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。     

游离脂肪酸刺激核因子NF-kBp65核转位诱导3T3-L1脂肪细胞胰岛素抵抗的分子机制

目的:研究游离脂肪酸对3T3-L1脂肪细胞核因子NF-kBp65表达及转位的影响,探讨游离脂肪酸诱导胰岛素抵抗的分子机制.方法:诱导成熟的3T3-L1脂肪细胞与0.3,0.5,1.0 mmol/L的软脂酸(PA)培养6-24h,用葡萄糖氧化酶法检测培液中的葡萄糖消耗量,以2-脱氧-[~3H]-D-葡萄糖摄入法观察葡萄糖的转运率,用Western blot检测总NF-kBp65蛋白及核NF-kBp65蛋白的表达,用激光扫描共聚焦(CLSM)对NF-kBp65进行定位显示.0.3-1.0 mmol/L软脂酸作用6-24 h后,3T3-L1脂肪细胞的葡萄糖消耗明显减少(3.03±0.34,2.71±0.36,2.64±0.25 mmol/L),呈时间剂量依赖效应,其作用不需要胰岛素的存在;0.3-1.0 mmol/L软脂酸作用6-24 h显著减少3T3-L1脂肪细胞胰岛素刺激的葡萄糖转运率(64%,33%,32%),呈时间剂量依赖效应;核NF-kBp65蛋白表达明显增加,CLSM显示NF- kBp65核转位增加,但软脂酸对3T3-L1脂肪细胞总NF-kBp65蛋白的表达无明显影响.结论:游离脂肪酸可以诱导胰岛素抵抗,其分子机制可能与FFAs刺激NF-kB的活化转位调节相关基因的表达有关.     

游离脂肪酸刺激核因子NF-κBp65核转位诱导3T3-L1脂肪细胞胰岛素抵抗的分子机制

目的:研究游离脂肪酸对3T3-L1脂肪细胞核因子NF-κBp65表达及转位的影响,探讨游离脂肪酸诱导胰岛素抵抗的分子机制.方法:诱导成熟的3T3-L1脂肪细胞与0.3,0.5,1.0 mmol/L的软脂酸(PA)培养6-24 h,用葡萄糖氧化酶法检测培液中的葡萄糖消耗量,以2-脱氧-[3H]-D-葡萄糖摄入法观察葡萄糖的转运率,用Western blot检测总NF-κBp65蛋白及核NF-κBp65蛋白的表达,用激光扫描共聚焦(CLSM)对NF-κBp65进行定位显示.结果:0.3-1.0 mmol/L软脂酸作用6-24 h后,3T3-L1脂肪细胞的葡萄糖消耗明显减少(3.03±0.34,2.71±0.36,2.64±0.25 mmol/L),呈时间剂量依赖效应,其作用不需要胰岛素的存在;0.3-1.0 mmol/L软脂酸作用6-24 h显著减少3T3-L1脂肪细胞胰岛素刺激的葡萄糖转运率(64%,33%,32%),呈时间剂量依赖效应;核NF-κBp65蛋白表达明显增加,CLSM显示NF-κBp65核转位增加,但软脂酸对3T3-L1脂肪细胞总NF-κBp65蛋白的表达无明显影响.结论:游离脂肪酸可以诱导胰岛素抵抗,其分子机制可能与FFAs刺激NF-κB的活化转位调节相关基因的表达有关.     

脂联素球状结构域对胰岛素抵抗模型脂肪细胞及胰岛素信号转导的影响

将3T3-L1前脂肪细胞诱导分化为成熟的脂肪细胞,用软脂酸制备脂肪细胞胰岛素抵抗模型,不同浓度的脂联素球状结构域(globular domain of adiponectin,gAd)干预已经产生胰岛素抵抗的3T3-L1脂肪细胞,葡萄糖氧化酶法检测培养液中葡萄糖的消耗量,实时荧光定量PCR法检测胰岛素受体底物(IRS)-1、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(PKB)基因水平的变化,Western印迹检测IRS-1酪氨酸磷酸化水平.结果显示,与对照组相比,各实验组葡萄糖消耗量均显著增加(P＜0.01),且随着gAd浓度的增加,葡萄糖消耗量也逐渐增加;500 ng/ml gAd组及1 000 ng/ml gAd组IRS-1、PI3K、PKB的mRNA表达均比对照组显著增加(P＜0.05);同时,gAd可增加3T3-L1脂肪细胞胰岛素抵抗模型IRS-1酪氨酸磷酸化水平,且呈浓度依赖性.提示gAd能够促进3T3-L1脂肪细胞胰岛素抵抗模型葡萄糖的摄取,其机制可能与促进脂肪细胞胰岛素信号转导、改善胰岛素抵抗有关.     

软脂酸制备3T3-L1脂肪细胞胰岛素抵抗模型的方法

目的 用软脂酸(PA)诱导3T3-L1脂肪细胞,探讨用PA制备3T3-L1脂肪细胞胰岛素抵抗(IR)模型的方法.方法 将3T3-L1前脂肪细胞诱导分化为成熟的脂肪细胞,用油红O染色法鉴定细胞.用不同浓度的PA(0 mmol/L、0.25 mmol/L、0.5mmol/L、1.0 mmol/L)干预3T3-L1脂肪细胞24 h,收集各组细胞培养液,用葡萄糖氧化酶法测定各组细胞培养液葡萄糖的含量,观察PA对3T3-L1脂肪细胞糖摄取的影响.结果 0.25 mmol/L PA就可明显抑制成熟的3T3-L1脂肪细胞葡萄糖的摄取(P＜0.01),且呈浓度依赖性.与对照组相比,0.25 mmol/L PA组、0.5 mmol/L PA组、1.0 mmol/L PA组葡萄糖摄取率分别下降5.25％、10.29％、14.54％.结论 在胰岛素刺激下,0.25 mmol/L PA作用于3T3-L1脂肪细胞24 h就可诱导细胞产生IR,且随着浓度的增加其效果逐渐增强.     

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。     

JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢的影响

目的 探讨JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢相关基因的影响.方法 构建JAZF1小发夹RNA (shRNA)表达载体并转染3T3-L1细胞,实时荧光定量PCR(RT-QPCR)和蛋白印迹法检测JAZF1 mRNA和蛋白水平的表达;氢三放射示踪法检测3T3-L1细胞糖摄取率;蛋白印记法检测糖、脂代谢相关基因蛋白水平;油红O染色检测脂肪细胞甘油三酯(TG)含量变化.结果 成功构建JAZF1-shRNA;转染脂肪细胞48 h后,JAZF1 mRNA和蛋白水平明显低于对照组(P＜0.05);氢3放射性示踪法显示转染组葡萄糖摄取率明显降低(P＜0.05);PPAR-γ蛋白表达升高(P＜0.05),激素敏感脂肪酶(HSL)、内脏脂肪素(Visfatin)、胰岛素诱导基囚-2 (Insig-2)蛋白表达降低(均P＜0.05);油红O染色显示JAZF1转染组细胞内脂质积聚明显,比对照组升高约25％(P＜0.05).结论 JAZF1基因抑制可减少基础糖转运,增加脂质与胆固醇合成,减少脂质分解并减少相关脂肪细胞因子的表达.     

人白血病相关蛋白16基因对3T3-L1脂肪细胞葡萄糖摄取及过氧化物酶体增殖物激活受体γ活性的影响

目的探讨人白血病相关蛋白（LRP）16对3T3-L1脂肪细胞葡萄糖摄取及过氧化物酶体增殖物激活受体（PRAR）γ活性的影响。方法利用脂质体转染及慢病毒介导的RNA干扰技术构建LRP16过表达、抑制表达及对照细胞系。检测LRP16对细胞葡萄糖摄取的影响。Luciferase法检测LRP16对PPAR反应元件（PPRE）相对荧光素酶活性的影响。Western Blot法检测LRP16对PPARγ及葡萄糖转运蛋白（GluT）-4表达的影响。结果（1）成功构建LRP16过表达、抑制表达及对照细胞系。（2）过表达LRP16抑制细胞胰岛素刺激的葡萄糖摄取;抑制表达LRP16促进细胞胰岛素刺激的葡萄糖摄取。（3）LRP16剂量依赖性的抑制COS-7细胞PPRE的相对荧光素酶活性;（4）LRP16抑制脂肪细胞PPARγ及GluT-4蛋白表达。结论LRP16通过下调PPAR7活．眭抑制3T3-L1脂肪细胞葡萄糖摄取导致胰岛素抵抗。     

甘精、地特胰岛素对3T3-L1脂肪细胞瘦素mRNA表达的影响

目的 探讨不同浓度的人胰岛素、甘精胰岛素、地特胰岛素对体外3T3-L1脂肪细胞瘦素(leption)mRNA表达的影响.方法 通过不同浓度人胰岛素、甘精胰岛素、地特胰岛素刺激分化后的3T3-L1脂肪细胞,实时荧光定量PCR法测定瘦素mRNA表达.结果 随着人胰岛素、甘精胰岛素、地特胰岛素浓度的升高,瘦素mRNA表达逐渐升高,相同浓度的地特胰岛素、甘精胰岛素和人胰岛素对瘦素mRNA的促进作用有统计学意义(P<0.05).结论 体外人胰岛素、甘精胰岛素和地特胰岛素均可使分化后3T3-L1细胞瘦素表达增加;人胰岛素对3T3-L1细胞瘦素表达增加的作用最强,甘精胰岛素次之,地特胰岛素最弱.     

高糖诱导分化对3T3-L1成熟脂肪细胞线粒体形态和功能的影响

目的观察高糖诱导分化对3T3-L1成熟脂肪细胞葡萄糖转运以及线粒体功能的影响。方法 3T3-L1前体脂肪细胞分别在含25 mmol/L葡萄糖(高糖组)及5 mmol/L葡萄糖(低糖组)的DMEM培养基中诱导分化。采用油红"O"染色法观察细胞的分化程度,采用液闪仪检测成熟脂肪细胞对[3H]-2-脱氧葡萄糖的摄取率,采用透射电镜观察脂肪细胞的线粒体形态,生物发光法检测脂肪细胞内ATP。结果两组3T3-L1前体脂肪细胞均可分化为成熟脂肪细胞,高糖组成熟脂肪细胞体积及胞质内脂滴均较低糖组大;高糖组成熟脂肪细胞基础状态及胰岛素刺激下的葡萄糖摄取率均低于低糖组脂肪细胞;高糖组成熟脂肪细胞线粒体形态异常,细胞内ATP的含量为(63.00±2.48)nM/mg protein,低糖组为(102.00±1.39)nM/mg protein,两组比较,P<0.05。结论采用含25mmol/L或5 mmol/L葡萄糖的DMEM培养基培养,对3T3-L1前体脂肪细胞向成熟脂肪细胞的分化进程无明显影响;高糖诱导分化可致成熟脂肪细胞产生胰岛素抵抗和线粒体功能损伤。     

Advanced glycation end products attenuate cellular insulin sensitivity by increasing the generation of intracellular reactive oxygen species in adipocytes

Advanced glycation end products (AGE) have been observed in various pathological conditions especially in diabetes mellitus. However, it is unclear as to whether AGE are involved in insulin resistance in adipose tissues. In this study, we examined the effects of AGE on insulin sensitivity in adipocytes by examining the effects of AGE and its mechanisms on the glucose uptake in adipocytes and adipocyte differentiation. Glucose-, glyceraldehyde-, or glycolaldehyde-derived AGE inhibited the differentiation of 3T3-L1 cells. These AGE also inhibited the glucose uptake in the absence or presence of insulin, which were completely prevented by antibody against AGE or receptor for AGE (RAGE). The AGE increased the intracellular reactive oxygen species (ROS) generation in 3T3-L1 adipocytes, and the effects of AGE on glucose uptake were completely reversed by the treatment with an anti-oxidant, N-acetylcysteine. The AGE also induced the expression of monocyte chemoattractant protein-1, which has been implicated in the development of obesity-associated glucose intolerance, in 3T3-L1 adipocytes. Our present study suggests that AGE-RAGE interaction inhibits the glucose uptake through the overgeneration of intracellular ROS, thus indicating that it is involved in the development of obesity-related insulin resistance.     

Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment.

The HepG2-type glucose transporter (HepG2-GT) is expressed in 3T3-L1 fibroblasts and adipocytes. In contrast, the acutely insulin-regulatable glucose transporter (IRGT) is expressed only in the adipocytes. In the present study, the expression of the IRGT was shown to increase in parallel with the acquisition of acutely insulin-stimulated glucose uptake during differentiation of these cells, whereas the level of the HepG2-GT decreased during the course of differentiation in parallel with a decline in basal glucose uptake. We examined the effects of chronic insulin and tolbutamide treatment on glucose transporter activity in conjunction with the expression of these two glucose transporter species in 3T3-L1 adipocytes. Treatment of adipocytes with insulin, tolbutamide, or both agents in combination increased 2-deoxyglucose uptake, HepG2-GT protein, and HepG2-GT mRNA levels in parallel. The effect of combined insulin/tolbutamide administration on these three parameters was greater than the effect of either treatment alone. In contrast, these treatments either had no significant effect or decreased levels of IRGT protein and mRNA. We conclude that chronic treatment of 3T3-L1 adipocytes with insulin or tolbutamide increases glucose uptake primarily by means of a selective increase in the expression of the HepG2-GT. We suggest that part of the in vivo hypoglycemic effect of insulin and sulfonylureas may involve an increased expression of the HepG2-GT.     

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。     

Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes.

The cDNAs for two putative glucose transporters from mouse 3T3-L1 adipocytes were isolated and sequenced. One of these cDNAs encodes the murine homolog of the human hepG2/erythrocyte glucose transporter, termed GT1. GT1 mRNA is most abundant in mouse brain and is expressed in both 3T3-L1 preadipocytes and adipocytes. The other cDNA encodes a glucose transporter-like protein, termed GT2, that has a unique amino acid sequence and tissue distribution. GT2 cDNA encodes a protein with 63% amino acid sequence identity and a similar structural organization to GT1. GT2 mRNA is found at high levels in mouse skeletal muscle, heart, and adipose tissue, all of which exhibit insulin-stimulated glucose uptake. GT2 mRNA is absent from 3T3-L1 preadipocytes but is induced dramatically during differentiation into adipocytes. This increase in mRNA content correlates closely with the acquisition of insulin-stimulated glucose uptake. We propose that GT2 is an insulin-regulated glucose transporter.     

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.     

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。     

Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3-L1 adipocytes

Adipose tissue is an important insulin target organ, and 3T3-L1 cells are a model cell line for adipocytes. In this study, we have used lentivirus-mediated short hairpin RNA (shRNA) for functional gene knockdown in 3T3-L1 adipocytes to assess the molecular mechanisms of insulin signaling. We chose to target GLUT4 to validate this approach. We showed that lentiviruses efficiently delivered transgenes and small interfering RNA (siRNA) into fully differentiated 3T3-L1 adipocytes. We established a strategy for identifying efficient siRNA sequences for gene knockdown by transfecting 293 cells with the target gene fluorescent fusion protein plasmid along with a plasmid that expresses shRNA. Using these methods, we identified highly efficient siGLUT4 sequences. We demonstrated that lentivirus-mediated shRNA against GLUT4 reduced endogenous GLUT4 expression to almost undetectable levels in 3T3-L1 adipocytes. Interestingly, insulin-stimulated glucose uptake was only reduced by 50-60%, suggesting that another glucose transporter mediates part of this effect. When siGLUT1 was introduced into GLUT4-deficient adipocytes, insulin-stimulated glucose uptake was essentially abolished, indicating that both GLUT4 and GLUT1 contribute to insulin-stimulated glucose transport in 3T3-L1 adipocytes. We also found that GLUT4 knockdown led to impaired insulin-responsive aminopeptidase protein expression that was dependent on whether GLUT4 was knocked down in the differentiating or differentiated stage. We further found that GLUT4 expression was not required for adipogenic differentiation but was necessary for full lipogenic capacity of differentiated adipocytes. These studies indicate that lentiviral shRNA constructs provide an excellent approach to deliver functional siRNAs into 3T3-L1 adipocytes for studying insulin signaling and adipocyte biology.     

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。