抵抗素在3T3-L1前脂细胞分化前后表达量的变化

目的检测3T3-L1前脂细胞诱导分化为脂肪细胞前后抵抗素基因表达量的变化情况，为阐明抵抗素在细胞分化过程中所起作用并为研究其与胰岛素抵抗（珉）及2型糖尿病的相关性奠定基础。方法用地塞米松、甲基异丁基黄嘌呤与胰岛素联合诱导法抽提诱导分化前后细胞总RNA，半定量RT-PCR检测抵抗素基因表达量。结果抵抗素在3T3-L1细胞诱导前后表达量明显上升。结论抵抗素在3T3-L1细胞分化过程中表达量的提高，提示其很有可能在鼠脂肪细胞产生珉的过程中发挥积极作用。     

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.     

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.     

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。     

Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes.

A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expressed concomitant with mRNAs encoding adipocyte marker proteins. A factor(s) present in calf serum markedly activated expression of leptin by fully differentiated 3T3-L1 adipocytes. A 16-hr fast decreased (by approximately 85%) the leptin mRNA level of adipose tissue of lean (ob/+ or +/+) mice but had no effect on the approximately 4-fold higher level in obese (ob/ob) littermates. Since the mutation at the ob locus fails to produce the functional protein, yet its cognate mRNA is overproduced, it appears that leptin is necessary for its own downregulation. Leptin mRNA was also suppressed in adipose tissue of rats during a 16-hr fast and was rapidly induced during a 4-hr refeeding period. Insulin deficiency provoked by streptozotocin also markedly down-regulated leptin mRNA and this suppression was rapidly reversed by insulin. These results suggest that insulin may regulate the expression of leptin.     

Tumour necrosis factor-alpha exerts dual effects on human adipose leptin synthesis and release

The acute and chronic effects of tumour necrosis factor-alpha (TNF-alpha) on leptin production by human preadipocytes, differentiated preadipocytes, and mature adipocytes have been examined by competitive RT-PCR of leptin mRNA and by western blotting. In preadipocytes, secreted leptin was detectable after 5-day incubation in differentiation medium and this increased 4-fold by day 20. TNF-alpha blocked leptin synthesis during differentiation. In differentiated preadipocytes and mature adipocytes, TNF-alpha treatment resulted in time-dependent decreases in mRNA for leptin and glycerol-3-phosphate dehydrogenase (G3PD). In contrast, TNF-alpha (4-8-h treatment) resulted in a 4-fold increase in leptin release. This effect was lost at 24 h and leptin accumulation in culture medium was decreased 24-48 h after TNF-alpha addition. We conclude that TNF-alpha stimulates the release of preformed leptin from human mature adipocytes and existing differentiated preadipocytes, which may contribute to obesity/infection-linked hyperleptinemia, and that TNF-alpha inhibits leptin synthesis via inhibition of preadipocyte differentiation and induction of adipocyte dedifferentiation.     

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。     

Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells

OBJECTIVE: To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA). METHODS: Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques. RESULTS: The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells. CONCLUSION: The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.     

chemerin及其受体在小鼠体内外表达的研究

目的观察chemerin及其受体chemerinR基因在小鼠各脏器中的表达谱以及两者在3T3-L1脂肪细胞诱导分化过程中表达水平的变化。方法提取正常小鼠肝脏、脂肪、胃、脾脏、肾脏、心脏、骨骼肌等脏器组织中总RNA,采用半定量逆转录PCR技术检测其中chemerin及chemerinR基因水平;体外培养3T3-L1脂肪细胞,应用1-甲基-3-异丁基黄嘌呤（MIX）、地塞米松、胰岛素诱导其分化,采用适时PCR技术检测诱导分化不同时间脂肪细胞中chemerin及chemerinR基因的表达水平。结果 chemerin及chemerinR基因在小鼠体内广泛表达,以脂肪组织和肝脏为甚;二者低表达于3T3-L1前脂肪细胞中,并随前脂肪细胞诱导分化成熟表达水平呈逐渐上调趋势。结论 chemerin及其受体基因可能有利于脂肪细胞的分化成熟。     

ACTH and alpha-MSH inhibit leptin expression and secretion in 3T3-L1 adipocytes: model for a central-peripheral melanocortin-leptin pathway

Leptin is the 167 amino-acid protein product of the Lep (obese) gene that is released predominantly from adipose tissue and circulates at levels related to the amount of fat. Leptin expression is hormonally regulated: insulin and glucocorticoids are stimulators, while inhibitors include beta-adrenergic agonists and testosterone. Recently, adenylate cyclase-coupled melanocortin receptors have been identified in murine adipose tissue, the 3T3-L1 adipocyte cell line, and in human fat tissue. These studies prompted us to evaluate the effects of pro-opiomelanocortin (POMC)-derived peptides on leptin production and expression in 3T3-L1 adipocytes in culture. 3T3-L1 pre-adipocytes differentiated by the insulin/indomethacin (I/I) method produced leptin at levels that were two times higher than those obtained in cells differentiated by the more traditional insulin/dexamethasone/isobutylmethylxanthine (I/D/M) method. By RT-PCR studies, 3T3-L1 cells expressed both the melanocortin 2 receptors (MC2-R) and melanocortin 5 receptors (MC5-R) isoforms of the melanocortin receptor at an early stage of differentiation. When I/I differentiated 3T3-L1 adipocytes were incubated with different concentrations of dibutyryl cAMP (db-cAMP) or POMC-derived peptides (ACTH and alpha-MSH), ACTH and alpha-MSH stimulated cAMP production after 30 min (2-fold increase) associated with a dose-dependent inhibition of leptin secretion (ACTHz.Gt;alpha-MSH; IC(50)=3.2+/-0.4 SE and 36+/-5 nM, respectively), maximal after 3 h of incubation (30% inhibition). In addition, 100 nM ACTH and alpha-MSH induced a 60% reduction in leptin expression by RT-PCR. Incubation of cells with 0.5 mM db-cAMP led to a more prominent inhibition of leptin expression and secretion (up to 80% at 1 and 24 h, respectively). The ACTH and alpha-MSH inhibitory effects on leptin secretion were mediated by activation of the MC2-R and MC5-R and were reversed by the MC-R antagonists ACTH(11-24) and ACTH(7-38). In summary, we have shown that POMC-peptides are potent inhibitors of leptin expression and production in 3T3-L1 adipocytes. The finding of ACTH/alpha-MSH receptor-induced inhibition of leptin production and expression in adipocytes support the possibility that there is a control mechanism for modulation of adipose tissue function via a melanocortin-leptin axis.     

Methylation of B-hordein genes in barley endosperm is inversely correlated with gene activity and affected by the regulatory gene Lys3.

The methylation status of B-hordein genes in the developing barley endosperm was analyzed by digestion with methylation-sensitive restriction enzymes. Southern blotting revealed specific demethylation of Hpa II sites in DNA from wild-type endosperm, whereas leaf DNA and lys3a mutant endosperm DNA were highly methylated at these sites. Similar methylation patterns were observed at an Ava I site situated at position -260 in the B-hordein promoter. This differential methylation was confirmed by genomic sequencing with ligation-mediated PCR. The analyzed sequence covers most of the B-hordein promoter and includes 10 CpGs from the promoter and 4 CpGs from the adjacent coding region. These sites were all hypomethylated in wild-type endosperm, whereas--except for three partially methylated sites--full methylation was seen in leaf DNA. The four sites in the coding region were partially methylated in lys3a endosperm DNA, but the promoter sites remained highly methylated. The possible role of methylation in the regulatory function of the Lys3 gene product is discussed.     

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.     

Mechanism of adult primitive mesenchymal ST-13 preadipocyte differentiation

Convincing evidence supports the idea that adipogenesis occurs throughout the life of organisms. However, little is known about the adipogenesis program for adult adipocytes. We examine this issue using mouse adult primitive mesenchymal ST-13 preadipocytes that express the peroxisome proliferator-activated receptor-gamma (PPARgamma) gene while in a predifferentiated state. The gene expression of PPARgamma was sustained throughout differentiation when ST-13 preadipocytes were induced to become adipocytes by a PPARgamma ligand. However, the differentiation of pluripotent C3H10T1/2 stem cells and 3T3-L1 embryonic fibroblastic cells was associated with enhanced expression of the PPARgamma gene. Immunoblotting analysis revealed that C3H10T1/2 and 3T3-L1 cells expressed low levels of PPARgamma1 from the early stage, and the amount increased during differentiation, whereas PPARgamma2 appeared at the late stage. In contrast, ST-13 preadipocytes expressed an appreciable amount of PPARgamma1 that significantly decreased on differentiation, and a small amount of PPARgamma2 appeared late in the differentiation process. Furthermore, the standard hormone cocktail containing dexamethasone, methylisobutylxanthine, and insulin induced an increase in PPARgamma1 protein only at the early stage, and a low level of PPARgamma2 protein appeared late in ST-13 cells. However, levels of both PPARgamma1 and PPARgamma2 proteins were significantly induced within 2 d in 3T3-L1 cells in this hormonal adipogenesis. Moreover, exposing ST-13 preadipocytes to dexamethasone and insulin induced differentiation, but failed to induce adipogenesis in 3T3-L1. Adipogenesis in adult rat primary preadipocytes was also induced in a similar manner to that of ST-13. Our results indicate that ST-13 cells and primary preadipocytes derived from adults possess an adipogenesis program distinct from that of 3T3-L1 and C3H10T1/2 cells, and that it may represent the adipogenesis program for adult-specific adipocytes.     

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.