猪成熟脂肪细胞分离培养及去分化研究

成熟脂肪去分化技术可为研究脂肪细胞分化提供均一的前体脂肪细胞。本研究分离培养了猪成熟脂肪细胞,并去分化为前体脂肪细胞。本实验采用Ⅱ胶原酶消化后离心分离1~3日龄仔猪(Susscrofa)皮下脂肪组织,天花板法培养获得成熟脂肪细胞。显微镜下观察脂肪细胞去分化形态学变化,并在成脂诱导培养液的作用下诱导再分化。采用油红O染色法检测分化不同时期细胞脂滴聚集效率,脂滴的累积随诱导的进行不断增加。RT-PCR检测成熟脂肪细胞标志基因过氧化物酶体增殖物活化受体(PPARγ)和和脂肪酸结合蛋白4(FABP4)的mRNA相对表达量,分化早期基因表达水平较低,其表达水平在分化过程中持续增高,在分化后期PPARγ相对表达量与诱导分化前增加了2.8倍,FABP4增加了约62倍(差异显著P<0.05)。说明去分化获得的前体脂肪细胞在成脂诱导培养液作用下,可有效地分化为成熟脂肪细胞。本研究优化了猪成熟脂肪细胞分离和培养体系,并通过去分化获得具有再分化能力的前体脂肪细胞,为进一步深入研究猪脂肪细胞分化与代谢提供技术平台。     

Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways

Cinnamaldehyde (CA), one of the active components of cinnamon, has been known to exert several pharmacological effects such as anti-inflammatory, antioxidant, antitumor, and antidiabetic activities. However, its antiobesity effect has not been reported yet. This study investigated the antidifferentiation effect of CA on 3T3-L1 preadipocytes, and the antiobesity activity of CA was further explored using high-fat-diet-induced obese ICR mice. During 3T3-L1 preadipocytes were differentiated into adipocytes, 10-40 μM CA was treated and lipid contents were quantified by Oil Red O staining, along with changes in the expression of genes and proteins associated with adipocyte differentiation and adipogenesis. It was found that CA significantly reduced lipid accumulation and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding proteins α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) in concentration-dependent manners. Moreover, CA markedly up-regulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), and these effects were blunted in the presence of AMPK inhibitor, compound C. In the animal study, weight gains, insulin resistance index, plasma triglyceride (TG), nonesterified fatty acid (NEFA), and cholesterol levels in the 40 mg/kg of CA-administered group were significantly decreased by 67.3, 55, 39, 31, and 23%, respectively, when compared to the high-fat diet control group. In summary, these results suggest that CA exerts antiadipogenic effects through modulation of the PPAR-γ and AMPK signaling pathways.     

Antiadipogenic effect of dietary apigenin through activation of AMPK in 3T3-L1 cells

Adipocyte differentiation (adipogenesis) is a complex process including the coordinated changes in hormone sensitivity and gene expression in response to various stimuli. Natural compounds are known to be involved in the regulation of this process. Here we investigated the effects of dietary apigenin, a plant flavonoid, on adipogenesis. Apigenin suppressed adipocyte differentiation of mouse adipocytic 3T3-L1 cells and reduced the accumulation of intracellular lipids. Quantitative PCR and Western blot analyses revealed that apigenin decreased the levels of peroxisome proliferator-activated receptor γ and its target genes such as fatty acid binding protein 4 (aP2) and stearoyl-CoA desaturase. Apigenin decreased or had no effect on the expression of lipolytic genes such as adipose triglyceride lipase, hormone sensitive lipase, and monoacyl glyceride lipase, thereby reducing glycerol release from adipocytes. Noteworthily, apigenin activated 5'-adenosine monophosphate-activated protein kinase (AMPK) in an apigenin dose-dependent manner, which activation is known to suppress adipogenesis. These results provide a novel insight into the molecular mechanism involved in the action of apigenin: the apigenin-induced activation of AMPK leads to decreased expression of adipogenic and lipolytic genes, thus suppressing adipogenesis in 3T3-L1 cells. Thus, dietary apigenin may contribute to lower body-fat content and body-weight gain through the activation of AMPK.     

IGF-I、GH和CLA调控脂肪细胞增殖分化和基因表达

为探讨IGF-I、GH、CLA对不同部位来源脂肪前体细胞的增殖和分化程度的影响，本研究以大鼠为试验模型，分别从附睾及皮下脂肪组织分离得到脂肪前体细胞，并在不同浓度的IGF-I、GH、 CLA处理后，用MTT法检测脂肪前体细胞的增殖，用油红O染色法检测脂肪前体细胞的分化聚脂程度结果表明：各剂量IGF-I和CLA均能够显著促进大鼠附睾及皮下脂肪前体细胞的增殖与分化，但GH对大鼠附睾及皮下脂肪前体细胞的增殖与分化均无明显作用。为进一步观察IGF-I和CLA对皮下脂肪组织的作用机制，试验还采用相对定量RT-PCR法检测细胞中PPARγ和C/EBPα的基因表达水平。研究发现，100ng/ml IGF-I可显著上调大鼠脂肪前体细胞中PPARγ 与C/EBPα的基因表达水平； 48ng/ml GH和100μM CLA可显著促进PPARγ的基因表达，但对C/EBPα mRNA表达无显著影响。上述结果提示IGF-I可能主要通过上调PPARγ 与C/EBPα基因的高水平表达进而促进大鼠脂肪前体细胞的分化，而CLA对脂肪前体细胞的作用在上调PPARγ的表达的同时，还作为PPARγ的配体激活该受体而发挥作用     

Conjugated linoleic acid regulates phosphorylation of PPARγ by modulation of ERK 1/2 and p38 signaling in human macrophages/fatty acid-laden macrophages

Stimulation of macrophages by a variety fatty acids causes activation of MAP kinases (MAPKs). The consequences arising from down-regulation of MAPKs may be a limitation in the activity of PPARγ, which is modulated by a modification catalyzed by these kinases. Phosphorylation of MAP kinases-ERK1/2 and p38 as well as PPARγ was determined by real-time polymerase chain reaction and Western blotting in human macrophages cultured with conjugated linoleic acids (CLAs). We demonstrated that CLA isomers alter MAP kinase phosphorylation and PPARγ activation. Phosphorylation of ERK1/2 was diminished in cells cultivated with cis-9,trans-11 CLA, whereas phosphorylation of p38 was reduced by trans-10,cis-12 CLA. PPARγ was phosphorylated mainly by ERK1/2, and consequently, PPARγ phosphorylation was suppressed mainly by cis-9,trans-11 isomer. In human adipocytes, cis-9,trans-11 C 18:2 raised the activation of PPAR and several of its downstream target genes. We suggest that a similar process may also occur in human macrophages.     

Inhibitory effect of α-lipoic acid on platelet aggregation is mediated by PPARs

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, β/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.     

雷帕霉素靶蛋白(mTOR)信号通路参与沉默信息调节因子1(Sirt1)抑制小鼠脂肪沉积

探讨沉默信息调节因子1(Sirt1)对小鼠脂肪沉积的抑制作用和雷帕霉素靶蛋白(mTOR)信号通路的影响。用Sirt1的激动剂白藜芦醇(100mg·kg-·1d-1)和抑制剂尼克酰胺(500mg·kg-·1d-1)灌胃处理小鼠(Mus mussulus)15d,记录小鼠体质量变化,测定小鼠皮下脂肪、附睾脂肪和肾周脂肪的沉积量,试剂盒测定血脂指标,同时利用Real-timePCR分析与脂肪生成密切相关的转录因子过氧化物酶体增殖物激活受体γ(PPARγ)、固醇调节元件结合蛋白1(SREBP1)和脂解基因甘油三酯水解酶(ATGL)、激素敏感性脂肪酶(HSL)、围脂滴蛋白(Perilipin)及生脂基因脂肪酸合成酶(FAS)mRNA的表达水平,检测mTOR通路关键因子雷帕霉素靶蛋白(mTOR)、核糖体S6蛋白激酶1(S6K1)和真核启动因子4E结合蛋白1(4EBP1)mRNA表达水平。与对照组相比,白藜芦醇处理组小鼠体质量增加量和体脂含量均显著降低(P<0.01),血清中甘油三酯(TG)、总胆固醇(TC)和低密度脂蛋白(LDL-C)浓度均显著降低(P<0.01),而高密度脂蛋白(HDL-C)浓度升高(P<0.01),mTOR通路关键因子mTOR、4EBP1和S6K1mRNA表达水平降低(P<0.01),脂代谢相关基因PPARγ、SREBP1及成脂基因FASmRNA表达水平显著降低(P<0.01),脂解相关基因ATGL、HSL和PerilipinmRNA表达水平显著升高(P<0.01);烟酰胺处理组小鼠体质量、附睾脂肪以与皮下脂肪沉积量增加缓慢(P>0.05),肾周脂肪沉积量增加(P<0.05),血清中LDL-C浓度升高(P<0.05),HDL-C浓度降低(P<0.01),mTOR通路关键因子mTOR和4EBP1mRNA表达水平升高(P<0.01),而脂代谢调控相关因子PPARγ和SREBP1mRNA水平升高(P<0.05),ATGLmRNA表达量显著降低(P<0.05),FAS、HSL和PerilipinmRNA表达量变化不显著(P>0.05)。表明激活Sirt1可减少脂肪合成,增加脂肪分解,从而降低体脂沉积,而mTOR信号通路参与这个过程。     

鸡γ-干扰素基因(ChIFN-γ)的瞬时表达及其抗病毒活性检测

采用PCR技术从质粒pET-ChIFN-γ中扩增得到鸡(Gallus gallus)γ-干扰素(chicken interferon gamma,ChIFN-γ)基因,将其亚克隆到真核表达载体pCAGGS中.将鉴定正确的克隆命名为pCAGGS-ChIFN-γ,体外转染鸡胚成纤维(CEF)细胞,24 h后经间接免疫荧光(IFA)和免疫印迹(Western blot)检测,表达蛋白具有良好的免疫原性.然后利用表达绿色荧光蛋白(GFP)的重组水疱口炎病毒(VSV*GFP)在CEF细胞上检测表达的ChIFN-γ抗病毒活性(AVA),经检测其抗病毒活性为2×10~3AU/mL,并且其活性可被抗鸡IFN-γ的多克隆抗体阻断.     

Effect of eriodictyol on glucose uptake and insulin resistance in vitro

Eriodictyol [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4-one] is a flavonoid with anti-inflammatory and antioxidant activities. Because inflammation and oxidative stress play critical roles in the pathogenesis of diabetes mellitus, the present study was designed to explore whether eriodictyol has therapeutic potential for the treatment of type 2 diabetes. The results show that eriodictyol increased insulin-stimulated glucose uptake in both human hepatocellular liver carcinoma cells (HepG2) and differentiated 3T3-L1 adipocytes under high-glucose conditions. Eriodictyol also up-regulated the mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and adipocyte-specific fatty acid-binding protein (aP2) as well as the protein levels of PPARγ2 in differentiated 3T3-L1 adipocytes. Furthermore, it reactivated Akt in HepG2 cells with high-glucose-induced insulin resistance. This response was strongly inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, indicating that eriodictyol increased Akt phosphorylation by activating the PI3K/Akt pathway. These results imply that eriodictyol can increase glucose uptake and improve insulin resistance, suggesting that it may possess antidiabetic properties.     

α-Mangostin, a dietary xanthone, induces autophagic cell death by activating the AMP-activated protein kinase pathway in glioblastoma cells

This study is the first to investigate the anticancer effects of α-mangostin in human glioblastoma cells. α-Mangostin decreases cell viability by inducing autophagic cell death but not apoptosis. Pretreatment of cells with the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin or knockdown beclin-1, resulted in the suppression of α-mangostin-mediated cell death. We also found that liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling is a critical mediator of α-mangostin-induced inhibition of cell growth. Activation of AMPK induces α-mangostin-mediated phosphorylation of raptor, which subsequently associates with 14-3-3γ and results in the loss of mTORC1 activity. The phosphorylation of both downstream targets of mTORC1, p70 ribosomal protein S6 kinase (p70S6 kinase) and 4E-BP1, is also diminished by activation of AMPK. Furthermore, the inhibition of AMPK expression with shRNAs or an inhibitor of AMPK reduced α-mangostin-induced autophagy and raptor phosphorylation, supporting the theory that activation of AMPK is beneficial to autophagy. A further investigation revealed that α-mangostin also induced autophagic cell death in transplanted glioblastoma in nude mice. Together, these results suggest a critical role for AMPK activation in the α-mangostin-induced autophagy of human glioblastoma cells.     

Investigation of heat treating conditions for enhancing the anti-inflammatory activity of citrus fruit (Citrus reticulata) peels

In traditional Chinese medicine, dried citrus fruit peels are widely used as remedies to alleviate coughs and reduce phlegm in the respiratory tract. Induction of inducible nitric oxide synthase (iNOS) in inflammatory cells and increased airway production of nitric oxide (NO) are well recognized as key events in inflammation-related respiratory tract diseases. Despite the fact that the enhancing effect of heat treatment on the antioxidant activity of citrus fruit peels has been well documented, the impact of heat treatment on citrus peel beneficial activities regarding anti-inflammation is unclear. To address this issue, we determined the anti-inflammatory activities of heat-treated citrus peel extracts by measuring their inhibitory effect upon NO production by lipopolysaccharide-activated RAW 264.7 macrophages. Results showed that the anti-inflammatory activity of citrus peel was significantly elevated after 100 degrees C heat treatment in a time-dependent fashion during a period from 0 to 120 min. Inhibition of iNOS gene expression was the major NO-suppressing mechanism of the citrus peel extract. Additionally, the anti-inflammatory activity of citrus peel extract highly correlated with the content of nobiletin and tangeretin. Conclusively, proper and reasonable heat treatment helped to release nobiletin and tangeretin, which were responsible for the increased anti-inflammatory activity of heat-treated citrus peels.