator of AM expression in adipocytes, raising the possibility that in addition to its known actions in causing insulin resistance, TNF-alpha may also have actions against insulin resistance through AM and resistin.     

Resistin promotes 3T3-L1 preadipocyte differentiation

OBJECTIVE: To investigate the relationship between resistin (a potential link between obesity and type 2 diabetes) and preadipocyte differentiation. DESIGN: A rat resistin expression vector was transfected into 3T3-L1 preadipocytes and differentiation was compared between normal 3T3-L1 cells, rat resistin-transfected cells and non-transfected cells grown in conditioned medium taken from resistin-expressing cultures. METHODS: The rat resistin gene was inserted into the pDual GC and pEFGP-N2 expression vectors for examination of the effects of resistin overexpression in 3T3-L1 cells before and after differentiation was stimulated with 3-isobutyl-1-methyxanthine (MIX), insulin and dexamethasone (DEX). Smaller conserved fragments were inserted into short interference RNA (siRNA) expression vectors, for examination of the effect of targeted resistin inhibition on differentiation of resistin-overexpressing 3T3-L1 cells. RESULTS: Prior to stimulation, the resistin-transfected 3T3-L1 cells contained many more small lipid droplets than did non-transfected 3T3-L1 cells. Following stimulation, differentiation in the resistin-transfected 3T3-L1 cells was dramatically promoted, especially in the early stages. Stimulation of differentiation was also observed in non-transfected 3T3-L1 cells grown in resistin protein-containing conditioned medium. The expression of adipocyte differentiation-associated markers such as CCAAT enhancer binding protein (C/EBPalpha), retinoid X receptor (RXRalpha) and lipoprotein lipase (LPL) was upregulated in resistin-overexpressing cells, whereas expression of preadipocyte factor-1 (Pref-1), an inhibitor of preadipocyte differentiation, was downregulated. In addition, expression of two of the three tested siRNAs inhibited the adipoconversion process, providing further evidence that resistin promotes the differentiation of preadipocytes to adipocytes. CONCLUSION: Resistin can promote preadipocyte differentiation. Based on this, we propose that resistin may be an important candidate mediator of obesity-induced insulin resistance.     

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.     

A paradox： Insulin inhibits expression and secretion of resistin which induces insulin resistance

AIM： To confirm whether insulin regulates resistin expression and secretion during differentiation of 3T3-L1 preadipocytes and the relationship of resistin with insulin resistance both in vivo and in vitro. METHODS： Supernatant resistin was measured during differentiation of 3T3-L1 preadipocytes. L6 rat myoblasts and hepatoma cell line H4IIE were used to confirm the cellular function of resistin. Diet-induced obese rats were used as an insulin resistance model to study the relationship of resistin with insulin resistance. RESULTS： Resistin expression and secretion were enhanced during differentiation 3T3-L1 preadipocytes. This cellular differentiation stimulated resistin expression and secretion, but was suppressed by insulin. Resistin also induced insulin resistance in H4IIE hepatocytes and L6 myoblasts. In diet-induced obese rats, serum resistin levels were negatively correlated with insulin sensitivity, but not with serum insulin. CONCLUSION： Insulin can inhibit resistin expression and secretion in vitro, but insulin is not a major regulator of resistin in vivo. Fat tissue mass affects insulin sensitivity by altering the expression and secretion of resistin.     

Exendin-4对脂肪细胞分化及糖脂代谢相关基因mRNA表达的影响

目的探讨Exendin-4对3T3-L1前脂肪细胞的分化及糖脂代谢相关基因mRNA表达的影响。方法体外培养3T3-L1前脂肪细胞,在脂肪细胞分化成熟过程中的不同时期分别用Exendin-4等干预,采用油红O染色,观察脂肪细胞分化及脂质积聚情况;采用荧光定量PCR检测脂肪细胞糖脂代谢标志基因GLUT-4、PPARγ、HSLmRNA表达水平。酶法测定脂肪细胞的甘油三酯含量。结果分化成熟的脂肪细胞经油红O染色可见细胞质内大片脂滴呈亮红色,而未分化细胞不被油红O染色。在脂肪细胞分化第0天和第6天用Exendin-4干预,脂肪细胞内TG的含量较空白组增加（P〈0．01）,GLUT-4、HSL、PPARγ mRNA的表达上调（P〈0．01）;在脂肪细胞分化的第12天干预,Exendin-4对肪细胞分化及相关基因mRNA的表达与空白组无明显差异。结论Exendin-4促进脂肪细胞的分化并上调糖脂代谢相关基因GLUT-4、PPARγ、HSL mRNA表达,可能为Exendin-4抗糖尿病的部分作用机制。     

胰升血糖素样肽1对3T3-L1前脂肪细胞增殖及分化的影响

目的探讨胰升血糖素样肽1（GLP-1）对3T3-L1前脂肪细胞增殖及分化的影响。方法在3T3-L1前脂肪细胞增殖和分化的不同阶段添加不同浓度梯度的GLP-1（7—36）,使用XTT比色法测定细胞增殖情况,油红O脂肪染色、异丙醇萃取法评价细胞分化情况,RT-PCR法测定不同分化阶段PPAR-ymRNA表达水平。结果高浓度GLP-1（10^-9～10^-7mool／L）能够减弱3T3-L1前脂肪细胞的增殖能力;GLP-1在10^-11～10^-8mmoL／浓度梯度均存在抑制3T3-L1前脂肪细胞向脂肪细胞分化的作用,但对分化过程中PPARymRNA的表达水平均未见显著影响。结论本研究发现GLP-1能够抑制3T3-L1前脂肪细胞增殖及分化,提示脂肪细胞可能也是GLP-1减轻体重作用的潜在靶点。     

GW4064, a farnesoid X receptor agonist,upregulates adipokine expression in preadipocytes and HepG2 cells

AIM: To investigate the effect of GW4064 on the expression of adipokines and their receptors during differentiation of 3T3-L1 preadipocytes and in HepG2 cells.METHODS: The mRNA expression of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor-gamma 2 (PPAR-γ2), adiponectin, leptin, resistin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and the long isoform of leptin receptor (OB-Rb) and protein levels of adiponectin, leptin, and resistin were determined using fluorescent real-time PCR and enzyme linked immunosorbent assay, respectively, on days 0, 2, 4, 6, and 8 during the differentiation of 3T3-L1 preadipocytes exposed to GW4064. Moreover, mRNA expression of AdipoR2 and OB-Rb was also examined using fluorescent real-time PCR at 0, 12, 24, and 48 h in HepG2 cells treated with GW4064.RESULTS: The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, resistin, AdipoR1, AdipoR2, and OB-Rb and protein levels of adiponectin, leptin, and resistin increased along with differentiation of 3T3-L1 preadipocytes (P < 0.05 for all). The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, and AdipoR2 in 3T3-L1 preadipocytes, and AdipoR2 and OB-Rb in HepG2 cells was significantly increased after treatment with GW4064, when compared with the control group (P < 0.05 for all). A similar trend was observed for protein levels of adipokines (including adiponectin, leptin and resistin). However, the expression of resistin, AdipoR1, and OB-Rb in 3T3-L1 cells did not change after treatment with GW4064.CONCLUSION: The FXR agonist through regulating, at least partially, the expression of adipokines and their receptors could offer an innovative way for counteracting the progress of metabolic diseases such as nonalcoholic fatty liver disease.