Mature breast adipocytes promote breast cancer cell motility

Adipocytes express substances involved in both normal physiology and pathological processes. One such adipocyte protein is the Serpin (serine protease inhibitor) plasminogen activator inhibitor-1 (PAI-1). PAI-1 functions to inhibit urokinase type plasminogen activator (uPA) though PAI-1 itself is also implicated in breast cancer progression. While the role of adipocytes in breast cancer development is not fully understood, obesity is a known risk factor associated with breast cancer. Thus, we characterized adipocytes from breast and omental tissues for PAI-1 and uPA, and the influence of adipocytes on breast cancer cell motility. Using preadipocyte cells from breast and omental adipose tissue, we differentiated each site into mature adipocytes. PAI-1 protein was found in breast adipocytes>omental preadipocytes>omental adipocytes>breast preadipocytes. Interestingly, uPA protein was not detected in any of these cell types. We then incubated breast adipocyte conditioned media (Adip-CM) and preadipocyte conditioned media (PreAdip-CM) on both normal (MCF-10A) and malignant (MCF-10CA1) breast epithelial cell lines. Adip-CM, but not PreAdip-CM, (a) increased cell motility in both MCF-10A and MCF-10CA1 cells; (b) increased cell-associated uPA activity in both cell lines; (c) increased phosphorylated-Akt levels in MCF-10CA1 cells; and (d) gene array profiles show altered expression of several genes associated with cancer adhesion, metastasis and signaling. Our results suggest that mature breast adipocytes are capable of altering the epithelial cell phenotype, producing a more motile cell type and further provide a potential link between obesity and risk of breast cancer.     

Oxygen consumption in undifferentiated versus differentiated adipogenic mesenchymal precursor cells

To date, no adequate implant material for the correction of soft tissue defects such as after extensive deep burns, tumor resections or in congenital defects is available. A biohybrid composed of viable adipose precursor cells and an optimised matrix could help towards a solution. Morphologically, preadipocytes resemble fibroblasts and have not yet built a large cytoplasmic lipid droplet as found in differentiated adipocytes. Additionally, preadipocytes are smaller than mature adipocytes allowing a quicker revascularization after transplantation. Furthermore, transplanted preadipocytes can form adipose tissue in vivo whereas the transplantation of mature adipocytes often gives poor results, i.e. oil cysts or shrinkage of the transplant. Since these observations point to differences in metabolic activity between preadipocytes and adipocytes, we investigated the oxygen consumption of preadipocytes stimulated to undergo differentiation, and fibroblasts, by measuring the respiration with a Clark-type oxygen electrode. Preadipocytes had a significantly lower oxygen consumption than mature adipocytes. This advantage in respiration and the better revascularization of undifferentiated adipose tissue cells allow the development of innovative transplants and point to preadipocytes as promising tool to improve transplantations in adipose tissue reconstruction.     

Multiple symmetric lipomatosis. Ultrastructural investigation of the tissue and preadipocytes in primary culture

Multiple symmetric lipomatosis (MSL) is characterized by enlarging, painless fat deposits in the neck and upper trunk. The pathogenesis of MSL is unknown. Owing to localization of MSL fat deposits in the neck and interscapular region, it has been suggested that they could originate from brown fat. However, the histological appearance of MSL adipose tissue is that of white fat, with prevailing monovacuolar adipocytes. Nevertheless, MSL adipocytes are smaller than adipocytes of the common white adipose tissue and show peculiar metabolic features. The ultrastructure of MSL lesions has been not described. The present work investigated the ultrastructural morphology of MSL adipose tissue and lipomatous adipocyte precursors maintained in long-term culture. Samples of lipomatous tissue were obtained from patients affected with MSL undergoing surgical lipectomy. Portion of the tissue was processed for electron microscopy; the rest was digested with collagenase, and isolated preadipocytes from the stromal-vascular fraction were cultured up to 15 days. Cultured cells were prepared for electron microscopy in situ and their morphology compared with human white adipose tissue preadipocytes and rat brown preadipocytes cultured in parallel. Results show the following. 1) Adipocytes of MSL are not monovacuolar and resemble the largest adipocytes that can be found in rat and human brown fat. 2) Some morphological features of MSL adipocyte precursors resemble brown adipocyte more than white: cultured MSL preadipocytes transiently develop large mitochondria with parallel cristae resembling those of the brown fat cell and maintain a multivacuolar lipid deposit in culture, i.e. a typical feature of brown preadipocytes. 3) Some morphological features suggest a neoplastic nature of MSL adipocytes. Taken together, these findings suggest that MSL is a neoplastic disease which could originate in brown fat.     

Establishment of the Insulin Resistance Induced by Inflammatory Response in 3T3-L1 Preadipocytes Cell Line

In the light of given recent reports, insulin resistance related to inflammation is characterized by increasing a diverse array of pro-inflammatory cytokines. In this study, hypothesizing that 3T3-L1 non-differentiated preadipocytes cell line as a cell model could be used to investigate this linkage, the aim is to determine whether the preadipocytes induced by different inflammatory responses could cause insulin resistance. This paper has determined the time and concentration-dependent effects of insulin on glucose consumption in the 3T3-L1 non-differentiated preadipocytes. Glucose consumption has also been assayed in the preadipocytes which are treated with LPS and CM originated from LPS-activated RAW264.7. Then protein level of each group has been measured by coomassie brilliant blue protein kit. Furthermore, secretion levels of IL-6 and TNF-alpha are measured by ELISA in the supernatant of RAW264.7 and preadipocytes. Finally, the mRNA expressions for IL-6, TNF-alpha and PPARgamma has been assessed by RT-PCR. The results show that administration of LPS and CM both can increase releases of IL-6 and TNF-alpha, as well as gene expression of IL-6 mRNA; this change is accompanied with suppression of PPARgamma mRNA activation in 3T3-L1 undifferentiated preadipocytes. In conclusion, our results suggest that in preadipocytes, pro-inflammatory cytokines can result in insulin resistance, and deserve further investigation to be helpful for treatment and revealing mechanisms of T2DM.     

Macrophages produce IL-33 by activating MAPK signaling pathway during RSV infection

It has been reported that RSV infection can enhance IL-33 production in lung macrophages. However, little is known about specific signaling pathways for activation of macrophages during RSV infection. In the present study, by using real-time RT-PCR as well as western blot assay, it became clear that RSV infection can enhance not only the expression of mRNAs for MAPK molecules (including p38, JNK1/2, and ERK1/2), but also the levels of MAPK proteins in lung macrophages as well as RAW264.7 cells. Furthermore, infection with RSV resulted in an increased level of phosphorylated MAPK proteins in RAW264.7 cells, suggesting that MAPK signaling pathway may participate in the process of RSV-induced IL-33 secretion by macrophages. In fact, the elevated production of IL-33 in RAW264.7 was attenuated significantly by pretreatment of the cells with special MAPK inhibitor before RSV infection, further confirming the function of MAPKs pathway in RSV-induced IL-33 production in macrophages. In contrast, the expression of NF-κB mRNA as well as the production of NF-κB protein in lung macrophages and RAW264.7 cells was not enhanced markedly after RSV infection. Moreover, RSV infection failed to induce the phosphorylation of NF-κB in RAW264.7 cells, suggesting that NF-κB signaling pathway may be not involved in RSV-induced IL-33 production in macrophages. Conclusion, these results indicate that RSV-induced production of IL-33 in macrophages is dependent on the activation of MAPK signaling pathway.     

Deficiency of Phenylethanolamine N-Methyltransferase in Norepinephrine-Producing Pheochromocytoma

Pheochromocytoma is a catecholamine (CA)-producing tumor that is classified into two types: the norepinephrine (NE) and the mixed NE and epinephrine type (E-type) from plasma CA levels. Phenylethanolamine N-methyltransferase (PNMT) is the terminal enzyme in CA synthesis; it catalyzes the synthesis of E from NE. It is not known whether the absence of immunoreactive PNMT is paralleled by a lack of PNMT mRNA. The mRNA of tyrosine hydroxylase (TH) and PNMT in seven pheochromocytomas, five NE-type and two E-type tumors, were examined by Northern blot analysis andin situ hybridization (ISH) technique. TH mRNA was detected in all tumors but PNMT mRNA was limited only to the E-type tumors. In addition to our previous immunohistochemical study of 70 pheochromocytomas and paragangliomas in which all pheochromocytomas had cells immunoreactive to TH, but PNMT was expressed only in E-type, we concluded that NE-type pheochromocytoma lacks PNMT both at the mRNA and protein levels, resulting in an inability to produce E. The essential difference between NE-type and E-type pheochromocytoma is that the NE-type lacks PNMT.     

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.     

Adipogenesis of human adipose-derived stem cells within three-dimensional hollow fiber-based bioreactors

To further differentiate adipose-derived stem cells (ASCs) into mature adipocytes and create three-dimensional (3D) adipose tissue in vitro, we applied multicompartment hollow fiber-based bioreactor technology with decentral mass exchange for more physiological substrate gradients and integral oxygenation. We hypothesize that a dynamic 3D perfusion in such a bioreactor will result in longer-term culture of human adipocytes in vitro, thus providing metabolically active tissue serving as a diagnostic model for screening drugs to treat diabetes. ASCs were isolated from discarded human abdominal subcutaneous adipose tissue and then inoculated into dynamic 3D culture bioreactors to undergo adipogenic differentiation. Insulin-stimulated glucose uptake from the medium was assessed with and without TNF-alpha. 3D adipose tissue was generated in the 3D-bioreactors. Immunohistochemical staining indicated that 3D-bioreactor culture displayed multiple mature adipocyte markers with more unilocular morphologies as compared with two-dimensional (2D) cultures. Results of real-time polymerase chain reaction showed 3D-bioreactor treatment had more efficient differentiation in fatty acid-binding protein 4 expression. Repeated insulin stimulation resulted in increased glucose uptake, with a return to baseline between testing. Importantly, TNF-alpha inhibited glucose uptake, an indication of the metabolic activity of the tissue. 3D bioreactors allow more mature adipocyte differentiation of ASCs compared with traditional 2D culture and generate adipose tissue in vitro for up to 2 months. Reproducible metabolic activity of the adipose tissue in the bioreactor was demonstrated, which is potentially useful for drug discovery. We present here, to the best of our knowledge for the first time, the development of a coherent 3D high density fat-like tissue consisting of unilocular structure from primary adipose stem cells in vitro.     

Rab7负向调控巨噬细胞中CpG诱导的细胞因子的表达

目的:研究Rab7过表达及失活突变(Rab7T22N)对CpG刺激的RAW264.7巨噬细胞中分泌细胞因子的影响。方法:采用RT-PCR和Real-time PCR检测RAW264.7细胞中Rab7在CpG刺激下表达模式。将Rab7真核表达质粒及失活突变质粒Rab7T22N通过脂质体法转染RAW264.7细胞,G418稳定筛选,Western法鉴定表达效果。用CpG刺激稳定表达Rab7的RAW264.7细胞系,RT-PCR和Real-time PCR检测细胞因子IL-6、IL-1β、IFN-β的表达量变化。结果:CpG刺激RAW264.7后,Rab7 mRNA表达水平逐渐增高,在8小时达到高峰,表达增高近4倍。Rab7过表达后,CpG刺激后产生的IL-6、IL-1β、IFN-β显著降低。巨噬细胞中Rab7失活突变后,在CpG刺激后IL-6、IL-1β、IFN-β的表达又显著增加。结论:CpG促进RAW264.7巨噬细胞中Rab7 mRNA的表达,Rab7抑制了CpG刺激的巨噬细胞中IL-6、IL-1β、IFN-β的表达,该抑制作用的发挥与其酶活性的GTP结合有关。该研究为进一步阐明Rab7在CpG/TLR9信号通路中的作用奠定了基础。     

CCR1参与肺癌分泌因子诱导破骨细胞生成

目的建立肺癌细胞与单核/巨噬细胞间接共培养模型,观察在肺癌细胞条件培养基作用下抑制前体破骨细胞(RAW264.7)的CCR1对破骨细胞生成的影响。方法收集肺癌细胞A549的条件培养基,将条件培养基以不同浓度加入RAW264.7培养基中共培养,在共培养体系中加或不加CCR1特异性拮抗剂BX471。细胞培养至第5天进行TRAP染色,提取不同条件作用后RAW264.7细胞的总RNA,采用Real-time PCR检测CCR1及破骨细胞标志基因cathepsin K、TRAP mRNA的表达,使用细胞免疫荧光和Western blot检测不同时期RAW264.7细胞中CCR1及Cathepsin K蛋白的表达量。结果加入A549细胞条件培养基的RAW264.7细胞TRAP阳性细胞显著增多,条件培养基明显上调RAW264.7的CCR1、Cathepsin K、TRAP的表达且呈浓度依赖,CCR1特异性拮抗剂BX471可抑制肺癌分泌因子对破骨细胞的活化。结论肺癌细胞分泌因子可促进破骨前体细胞向破骨细胞分化,CCR1参与了肺癌细胞分泌因子对破骨细胞的活化过程。     

Two alternative procedures for isolating adipofibroblasts from sheep skeletal muscle

Methods to isolate cells used in the study of adipocyte differentiation are lengthy, potentially damaging to the cells collected, and usually result in a mixed group of cells which are difficult to define clearly. Additionally, much work done on the differentiation of fibroblasts to preadipocytes or preadipocytes to adipocytes has relied on the use of populations of cells which are either embryonic in origin or from genetically unique animals. We therefore report two simple and rapid protocols for obtaining relatively pure populations of preadipocytes from perimuscular fat and from intramuscular fat depots of normal sheep skeletal muscle. In the first procedure, a finely minced preparation of perimuscular adipose tissue is placed directly into flasks for ceiling culture. During the second procedure, free-floating adipocytes, resulting from the enzymatic preparation of satellite cells from skeletal muscle, are placed into ceiling culture. Cells from both isolation procedures attach, grow, and are later harvested for use. These cells demonstrate proliferation and differentiation abilities of normal preadipocytes. Cell populations may be expanded for use in cloning pure populations of adipocytes or used directly in studies to identify mechanisms of adipocyte development and intercellular communication with myogenic cells.     

4E-BP1 regulates the differentiation of white adipose tissue

4E Binding protein 1 (4E-BP1) suppresses translation initiation. The absence of 4E-BP1 drastically reduces the amount of adipose tissue in mice. To address the role of 4E-BP1 in adipocyte differentiation, we characterized 4E-BP1^−/− mice in this study. The lack of 4E-BP1 decreased the amount of white adipose tissue and increased the amount of brown adipose tissue. In 4E-BP1^−/− MEF cells, PPARγ coactivator 1 alpha (PGC-1α) expression increased and exogenous 4E-BP1 expression suppressed PGC-1α expression. The level of 4E-BP1 expression was higher in white adipocytes than in brown adipocytes and showed significantly greater up-regulation in white adipocytes than in brown adipocytes during preadipocyte differentiation into mature adipocytes. The amount of PGC-1α was consistently higher in HB cells (a brown preadipocyte cell line) than in HW cells (a white preadipocyte cell line) during differentiation. Moreover, the ectopic over-expression of 4E-BP1 suppressed PGC-1α expression in white adipocytes, but not in brown adipocytes. Thus, the results of our study indicate that 4E-BP1 may suppress brown adipocyte differentiation and PGC-1α expression in white adipose tissues.     

Bacterial Cell Wall Constituents Induce Hepcidin Expression in Macrophages Through MyD88 Signaling

Hepcidin is a key regulator of iron recycling by macrophages that is synthesized mainly by hepatocytes but also by macrophages. However, very little is known about the molecular regulation of hepcidin in macrophages. In the present study, we investigated hepcidin regulation in the RAW264.7 macrophage cell line and in murine peritoneal macrophages stimulated with different Toll-like receptor (TLR) ligands. We found that TLR-2 and TLR-4 ligands activated hepcidin expression in RAW264.7 cells and in wild-type murine peritoneal macrophages, but not in murine peritoneal macrophages isolated from TLR2(-/-), TLR-4-deficient or MyD88(-/-) mice. IL-6 production by RAW264.7 cells stimulated with lipopolysaccharide (LPS, TLR4 ligand) was enhanced by high amounts of iron present in the culture medium. We conclude that hepcidin expression in macrophages is regulated mainly through TLR2 and TLR4 receptors via the MyD88-dependent signaling pathway and that autocrine regulation of iron accumulation in macrophages by hepcidin may affect the levels of proinflammatory cytokine production.     

氧化剂对RAW264.7细胞M-CSF

目的:揭示氧化应激对巨噬细胞巨噬细胞集落刺激因子(M-CSF)基因表达的影响。方法:采用逆转录多聚酶链反应(RT-PCR)技术,观察氧化剂叔丁基氢过氧化物(tbOOH)对RAW264.7细胞M-CSF mRNA表达的影响。结果:一定浓度的tbOOH(1.5×10^-4 mol/L)能够诱导RAW264.7细胞M-CSF mRNA表达,且随tbOOH浓度的增加,其对M-CSF mRNA的诱导作用增强;另外,环己酰亚胺(cycloheximide)及acetovanilone可减弱tbOOH对RAW264.7细胞M-CSF表达的诱导。结论:tbOOH可诱导RAW264.7细胞M-CSF mRNA的表达,且作用在转录水平,该过程中有新的蛋白质合成及内源性活性氧产生的参与。     

Effects of Lycium barbarum polysaccharides with different molecular weights on function of RAW264.7 macrophages

The present study was aimed at investigating the effects of four LBP fractions with different molecular weights (MWs), designated LBP2, LBP3, LBP4 and LBP5, on RAW264.7 macrophages function. Results showed that LBP fractions could significantly enhance the expression of CD86 and MHC-II molecules on RAW264.7 macrophages. LBP3, LBP4 and LBP5 could enhance the production of ROS, NO, TNF-α and IL-6, and the phagocytosis of RAW264.7 macrophages. LBP2 with an MW of larger than 350?kDa could only enhance the secretion of TNF-α. LBP3 enhanced the RAW264.7 macrophages function in a dose-dependent manner and also enhanced the iNOS mRNA expression in the cells. These results demonstrated that the immunomodulatory activity of LBP on RAW264.7 macrophages was closely related to its MW. It indicated that fractions with an MW smaller than 350?kDa were the main active fractions of LBP in enhancing macrophages function.     

Elucidating the Preadipocyte and Its Role in Adipocyte Formation: a Comprehensive Review

Adipogenesis is a complex process whereby the multipotent adipose-derived stem cell is converted to a preadipocyte before terminal differentiation into the mature adipocyte. Preadipocytes are present throughout adult life, exhibit adipose fat depot specificity, and differentiate and proliferate from distinct progenitor cells. The mechanisms that promote preadipocyte commitment and maturation involve numerous protein factor regulators, epigenetic factors, and miRNAs. Detailed characterization of this process is currently an area of intense research and understanding the roles of preadipocytes in tissue plasticity may provide insight into novel approaches for tissue engineering, regenerative medicine and treating a host of obesity-related conditions. In the current study, we analyzed the current literature and present a review of the characteristics of transitioning adipocytes and detail how local microenvironments influence their progression towards terminal differentiation and maturation. Specifically, we detail the characterization of preadipocyte via surface markers, examine the signaling cascades and regulation behind adipogenesis and cell maturation, and survey their role in tissue plasticity and health and disease.     

PLIN2通过调控SREBP2促进RAW264.7巨噬细胞脂质蓄积

目的:探讨脂滴包被蛋白2(PLIN2)是否通过调控固醇调节元件结合蛋白2(SREBP2)影响小鼠RAW264.7巨噬细胞中脂质蓄积.方法:采用氧化低密度脂蛋白(ox-LDL)处理RAW264.7巨噬细胞不同时间(0、6、12、24和48 h),Western blot检测PLIN2和SREBP2蛋白表达水平;过表达或敲...     

小鼠巨噬细胞内表达结核分枝杆菌CFP10-ESAT6融合蛋白对细胞增殖和凋亡的影响

 目的：建立培养滤液蛋白10-早期分泌性抗原靶6（CFP10-ESAT6）真核表达质粒并转染RAW2647巨噬细胞,观察胞内表达CFP10-ESAT6对细胞活性及凋亡的影响,并初步探讨其机制。方法：将CFP10-ESAT6融合基因插入真核表达质粒pEGFP-N1,构建重组质粒,转染RAW264.7巨噬细胞。采用MTT的方法测定CFP10-ESAT6融合蛋白对RAW264.7巨噬细胞活性的影响,采用结核分枝杆菌19 kD脂蛋白和十字孢碱（staurosporine）处理RAW264.7巨噬细胞,并以流式细胞术检测细胞凋亡率以及Toll样受体2（TLR2）的表达。结果：成功构建了重组质粒pEGFP-N1/CFP10-ESAT6并转染至RAW264.7巨噬细胞;与对照组相比,胞内表达CFP10-ESAT6不能影响巨噬细胞的活性,但可以明显抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡(P＜0.05),并显著抑制了TLR2的表达(P＜0.05)。结论：巨噬细胞内表达的CFP10-ESAT6融合蛋白不具有细胞毒性作用,但可以通过下调TLR2的表达来抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡。     

Phosphatidylcholine-specific phospholipase C regulates activation of RAW264.7 macrophage-like cells by lipopeptide JBT3002

Phospholipase activities are thought to be involved in the activation of macrophages by lipopolysaccharide (LPS). Because our previous studies showed that the synthetic lipopeptide JBT3002 might activate macrophages via signaling pathways similar to those used by LPS, we investigated whether phospholipase activities are required for activation of macrophages by JBT3002. Treatment of RAW264.7 murine macrophage-like cells with JBT3002 stimulated expression of both inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner. The JBT3002-induced production of nitric oxide and TNF-alpha was significantly inhibited by tricyclodecan-9-yl xanthogenate (D609), a selective inhibitor of phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). JBT3002-induced expression of steady-state mRNA for both iNOS and TNF-alpha was inhibited by D609. Cells treated with JBT3002 had greater production of diacylglycerol (DAG) in 2 min, which lasted for at least 30 min and could be blocked by D609. Activation of RAW264.7 cells was not affected by butanol, a PC-specific phospholipase D inhibitor, and treatment with JBT3002 did not affect phosphatidic acid formation. RAW264.7 cells treated with DAG analogue 1-oleoyl-2-acetyl-sn-glycerol, in the presence of interferon-gamma, produced TNF-alpha. These results suggested that activation of RAW264.7 cells by JBT3002 requires PC-PLC activity.