促酰化蛋白对脂肪细胞分化中脂滴相关蛋白TIP47表达的影响

研究促酰化蛋白（acylation stimulating protein, ASP）在3T3-L1脂肪细胞分化中对脂滴相关蛋白TIP47(tail-interacting protein 47 kD)表达的影响,从而探讨ASP在成脂方面的重要意义．用免疫荧光染色法观察3T3-L1前脂肪细胞中TIP47的表达定位;采用经典激素鸡尾酒法诱导分化3T3-L1前脂肪细胞,用RT-PCR和Western 印迹方法检测诱导分化的3T3-L1脂肪细胞中TIP47 mRNA和蛋白表达;在分化过程中不同时点,对诱导分化中的3T3-L1脂肪细胞分别给予胰岛素和ASP处理,并设立相应空白对照,用RT-PCR和Western印迹方法检测TIP47 mRNA和蛋白表达． 结果显示,3T3-L1前脂肪细胞中TIP47主要在胞浆内表达;诱导分化过程中的3T3-L1脂肪细胞TIP47 mRNA和蛋白的表达水平呈时间依赖性降低;ASP对诱导分化的3T3-L1脂肪细胞中TIP47 mRNA和蛋白表达有显著的上调作用,但随着分化至48 h,其上调作用已不明显;胰岛素仅在分化的0 d对脂肪细胞中TIP47 mRNA和蛋白表达有上调作用,之后基本无影响．结果提示,ASP促成脂作用可能与其调节脂滴相关蛋白TIP47的表达密切相关,从而为认识及防治肥胖症开拓新的思路．     

槟榔碱对3T3-L1脂肪细胞脂代谢的影响

目的：观察槟榔碱对3T3-L1脂肪细胞脂代谢的影响并探讨其可能机制。方法：采用经典的"鸡尾酒"法诱导3T3-L1前脂肪细胞分化成熟,随后用不同浓度的槟榔碱（0、25、50、100 μmol/L）处理成熟脂肪细胞72 h。72 h后,四甲基偶氮唑盐（MTT）法检测细胞的活性;油红O染色观察胞浆内脂滴情况;Western blot检测脂肪酸合成酶（FAS）、甘油三酯脂肪酶（ATGL）、激素敏感性脂肪酶（HSL）蛋白表达。结果：诱导分化成熟的脂肪细胞胞浆内可见大量脂滴;MTT显示：0~100 μmol/L槟榔碱对脂肪细胞活力无显著影响;油红O染色后脂质含量测定结果表明槟榔碱能减少成熟脂肪细胞中脂质含量;Western blot结果显示：与0 μmol/L组（对照组）相比,槟榔碱可显著降低脂肪细胞内FAS的蛋白表达,增加ATGL和HSL的蛋白表达;其中以50 μmol/L组最为显著。结论：槟榔碱使脂肪细胞脂解增强,可能与降低脂质合成关键酶FAS的表达,增加脂质分解代谢关键酶ATGL和HSL的表达有关。     

人源FGF-21在脂肪细胞糖代谢中的作用

近年来研究发现,成纤维细胞生长因子(FGF)-21是一种新的代谢调节因子.为了深入研究人源FGF-21（hFGF-21）的生物活性,本实验利用SUMO高效表达载体,高效表达成熟的hFGF-21,并利用小鼠3T3-L1脂肪细胞检测hFGF-21的糖代谢活性．实验结果表明,hFGF-21可促进脂肪细胞的葡萄糖吸收,且葡萄糖吸收效率呈剂量依赖性．hFGF-21作用4 h即可促进脂肪细胞糖吸收,其活性可持续24 h以上．hFGF-21与胰岛素共同作用的葡萄糖吸收效果,明显优于它们的单独作用结果,说明hFGF-21与胰岛素发挥协同作用．脂肪细胞经hFGF-21预处理后,显著增加了胰岛素促进脂肪细胞吸收葡萄糖的效率,说明hFGF-21可以增加胰岛素的敏感性．本实验为临床应用hFGF-21治疗糖尿病,增加胰岛素敏感性提供了依据．     

共轭亚油酸对脂肪代谢相关基因表达的影响

本文利用实时定量PCR的测定方法,分析了两种共轭亚油酸(CLA)异构体对3T3-L1小鼠前脂肪细胞脂肪代谢相关基因表达的影响。本研究CLA异构体的处理浓度和时间为75.4μmol/L,8 d,测定了与能量代谢、细胞凋亡、脂肪酸氧化作用和脂解作用相关的多种基因的mRNA水平。结果显示:两种异构体均能够显著提高UCP1、UCP3、Perilipin和PPARα的mRNA水平,而抑制UCP2的表达水平(P<0.01)。与cis-9t,rans-11CLA相比t,rans-10c,is-12 CLA显著提高PKA(P<0.05)、CPT-1和TNF-α(P<0.01)的mRNA水平。与对照组相比,两种CLA异构体处理组均对HSL、ATGL、ACO和Leptin的基因表达无显著影响。     

Phenols from Potentilla anserina L. Improve Insulin Sensitivity and Inhibit Differentiation in 3T3-L1 Adipocytes in Vitro

Potentilla anserina L., a well-known perennial herb, is widely used in traditional Tibetan medicine and used as a delicious food in humans. The present investigation reports on the activity of P. anserina phenols (PAP) in regulating glycolipid metabolism in 3T3-L1 adipocytes. Insulin sensitivity tests showed that PAP improved insulin-stimulated glucose uptake by promoting the phosphorylation of serine/threonine kinase Akt. Moreover, an assay involving the differentiation of 3T3-L1 preadipocytes demonstrated that PAP also decreased the accumulation of lipid droplets by suppressing the expression of adipokines during the differentiation process. In addition, the underlying mechanism from the aspects of energy metabolism and oxidative stress is also discussed. The improvement in energy metabolism was supported by an increase in mitochondrial membrane potential (MMP) and intracellular ATP. Amelioration of oxidative stress was supported by decreased levels of intracellular reactive oxygen species (ROS). In summary, our findings suggest that PAP can ameliorate the disorder of glycolipid metabolism in insulin resistant 3T3-L1 adipocytes by improving energy metabolism and oxidative stress and might be an attractive candidate for the treatment of diabetes.     

Live Cell Multicolor Imaging of Lipid Droplets with a New Dye, LD540

A lipophilic dye based on the Bodipy fluorophore, LD540, was developed for microscopic imaging of lipid droplets. In contrast to previous lipid droplet dyes, it can spectrally be resolved from both green and red fluorophores allowing multicolor imaging in both fixed and living cells. Its improved specificity, brightness and photostability support live cell imaging, which was used to demonstrate by two-color imaging lipid droplet motility along microtubules.     

Rosiglitazone Modulates Insulin-Induced Plasma Membrane Area Changes in Single 3T3-L1 Adipocytes

The antioxidant activity of mitochondria-targeted small molecules, SkQ1 and MitoQ (conjugates of a lipophilic decyltriphenylphosphonium cation with an antioxidant moiety of a plastoquinone and ubiquinone, respectively), was studied in aqueous solution and in a lipid environment, i.e., micelles, liposomes and planar bilayer lipid membranes. Reactive oxygen species (ROS) were generated by azo initiators or ferrous ions with or without tert-butyl-hydroperoxide (t-BOOH). Chemiluminescence, fluorescence, oxygen consumption and inactivation of gramicidin peptide channels were measured to detect antioxidant activity. In all of the systems studied, SkQ1 was shown to effectively scavenge ROS. The scavenging was inherent to the reduced form of the quinone (SkQ1H(2)). In the majority of the above model systems, SkQ1 exhibited higher antioxidant activity than MitoQ. It is concluded that SkQ1H(2) operates as a ROS scavenger in both aqueous and lipid environments, being effective at preventing ROS-induced damage to membrane lipids as well as membrane-embedded peptides.     

Regulation of Apelin and Its Receptor Expression in Adipose Tissues of Obesity Rats with Hypertension and Cultured 3T3-L1 Adipocytes

The apelin/APJ system has been implicated in obesity-related hypertension. We investigated the mechanism responsible for the pathogenesis of obesity-related hypertension with a special focus on the crosstalk between AngII/its type 1 receptor (AT1R) signaling and apelin/APJ expression. Sprague-Dawley rats fed a high-fat (obesity-related hypertension, OH) or normal-fat diet (NF) for 15 weeks were randomly assigned to one of two groups and administered vehicle or perindopril for 4 weeks. Compared to the NF rats, the OH rats showed lower levels of plasma apelin and apelin/APJ mRNAs of perirenal adipose tissues, and these changes were restored by perindopril. Administration of the AT1R antagonist olmesartan resulted in the restoration of the reduction of apelin and APJ expressions induced by AngII for 48 h in 3T3-L1 adipocytes. Among several inhibitors for extracellular signal-regulated kinases 1/2 (ERK1/2) PD98059, p38 mitogen-activated protein kinase (p38MAPK) SB203580 and phosphatidylinositol 3-kinase (PI3K) {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, the latter showed an additive effect on AngII-mediated inhibitory effects. In addition, the levels of p-Akt, p-ERK and p38MAPK proteins were decreased by long-term treatment with AngII (120 min), and these changes were restored by Olmesartan. Apelin/APJ appears to be impaired in obesity-related hypertension. The AngII inhibition-mediated beneficial effects are likely attributable, at least in part, to restoration of p38/ERK-dependent apelin/APJ expression in diet-induced obesity-related hypertension.     

Optimization of Xylanase Production by Thermomyces Lanuginosus in Tomato Seed Meal Using Response Surface Methodology

Summary A 3² central composite experimental design was performed with the aim of optimizing xylanase production by Thermomyces lanuginosus grown on corn cobs in submerged cultures. Xylanase production was first tested on different nitrogen sources (tomato skin, tomato seed meal, corn steep liquor, meat peptone, bacto-tryptone and yeast extract). Tomato seed meal was the selected substrate to test the effect of two variables on xylanase production (corn cobs and tomato seed meal concentrations). A second-order quadratic model and a response surface method showed that the optimum condition for xylanase production was corn cobs 4.6% (w/v) and tomato seed meal 2.1% (w/v). The optimum conditions found were transferred to 7-l bioreactors, where activities as high as 1630 U/ml were obtained.     

Pacer Is a Mediator of mTORC1 and GSK3-TIP60 Signaling in Regulation of Autophagosome Maturation and Lipid Metabolism

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Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: A Monte Carlo simulation

Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.     

Coordinated reprogramming of metabolism and cell function in adipocytes from proliferation to differentiation

Adipose tissue plays a major role in regulating lipid and energy homeostasis by storing excess nutrients, releasing energetic substrates through lipolysis, and regulating metabolism of other tissues and organs through endocrine and paracrine signaling. Adipocytes within fat tissues store excess nutrients through increased cell number (hyperplasia), increased cell size (hypertrophy), or both. The differentiation of pre-adipocytes into mature lipid-accumulating adipocytes requires a complex interaction of metabolic pathways that is still incompletely understood. Here, we applied parallel labeling experiments and ¹³C-metabolic flux analysis to quantify precise metabolic fluxes in proliferating and differentiated 3T3-L1 cells, a widely used model to study adipogenesis. We found that morphological and biomass composition changes in adipocytes were accompanied by significant shifts in metabolic fluxes, encompassing all major metabolic pathways. In contrast to proliferating cells, differentiated adipocytes 1) increased glucose uptake and redirected glucose utilization from lactate production to lipogenesis and energy generation; 2) increased pathway fluxes through glycolysis, oxidative pentose phosphate pathway and citric acid cycle; 3) reduced lactate secretion, resulting in increased ATP generation via oxidative phosphorylation; 4) rewired glutamine metabolism, from glutaminolysis to de novo glutamine synthesis; 5) increased cytosolic NADPH production, driven mostly by increased cytosolic malic enzyme flux; 6) increased production of monounsaturated C16:1; and 7) activated a mitochondrial pyruvate cycle through simultaneous activity of pyruvate carboxylase, malate dehydrogenase and malic enzyme. Taken together, these results quantitatively highlight the complex interplay between pathway fluxes and cell function in adipocytes, and suggest a functional role for metabolic reprogramming in adipose differentiation and lipogenesis.     

Ghrelin Augments the Expressions and Secretions of Proinflammatory Adipokines,VEGF120 and MCP‐1, in Differentiated 3T3‐L1 Adipocytes

Ghrelin is a physiological‐active peptide with growth hormone‐releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin‐direct‐effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3‐L1 adipocytes stimulated with ghrelin in vitro . The stimulation with 50 nmol/L ghrelin for 24 h resulted in the significant 1.9‐fold increase on vascular endothelial growth factor‐120 (VEGF₁₂₀) releases (p < 0.01) and the 1.7‐fold on monocyte chemoattractant protein‐1 (MCP‐1) (p < 0.01) from 3T3‐L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, IL‐10 and adiponectin secretions. In addition, Akt phosphorylation on Ser473 and c‐Jun NH₂‐terminal protein kinase (JNK) phosphorylation on Thr183/Tyr185 were markedly enhanced 1.4‐fold (p < 0.01) and 1.6‐fold (p < 0.01) in the ghrelin‐stimulated adipocytes, respectively. Furthermore, the treatment with LY294002 (50 μmol/L) and Wortmannin (10nmol/L), inhibitors of phosphatidylinositol 3‐kinase (PI3K), significantly decreased the amplified VEGF₁₂₀ secretion by 29% (p < 0.01) and 28% (p < 0.01) relative to the cells stimulated by ghrelin alone, respectively, whereas these inhibitors had no effects on increased MCP‐1 release. On the other hand, JNK inhibitor SP600125 (10 μmol/L) clearly reduced the increased MCP‐1, but not VEGF₁₂₀, release by 35% relative to the only ghrelin‐stimulated cells (p < 0.01). In conclusion, ghrelin can enhance the secretions of proinflammatory adipokines, VEGF₁₂₀ and MCP‐1, but fails to affect IL‐10 and adiponectin which are considered to be anti‐inflammatory adipokines. Moreover, this augmented VEGF₁₂₀ release is invited through the activation of PI3K pathways and the MCP‐1 is through JNK pathways. Consequently, our results strongly suggest that ghrelin can induce the development of diabetes via its direct‐action in peripheral tissues as well as via in CNS. J. Cell. Physiol. 230: 199–209, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

c-Jun N-terminal kinase is involved in the suppression of adiponectin expression by TNF-alpha in 3T3-L1 adipocytes

Adiponectin, one of adipokines that is secreted from adipocytes, plays an important role in the regulation of glucose and lipid metabolism. Paradoxically, serum concentrations of adiponectin are decreased in obese and type 2 diabetic patients, although it is produced in adipose tissue. On the other hand, plasma TNF-alpha levels are increased in such subjects. In the present study, the mechanism by which adiponectin is regulated by TNF-alpha was investigated. The decreased adiponectin mRNA levels by TNF-alpha were partially recovered by treatment with a c-Jun N-terminal kinase (JNK) inhibitor or the PPAR-gamma agonist rosiglitazone in 3T3-L1 adipocytes. Interestingly, however, cotreatment with the JNK inhibitor and rosiglitazone led to a recovery of TNF-alpha-mediated adiponectin suppression to the control level. The JNK inhibitor regulated the expression of adiponectin by the increase of PPAR-gamma DNA binding activity and the recovery of its mRNA expression while rosiglitazone acted via a PPAR-gamma independent pathway which remains to be elucidated. These findings suggest that the JNK signaling pathway, activated by TNF-alpha, is involved in the regulation of adiponectin expression.