黄芩素对猪前体脂肪细胞增殖分化的影响

研究黄芩素(BAI)对猪前体脂肪细胞增殖分化的影响,并探讨其可能的作用机制。原代培养猪前体脂肪细胞,采用油红O染色观察细胞分化的形态学变化;MTT检测细胞增殖状况;油红O染色提取定量分析细胞内脂肪生成及细胞分化程度;分光光度法测定脂肪酸合酶(FAS)的活性;逆转录-聚合酶链反应(RT-PCR)检测分化特异基因过氧化物酶体增殖物激活受体γ2(PPARγ2)mRNA表达变化。结果显示,前体脂肪细胞在分化成脂肪细胞的过程中,其形态由梭形变成椭圆形、圆形,细胞内充满大小不一的脂滴;BAI浓度在160～640μmol/L时显著抑制其增殖(P<0.05)、BAI浓度为40～320μmol/L时显著抑制PPARγ2mRNA表达和FAS的活性,并抑制细胞分化(P<0.05)。以上结果说明,BAI对前体脂肪细胞增殖分化均有一定抑制作用,BAI可能通过抑制PPARγ2mRNA表达和降低FAS活性,从而抑制猪前体脂肪细胞分化。     

Fsp27基因沉默载体的构建及其对细胞脂解的影响研究

构建脂肪特异性蛋白27(Fat-specific protein of 27,Fsp27)基因沉默载体,研究沉默Fsp27基因表达对3T3-L1细胞脂解的影响,并对其作用机制进行探究。采用RNAi技术,构建Fsp27基因真核干扰载体,下调Fsp27基因的表达。“鸡尾酒”法诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞。脂质体转染脂肪细胞,油红O染色脂滴,酶法测定细胞中甘油及甘油三酯的含量。Western blot法检测细胞中Fsp27、HSL、ATGL和PPARγ的蛋白表达。Western blot结果显示:阳性sh-Fsp27干扰载体均能有效下调Fsp27的表达,且伴随细胞内ATGL和PPARγ的表达量升高(P<0.05),其中sh-Fsp27-2的沉默效果最好;酶学方法检测结果显示:阳性sh-Fsp27干扰组细胞中甘油三酯含量下降,甘油含量升高(P<0.05);油红O染色结果发现:空白对照组与阴性对照组均有大脂滴堆积,阳性sh-Fsp27组小脂滴分布广泛,未见明显的大脂滴。sh-Fsp27-2组基因沉默载体的沉默效果最好,Fsp27基因沉默可以加快3T3-L1细胞的脂解速率,其主要是通过抑制脂滴融合和增强ATGL酶的水解来完成对脂解的调控。     

维生素C通过调控脂肪形成相关基因的转录促进猪前体脂肪细胞的增殖与分化

探讨维生素C（Vit C）诱导猪前体脂肪细胞增殖分化最佳浓度及在分化过程中,5种脂肪形成相关基因peroxisome proliferator activated receptor gamma(PPARγ)和retinoid X receptor alpha(RXRα),脂肪细胞分化标志基因lipoprotein lipase (LPL),生脂基因phosphoenolpyruvate carboxykinase(PEPCK)、stearoyl CoA desaturase(SCD) mRNA表达时序性的变化. 以3　d龄猪前体脂肪细胞为实验对象,用Vit C诱导猪前体脂肪细胞增殖分化,分别在增殖分化第2、4、6和8 d收获细胞,利用MTT测定其增殖程度;油红O染色提取法检测其脂肪含量;采用SQ RT PCR法检测脂肪生成相关基因PPARγ、RXRα、LPL、PEPCK和SCD mRNA表达的变化. 结果显示,PPARγ mRNA在诱导分化第2 d时有低水平表达,在诱导分化过程中表达量逐步升高,在终末分化阶段仍保持高水平表达;RXRα mRNA在诱导分化第2和4 d表达量很低,诱导分化第6 d时表达增加.在诱导分化第8 d,RXRα mRNA表达与第6 d相比差异不显著,直至终末分化. 脂肪细胞分化标志基因LPL在第2 d开始表达,第4和6 d逐步升高,在终末分化阶段仍保持高水平的表达;生脂基因PEPCK和SCD mRNA在第2和4 d开始表达,第6和8 d仍保持高水平的表达. 研究结果表明,100 μmol/L的Vit C促进猪前体脂肪细胞增殖能力最强;250 μmol/L Vit C能显著促进猪前体脂肪细胞分化. 其作用机制可能是通过对转录因子PPARγ和RXRα及标志基因LPL mRNA时序性表达的调控来进行的,促进生脂基因的表达,从而诱导脂肪细胞的分化.     

鸡PPARγ基因的表达特性及其对脂肪细胞增殖分化的影响

为分析鸡PPARγ基因的组织表达特性及其在脂肪细胞增殖和分化过程中的功能,文章以东北农业大学高、低腹脂双向选择品系肉鸡为实验材料,利用Western blotting方法,检测PPARγ基因的组织表达特性及其在高、低脂系肉鸡腹部脂肪组织间的表达差异;采用RNAi技术,在鸡原代脂肪细胞中抑制PPARγ基因的表达后,通过MTT和油红O提取比色的方法,研究鸡PPARγ基因对脂肪细胞增殖和分化的调控作用;利用Real-timePCR和Western blotting技术,分析PPARγ基因表达下调后,其他脂肪细胞分化转录因子以及与脂肪细胞分化相关的重要基因的表达变化情况。结果表明,PPARγ基因在7周龄高脂系肉鸡腹部脂肪组织、肌胃、脾脏、肾脏组织中表达量较高,在心脏中表达量较低,在肝脏、胸肌、腿肌、十二指肠中未检测到表达信号;与高脂系相比,PPARγ基因在5和7周龄低脂系肉鸡腹部脂肪组织中的表达量较低(P<0.05);PPARγ基因的表达量下降后,鸡脂肪细胞的增殖能力增强,分化能力减弱;同时,C/EBPα、SREBP1、A-FABP、Perilipin1、LPL、IGFBP-2基因的表达量均下降(P<0.05)。由此可见,PPARγ基因的表达可能与肉鸡腹部脂肪的沉积有一定的关系,该基因可能是调控鸡脂肪细胞增殖与分化的关键因子。     

miR-196a-5p对3T3-L1前脂肪细胞增殖和分化的影响效应

目的:探究miR-196a-5p对小鼠前体脂肪细胞增殖、分化的影响及其潜在的分子机制。方法:(1)构建小鼠肥胖模型,RT-PCR检测脂肪组织中miR-196a-5p表达量;(2)鸡尾酒法诱导3T3-L1前脂肪细胞分化,RT-PCR检测分化过程中miR-196a-5p的表达变化;(3)合成miR-196a-5p mimics和inhibitors转染3T3-L1细胞,以CCK8、EdU试剂盒检测miR-196a-5p对3T3-L1前脂肪细胞增殖的影响作用;(4)运用油红O染色、甘油三酯测定评估miR-196a-5p对3T3-L1细胞分化的影响;(5) RT-PCR检测miR-196a-5p对前脂肪细胞增殖、分化相关基因的影响;(6)结合前人文献,运用生物信息软件、萤光素酶报告系统对miR-196a-5p调控脂肪细胞分化的靶基因进行筛选和验证。结果:(1) miR-196a-5p在肥胖小鼠脂肪组织中高表达,在3T3-L1前脂肪细胞分化过程中先升高后下降;(2)与阴性对照组相比,mimics转染抑制了3T3-L1细胞增殖,inhibitors转染促进了3T3-L1细胞增殖;(3)与阴性对照组相比,mimics组积累了大量油红着色的脂滴,甘油三酯含量增多,而inhibitors组的脂滴少而小,甘油三酯含量相对降低;(4)与阴性对照组相比,mimics转染抑制了增殖标志基因Cyclin D1、Cyclin E、CDK2和CDK4表达,促进了分化标志基因PPARγ、C/EBPα、LPL、aP2等的表达,inhibitors转染则表现出与mimics转染相反的作用;(5) miR-196a-5p可显著抑制野生型MAP4K3和MAPK1 3'UTR萤光素酶活性,而突变绑定位点可废除该抑制效应。结论:miR-196a-5p不仅可抑制3T3-L1前脂肪细胞增殖,还可促进其诱导分化、沉积脂滴;miR-196a-5p可能通过靶向调节MAP4K3和MAPK1来介导3T3-L1前脂肪细胞分化。     

MiR-186-5p对3T3-L1前脂肪细胞增殖分化的影响研究

目的:探究miR-186-5p对小鼠3T3-L1前脂肪细胞增殖、分化的影响及其潜在的分子机制。方法:qRT-PCR检测miR-186-5p在不同周龄小鼠白色脂肪组织及3T3-L1前脂肪细胞增殖分化过程中的表达变化;通过脂质体将miR-186-5p mimics、inhibitors转染入增殖液或分化液培养的3T3-L1细胞后,利用CCK-8、EdU和qRT-PCR检测3T3-L1前脂肪细胞增殖变化,油红O染色观察其脂滴形态;通过生物信息软件TargetScan和双荧光报告系统分别对miR-186-5p靶基因进行预测和确认。结果:(1) miR-186-5p在1～6周龄小鼠的白色脂肪组织及3T3-L1前脂肪细胞自然分化过程中表达量均逐渐上调。(2)与阴性对照相比,mimics或inhibitors转染分别显著地促进或抑制了miR-186-5p的表达。(3)过表达miR-186-5p后,3T3-L1前脂肪细胞的增殖速率减慢,脂滴增大增多;而抑制miR-186-5p后,3T3-L1前脂肪细胞增殖速率增快,脂滴数量减少,且粒径变小。其中过表达miR-186-5p显著地降低了野生型Wnt5a和Mapk1 3'-UTR活性,而突变相应的绑定位点可解除该抑制作用。结论:miR-186-5p可抑制3T3-L1前脂肪细胞增殖,且通过直接靶向Wnt5a和Mapk1以促进其分化为成熟脂肪细胞。     

TNF-α影响胰岛素对原代培养大鼠脂肪细胞增殖及生脂基因的转录表达

采用RTPCR、MTT比色法和油红O染色提取法,分别测定外源TNFα、胰岛素对原代培养大鼠前体脂肪细胞增殖及脂肪生成和碳水化合物反应元件结合蛋白(ChREBP)、固醇调控元件结合蛋白(SREBP)1c、脂肪酸合酶(FAS)及乙酰辅酶A羧化酶(ACC)1基因转录表达的影响。结果表明,TNFα可有效抑制胰岛素对原代培养大鼠前体脂肪细胞增殖的促进作用,并通过抑制胰岛素对转录因子SREBP1c和脂肪酸合酶FAS及ACC1基因转录表达的促进作用,减少脂肪酸的生物合成,抑制成熟脂肪细胞的脂肪生成,证明TNFα是动物脂肪形成的有效抑制因子。此外,TNFα下调ChREBPmRNA表达,但胰岛素在低糖条件下对其表达没有明显影响     

猪前体脂肪细胞的分离培养

在比较组织块法和消化法分离培养猪前体脂肪细胞的基础上,通过对前体脂肪细胞增殖与分化过程中细胞的形态学变化、生长曲线、油红O染色以及脂肪细胞特异性标志基因脂蛋白脂酶（lipoprotein lipase,LPL）和过氧化物体增殖剂活化受体γ（peroxisome proliferators-activated receptor γ,PPARγ）表达的研究,证明用消化法可从猪的脂肪组织中分离获得大量的前体脂肪细胞,其生长旺盛,可见自发充脂;传代细胞经诱导培养后,充脂率大幅度提高,脂肪特异性标志基因表达增强。这为深入研究脂肪细胞增殖与分化以及猪体脂肪沉积提供了一个较好的模型。     

BAMBI通过促进ERK1/2磷酸化抑制猪前体脂肪细胞分化

为了研究BAMBI在猪前体脂肪细胞分化过程中的作用,构建了BAMBI慢病毒干扰载体,包装并感染猪前体脂肪细胞,采用油红O染色、油红O提取比色法检测猪前体脂肪细胞分化情况,采用Real-time qPCR、Western blotting检测成脂标志基因mRNA以及蛋白水平表达的变化情况。结果表明,BAMBI慢病毒干扰载体感染前体脂肪细胞后显著降低了BAMBI的表达,shRNA2干扰效率最高,达到了60%以上,干扰BAMBI后能增加猪脂肪细胞的脂质积累,增加了成脂标志基因过氧化物酶体增殖物激活受体γ(Peroxisome proliferator-activated receptorγ,PPARγ)和脂肪酸结合蛋白2(Adipocyte protein 2,ap2)的表达。此外,干扰BAMBI后ERK1/2的磷酸化水平减少了。这些结果表明,BAMBI可能通过促进ERK1/2的磷酸化抑制脂肪细胞分化。     

启动脂肪细胞脂动员过程的新成员ATGL

 过去近20年里,激素敏感脂酶（HSL）一直被认为是脂肪细胞脂动员过程中唯一的脂肪水解限速酶,但随着HSL基因敲除鼠的出现,其限速作用受到了质疑.脂肪甘油三酯脂酶（adipose triglyceride lipase,ATGL)是随后发现的启动脂动员的又一个脂肪分解酶.本文就ATGL基因的结构和功能特征、表达及其调控途径和影响因素等方面的研究进展进行了综述,并对今后的研究方向和应用做了展望.     

Inhibition of adipogenesis and induction of apoptosis and lipolysis by stem bromelain in 3T3-L1 adipocytes

The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBPα and PPARγ independent of C/EBPβ gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPARγ expression seems to stem from its ability to inhibit Akt and augment the TNFα pathway. The Akt-TSC2-mTORC1 pathway has recently been described for PPARγ expression in adipocytes. In our experiments, TNFα upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein G(i)α(1), as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPARγ-regulated adipogenesis pathway at all stages and by augmenting TNFα-induced lipolysis and apoptosis in mature adipocytes.     

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells’ lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

     

Dicer has a crucial role in the early stage of adipocyte differentiation, but not in lipid synthesis, in 3T3-L1 cells

Dicer is a rate-limiting enzyme for microRNA (miRNA) synthesis. To determine the effects of Dicer on adipogenesis, we performed stage-specific knockdown of Dicer using adenovirus encoding short-hairpin RNAi against Dicer in 3T3-L1 cells. When cells were infected with the adenovirus before induction of adipocyte differentiation, Dicer RNAi suppressed the gene expression of inducers of adipocyte differentiation such as PPARγ, C/EBPα, and FAS in 3T3-L1 cells during adipocyte differentiation. Concurrently, both adipocyte differentiation and cellular lipid accumulation were cancelled by Dicer RNAi when compared with control RNAi. Meanwhile, we addressed the roles of Dicer in lipid synthesis and accumulation in the final stages of differentiation. When the differentiated cells at day 4 after induction of differentiation were infected with adenovirus Dicer RNAi, cellular lipid accumulation was unchanged. Consistent with this, Dicer RNAi had no effects on the expression of genes related to cellular lipid accumulation, including PPARγ and FAS. Thus, Dicer controls proadipogenic genes such as C/EBPα and PPARγ in the early, but not in the late, stage of adipogenesis via regulation of miRNA synthesis.     

LSDP5 enhances triglyceride storage in hepatocytes by influencing lipolysis and fatty acid β-oxidation of lipid droplets

Lipid storage droplet protein 5 (LSDP5) is a lipid droplet-associated protein of the PAT (perilipin, adipophilin, and TIP47) family that is expressed in the liver in a peroxisome proliferator-activated receptor alpha (PPARα)-dependent manner; however, its exact function has not been elucidated. We noticed that LSDP5 was localized to the surface of lipid droplets in hepatocytes. Overexpression of LSDP5 enhanced lipid accumulation in the hepatic cell line AML12 and in primary hepatocytes. Knock-down of LSDP5 significantly decreased the triglyceride content of lipid droplets, stimulated lipolysis, and modestly increased the mitochondrial content and level of fatty-acid β-oxidation in the mitochondria. The expression of PPARα was increased in LSDP5-deficient cells and required for the increase in the level of fatty acid β-oxidation in LSDP5-deficient cells. Using serial deletions of LSDP5, we determined that the lipid droplet-targeting domain and the domain directing lipid droplet clustering overlapped and were localized to the 188 amino acid residues at the N-terminus of LSDP5. Our findings suggest that LSDP5, a novel lipid droplet protein, may contribute to triglyceride accumulation by negatively regulating lipolysis and fatty acid oxidation in hepatocytes.