Chemerin过表达致3T3-L1脂肪细胞葡萄糖消耗减少

目的 应用重组慢病毒构建3T3-L1脂肪细胞chemerin过表达模型并进一步探讨其对糖代谢的影响及可能机制.方法 构建鼠chemerin过表达重组慢病毒,并设对照慢病毒,感染3T3-L1细胞,实时定量聚合酶链反应（RT-PCR）法检测转染后chemerin表达水平;应用胰岛素、3-异丁基1-甲基黄嘌呤、地塞米松诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,油红O染色鉴定;诱导分化第8天加入慢病毒重组体,继续培养5d,葡萄糖氧化酶法检测各组葡萄糖消耗;RT-PCR法检测各组胰岛素受体底物1（IRS1）、胰岛素受体底物2（IRS2）、蛋白激酶B1 （Akt1）、叉头状转录因子O1 （FoxO1）基因表达水平;Western-blotting检测chemerin、丝氨酸/苏氨酸蛋白激酶（Akt）、磷酸化丝氨酸/苏氨酸蛋白激酶（pAkt）、FoxO1、磷酸化叉头状转录因子O1 （pFoxO1）蛋白水平.两组数据比较应用t检验.结果 Chemerin过表达慢病毒感染3T3-L1细胞72 h后细胞中可见红色荧光,RT-PCR结果显示：过表达组与空载对照组相比chemerin基因表达明显增加（分别为3.04±0.19比1.01±0.11,t=15.65,P＜0.05）;chemerin过表达组葡萄糖消耗减少[分别为（3.30± 1.44）比（6.07±1.15） mmol/L,t=-0.35,P＜0.05];RT-PCR结果显示：IRS1、IRS2基因水平无明显变化（均P＞0.05）,Akt1基因表达下降（分别为0.76±0.08比1.07±0.15,t=-3.11,P＜0.05）,FoxO1基因表达上调（分别为1.53±0.30与1.03±0.21,t=2.34,P＜0.05）.Western-blotting结果显示：Chemerin过表达后chemerin蛋白水平增加（相对表达量分别为1.08±0.06比0.72±0.03,t=-10.12;P＜0.05）;Akt、pAkt蛋白水平均降低（分别为0.74±0.21比1.23±0.20,0.58±0.17比0.92±0.07;t=2.81、3.17,均P＜0.05）,FoxO1蛋白水平升高（分别为1.04±0.09比0.76±0.14,t=-2.91,P＜0.05）、pFoxO1蛋白水平降低（分别为0.61±0.13比0.89±0.10,t=2.93,P＜0.05）.结论 Chemerin可能通过下调Akt1 mRNA使3T3-L1脂肪细胞葡萄糖消耗减少.     

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。     

外源性硫化氢对胰岛素抵抗脂肪细胞葡萄糖转运体4表达的影响

目的观察外源性硫化氢(H2S)对3T3-L1脂肪细胞胰岛素抵抗(IR)的影响,并探讨其机制。方法用高糖高胰岛素培养3T3-L1脂肪细胞,建立IR细胞模型,外源性H2S供体NaHS(10-5、10-4和10-3mol/L)处理IR 3T3-L1细胞12、24和48 h。MTT法检测细胞活力,葡萄糖氧化酶法检测培养液中的葡萄糖消耗量,2-脱氧-[3H]-D-葡萄糖摄入法检测葡萄糖的摄取。实时定量PCR和Western blot检测葡萄糖转运体4(Glut4)的表达。结果与对照组比较,IR模型组细胞葡萄糖消耗和摄取量以及Glut4 mRNA和蛋白的表达显著降低(均为P<0.05)。与对照组比较,所有浓度的NaHS均未影响细胞活力。与IR模型组比较,NaHS(10-4和10-3mol/L)处理24和48 h显著增加细胞葡萄糖消耗和摄取量以及Glut4 mRNA和蛋白的表达(均P<0.05)。结论外源性H2S改善了高糖高胰岛素诱导的脂肪细胞的IR,其机制可能与H2S上调Glut4的表达有关。     

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。     

胰岛素、葡萄糖对3T3-L1脂肪细胞脂肪水孔蛋白表达的调节

利用半定量RT PCR技术及Western印迹法研究胰岛素、葡萄糖对成熟脂肪细胞脂肪水孔蛋白 (AQPap)基因表达的影响。结果表明 ,胰岛素对AQPap的表达具有抑制作用 ;而高浓度葡萄糖则对AQPap的表达具有促进作用     

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 insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes.

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.     

Annexin II is a thiazolidinedione-responsive gene involved in insulin-induced glucose transporter isoform 4 translocation in 3T3-L1 adipocytes

The target genes of peroxisomal proliferator-activated receptor-gamma ligands that lead to insulin sensitization are not fully understood. In this study, we have found that the thiazolidinedione, troglitazone, increases expression of annexin II at both the mRNA and protein levels, raising the possibility that annexin II plays a role in insulin-stimulated glucose transporter isoform 4 (GLUT4) translocation and glucose transport. To assess this, we microinjected annexin II antibody or annexin II small interfering RNA into 3T3-L1 adipocytes and found that insulin-stimulated GLUT4 translocation was inhibited by 54 and 60%, respectively. Furthermore, microinjection of annexin II antibody inhibited constitutively active Galphaq (Q209L-Galphaq)-induced but not osmotic shock-induced GLUT4 translocation. When cells were cotransfected with wild-type annexin II, along with an enhanced green fluorescent protein-cmyc-GLUT4 construct, and the percentage of cells expressing cmyc-GLUT4 at the cell surface was measured by immunofluorescence microscopy, there was a marked increase in the ability of insulin to stimulate recruitment of cmyc-GLUT4 protein to the cell surface. In summary, our results show that annexin II is a newly described thiazolidinedione response gene involved in insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.     

Galanin inhibits leptin expression and secretion in rat adipose tissue and 3T3-L1 adipocytes

In addition to serving as a fat depot, adipose tissue is also considered as an important endocrine organ that synthesizes and secretes a number of factors. Leptin is an adipocyte-derived hormone that plays a vital role in energy balance. Expression of leptin is regulated by dietary status and hormones. In the present study, we report that galanin, an orexigenic peptide, inhibits leptin expression and secretion in rat adipose tissue and in 3T3-L1 adipocytes. Treatment with galanin (25 micro g/animal) induced approximately 46% down-regulation of leptin secretion at 15 min, followed by 40, 37 and 47% decreases in leptin secretion at 1, 2 and 4 h respectively. Although Northern blot analysis of adipose tissue from the same animals showed that leptin mRNA expression in adipose tissue was unaffected by galanin treatment for 2 h, galanin treatment for 4 h led to decline of leptin mRNA expression in a dose-dependent manner. Meanwhile, treating the rats with galanin had no effect on leptin mRNA expression in the hypothalamus. The inhibitory action of the galanin on leptin mRNA and protein levels was also observed in vitro. When incubated with 10 nM galanin for 48 h, leptin mRNA expression and protein secretion also decreased in 3T3-L1 adipocytes. On the other hand, galanin was found not only to express in rat adipose tissue, but also to increase about 8-fold after fasting. Based on these data, we speculate that increased galanin expression in rat adipose tissue after fasting may be involved in reducing leptin expression and secretion in fasting rats.     

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。     

Glutamine synthetase mRNA in cultured 3T3-L1 adipocytes. Complexity, content and hormonal regulation

Glutamine synthetase (GS) activity increases more than 100-fold during adipocyte differentiation of cultured 3T3-L1 cells. We now find that Northern hybridization analysis of RNA from 3T3-L1 adipocytes with a rat GS cDNA clone (pGSRK-1) yields two hybridizable GS RNAs of length 3.2 and 1.6 kilobases (kb). Densitometric analyses of autoradiographs of the Northern blots probed with pGSRK-1 indicate that the 3.2 kb GS-specific RNA is at least 4- to 5-fold more abundant than the 1.6 kb GS RNA. Analyses of both total and poly(A+)RNA from 3T3-L1 adipocytes yielded similar results. (It is noteworthy that an mRNA of 1.2 kb would be sufficient to encode the 42 500 Da GS subunit.) Quantitative dot-blot hybridization analysis indicates that dexamethasone increases GS mRNA while both insulin and dibutyryl cAMP decrease GS mRNA and/or prevent the dexamethasone-mediated increase. Our data suggest that there are at least two GS mRNAs in 3T3-L1 adipocytes and that they are regulated in parallel by dexamethasone, insulin and dibutyryl cAMP.     

Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes

Since the discovery of insulin roughly 80 yr ago, much has been learned about how target cells receive, interpret, and respond to this peptide hormone. For example, we now know that insulin activates the tyrosine kinase activity of its cell surface receptor, thereby triggering intracellular signaling cascades that regulate many cellular processes. With respect to glucose homeostasis, these include the function of insulin to suppress hepatic glucose production and to increase glucose uptake in muscle and adipose tissues, the latter resulting from the translocation of the glucose transporter 4 (GLUT4) to the cell surface membrane. Although simple in broad outline, elucidating the molecular intricacies of these receptor-signaling pathways and membrane-trafficking processes continues to challenge the creative ingenuity of scientists, and many questions remain unresolved, or even perhaps unasked. The identification and functional characterization of specific molecules required for both insulin signaling and GLUT4 vesicle trafficking remain key issues in our pursuit of developing specific therapeutic agents to treat and/or prevent this debilitating disease process. To this end, the combined efforts of numerous research groups employing a range of experimental approaches has led to a clearer molecular picture of how insulin regulates the membrane trafficking of GLUT4.     

Neuropeptide Y impairs insulin-stimulated translocation of glucose transporter 4 in 3T3-L1 adipocytes through the Y1 receptor

Neuropeptide Y (NPY) is expressed in adipose tissue and is involved in adipocyte metabolism. Although NPY impacts on glucose utilization in vivo, the underlying cellular mechanism is yet to be fully elucidated.In this study we investigated the effect of NPY on the insulin-stimulated translocation of glucose transporter 4 (GLUT4) from intracellular stores to the cell surface in vitro. Using cellular fractionation and immunofluorescence we analyzed the cellular localization and content of GLUT4 in 3T3-L1 adipocytes. Additionally we investigated the effect of NPY on insulin action in adipocyte cultures by assessing the phosphorylation of Akt and [³H]-deoxyglucose uptake.Our data suggest that in 3T3-L1 adipocytes NPY inhibits insulin-stimulated glucose uptake in a GLUT4-dependent manner. The insulin induced translocation of GLUT4 was attenuated by the Y1 receptor agonist [Phe(7),Pro(34)] pNPY, demonstrating an essential role of the Y1 receptor in GLUT4 translocation. Additionally, we observed an NPY dose-dependent impairment of Akt phosphorylation.This study provides evidence that NPY impairs the insulin sensitivity of adipocytes and suggests that the Y1 receptor could be a potential therapeutic target for type 2 diabetes.     

Heat shock modulates adipokines expression in 3T3-L1 adipocytes

Studies have demonstrated that heat shock is associated with alteration in energy metabolism. In this study, we investigated the effect of heat shock on gene expression and secretion of adiponectin and leptin, and gene expression of Hspa2 and Ppargamma in 3T3-L1 adipocytes. Compared with 37 degrees C, adiponectin mRNA was higher at 39 degrees C, and lower at 41 degrees C. Leptin mRNA was higher when adipocytes were exposed to 41 degrees C compared with 37 and 39 degrees C. Secretion of adiponectin increased at 39 degrees C, and when cells were exposed to 41 degrees C it was not detectable. Leptin secretion increased significantly at 41 degrees C, compared with 37 and 39 degrees C. Hspa2 mRNA was increased at 39 degrees C, and the highest level was reached at 41 degrees C. Ppargamma mRNA exhibited a substantial increase in a temperature-dependent manner. The study provides the first evidence of a possible direct effect of heat shock on adiponectin and leptin gene expression and secretion, and demonstrates that the expression of the two adipokines is differentially regulated at the temperatures tested.     

Effects of crude drugs on glucose uptake in 3T3-L1 adipocytes

In this study, various water-extracted crude drugs from Radix Asparagi, Radix Ginseng, Radix Scutellariae, Cortex Lycii Radicis, Cortex Phellodendri and Radix Ophiopogonis were investigated in their effects on [3H]-2-deoxyglucose uptake in 3T3-L1 adipocytes. Following treatment of cells with various crude drugs for 60 mim, the basal [3H]-2-deoxyglucose uptake in cultured 3T3-L1 cells was changed by Radix Asparagi from 140 pmole/min/mg protein of control to 513 (0.1 mg/ml), 201 (1 mg/ml) and 97 (10 mg/ml). Glucose uptake was changed to 324 (0.1 mg/ml), 146 (1 mg/ml) and 46 (10 mg/ml) with Radix Ginseng. In the presence of Radix Scutellariae, glucose uptake was changed to 215 (0.1 mg/ml), 213 (1 mg/ml) and 34 (10 mg/ml). In the presence of Cortex Lycii Radicis, glucose uptake was 230 (0.1 mg/ml), 188 (1 mg/ml) and 38 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, uptake was changed to 142 (0.1 mg/ml), 132 (1 mg/ml), 24 (10 mg/ml) and 489 (0.1 mg/ml), 374 (1 mg/ml), 344 (10 mg/ml), respectively. In insulin-stimulated cells, the [3H]-2-deoxyglucose uptake was changed by Radix Asparagi from 570 pmole/min/mg protein of the control to 816 (0.1 mg/ml), 674 (1 mg/ml) and 532 (10 mg/ml). After incubation with Radix Ginseng, the glucose uptake was changed to 254 (0.1 mg/mi), 123 (1 mg/mi) to 76 (10 mg/mi). In the presence of Radix Scutellariae, the glucose uptake was changed to 315 (0.1 mg/ml), 265 (1 mg/ml) and 33 (10 mg/ml). After incubation of Cortex Lycii Radicis, the uptake activity was changed to 281 (0.1 mg/ml), 248 (1 mg/ml) and 37 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, the activity of glucose uptake was measured as 747 (0.1 mg/ml), 523 (1 mg/ml), 33 (10 mg/ml) and 753 (0.1 mg/ml), 740 (1 mg/ml), and 421 (10 mg/ml), respectively. These results indicate that the water-extracted materials of Radix Asparagi and Radix Ophiopogonis increase the glucose uptake in basal and insulin-stimulated 3T3-L1 adipocytes.     

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。     

Stimulation of glucose transport by thyroid hormone in 3T3-L1 adipocytes: increased abundance of GLUT1 and GLUT4 glucose transporter proteins

In 3T3-L1 adipocytes we have examined the effect of tri-iodothyronine (T(3)) on glucose transport, total protein content and subcellular distribution of GLUT1 and GLUT4 glucose transporters. Cells incubated in T(3)-depleted serum were used as controls. Cells treated with T(3) (50 nM) for three days had a 3.6-fold increase in glucose uptake (P<0.05), and also presented a higher insulin sensitivity, without changes in insulin binding. The two glucose carriers, GLUT1 and GLUT4, increased by 87% (P<0.05) and 90% (P<0. 05), respectively, in cells treated with T(3). Under non-insulin-stimulated conditions, plasma membrane fractions obtained from cells exposed to T(3) were enriched with both GLUT1 (3. 29+/-0.69 vs 1.20+/-0.29 arbitrary units (A.U.)/5 microg protein, P<0.05) and GLUT4 (3.50+/-1.16 vs 0.82+/-0.28 A.U./5 microg protein, P<0.03). The incubation of cells with insulin produced the translocation of both glucose transporters to plasma membranes, and again cells treated with T(3) presented a higher amount of GLUT1 and GLUT4 in the plasma membrane fractions (P<0.05 and P<0.03 respectively). These data indicate that T(3) has a direct stimulatory effect on glucose transport in 3T3-L1 adipocytes due to an increase in GLUT1 and GLUT4, and by favouring their partitioning to plasma membranes. The effect of T(3) on glucose uptake induced by insulin can also be explained by the high expression of both glucose transporters.     

Interferon inhibits the conversion of 3T3-L1 mouse fibroblasts into adipocytes.

Confluent Swiss mouse 3T3-L1 fibroblasts slowly differentiate functionally and morphologically into adipocytes, a conversion hastened by insulin. The cells are sensitive (although less than L929 cells) to the antiviral action of mouse fibroblast interferons but not to interferons from heterologous species (human and chicken). Cultures stimulated with insulin in the presence of partially purified or electrophoretically pure mouse interferons have a much lower percentage of cells accumulating lipid than do insulin-treated control cultures. Interferon-treated cell cultures also contain much less triglyceride, cholesterol, and cholesterol esters than do replicate control cultures stimulated by insulin to differentiate. Increased de novo lipid biosynthesis that occurs during differentiation is inhibited, as determined by incorporation of [14C]acetate into lipids extractable by the Folch method. This incorporation is a sensitive bioassay of the antidifferentiation effect of interferon; less than 1 antiviral unit is inhibitory. Variously inactivated or mock interferon preparations as well as interferons from several heterologous species fail to inhibit 3T3-L1 adipocyte conversion. Interferon is inhibitory even when applied as long as 3 days after insulin stimulation. The effect of interferon does not appear to depend upon its competition with insulin for cell surface receptors. Because interferon can alter the program of events involved in conversion of 3T3-L1 fibroblasts into adipose cells, it may be able to affect the regulation of eukaryotic cell differentiation.     

肾上腺素和葡萄糖对3T3-L1脂肪细胞水孔蛋白mRNA表达的影响

目的：通过细胞培养研究肾上腺素、葡萄糖对成熟脂肪细胞水孔蛋白（aquaporin adipose,AQPap）基因表达的影响。了解AQPap基因表达的影响因素，探讨其在肥胖及糖尿病发病中所起作用。方法：用不同浓度（10^-6mol/L、10^－7mol/L、10^-8mol/L、10^-9mol/L）的肾上腺素刺激诱导分化第9天的3T3－L1细胞6h，另以不同浓度的葡萄糖（5．6mmol/L、11．2mmol/L、16．8mmol/L、33．6mmol/L）刺激细胞48h。提取细胞RNA。运用半定量RT－PCR技术检测AQPap mRNA表达量的变化。结果：与对照组相比，给予不同浓度的肾上腺素刺激分化成熟的3T3－L1细胞，其AQPap mRNA的表达量变化，差异无显著性意义（P＞0．05）。 葡萄糖浓度的升高使培养细胞AQPap mRNA的表达量显著增强（P＜0．05，16．8mmol/L葡萄糖刺激培养细胞48h,AQPap mRNA表达量约是对照组（葡萄糖浓度为5．6mmol/L）的3倍。结论：肾上腺素是一种脂解激素，它对脂肪细胞AQPap mRNA的表达无影响；高糖状态下AQPap mRNA表达明显增强。