Sample records for 3t3 feeder layer

  1. Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

    PubMed Central

    1982-01-01

    Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes. PMID:6182145

  2. Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

    PubMed Central

    Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

    2015-01-01

    Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum. PMID:25799110

  3. Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.

    PubMed

    Scafetta, Gaia; Siciliano, Camilla; Frati, Giacomo; De Falco, Elena

    2015-01-01

    The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems. PMID:25063497

  4. Layer-by-layer assembly of peptide based bioorganic-inorganic hybrid scaffolds and their interactions with osteoblastic MC3T3-E1 cells.

    PubMed

    Romanelli, Steven M; Fath, Karl R; Phekoo, Aruna P; Knoll, Grant A; Banerjee, Ipsita A

    2015-06-01

    In this work we have developed a new family of biocomposite scaffolds for bone tissue regeneration by utilizing self-assembled fluorenylmethyloxycarbonyl protected Valyl-cetylamide (FVC) nanoassemblies as templates. To tailor the assemblies for enhanced osteoblast attachment and proliferation, we incorporated (a) Type I collagen, (b) a hydroxyapatite binding peptide sequence (EDPHNEVDGDK) derived from dentin sialophosphoprotein and (c) the osteoinductive bone morphogenetic protein-4 (BMP-4) to the templates by layer-by-layer assembly. The assemblies were then incubated with hydroxyapatite nanocrystals blended with varying mass percentages of TiO2 nanoparticles and coated with alginate to form three dimensional scaffolds for potential applications in bone tissue regeneration. The morphology was examined by TEM and SEM and the binding interactions were probed by FITR spectroscopy. The scaffolds were found to be non-cytotoxic, adhered to mouse preosteoblast MC3T3-E1 cells and promoted osteogenic differentiation as indicated by the results obtained by alkaline phosphatase assay. Furthermore, they were found to be biodegradable and possessed inherent antibacterial capability. Thus, we have developed a new family of tissue-engineered biocomposite scaffolds with potential applications in bone regeneration. PMID:25842141

  5. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

    PubMed

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-12-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines. PMID:24807816

  6. Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

    PubMed

    Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

    2013-12-01

    Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63?), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63?(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ?Np63? than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-?3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture. PMID:23832306

  7. Inner ear stem cells derived feeder layer promote directional differentiation of amniotic fluid stem cells into functional neurons.

    PubMed

    Zong, Ling; Chen, Kaitian; Zhou, Wei; Jiang, Di; Sun, Liang; Zhang, Xuemei; Jiang, Hongyan

    2014-10-01

    Intact spiral ganglion neurons are required for cochlear implantation or conventional hearing amplification as an intervention for sensorineural hearing loss. Treatment strategies to replace the loss of spiral ganglion neurons are needed. Recent reports have suggested that amniotic fluid-derived stem cells are capable of differentiating into neuron-like cells in response to cytokines and are not tumorigenic. Amniotic fluid stem cells represent a potential resource for cellular therapy of neural deafness due to spiral ganglion pathology. However, the directional differentiation of amniotic fluid stem cells is undetermined in the absence of cytokines and the consequence of inner ear supporting cells from the mouse cochlea organ of Corti on the differentiation of amniotic fluid stem cells remains to be defined. In an effort to circumvent these limitations, we investigated the effect of inner ear stem cells derived feeder layer on amniotic fluid stem cells differentiation in vitro. An inner ear stem cells derived feeder layer direct contact system was established to induce differentiation of amniotic fluid stem cells. Our results showed that inner ear stem cells derived feeder layer successfully promoted directional differentiation of amniotic fluid stem cells into neurons with characteristics of functionality. Furthermore, we showed that Wnt signaling may play an essential role in triggering neurogenesis. These findings indicate the potential use of inner ear stem cells derived feeder layer as a nerve-regenerative scaffold. A reliable and effective amniotic fluid stem cell differentiation support structure provided by inner ear stem cells derived feeder layer should contribute to efforts to translate cell-based strategies to the clinic. PMID:25124154

  8. Bird Feeders

    NSDL National Science Digital Library

    Lawrence Hall of Science

    2010-01-01

    In this activity, learners build a bird feeder or feeders to attract birds for observation. The main feeder to be built is made from a large milk carton, but a suggestion is also included for a simple flat box or can lid feeder. Adult assistance or supervision may be needed depending on materials used to construct and hang the feeder.

  9. 3T3 cells in adipocytic conversion.

    PubMed

    O'Shea Alvarez, M S

    1991-01-01

    3T3 are murine cells of an established heteroploid cellular line. Some clones of this cellular line, when cultured under adequate conditions differentiate into adipocytes. During the process of differentiation, the cells undergo a change from the elongated fibroblastic shape to a round or oval form and accumulate small drops of lipids within their cytoplasma. These lipid drops fuse into one large drop which displaces the nucleus towards the periphery, giving the cell the aspect of a mature adipocyte of white adipose tissue. The cells not only change their morphology, but they also present important biochemical changes. They show a simultaneous increase in triglyceride synthesis and activity of lipogenic enzymes. There is also an increase in the response of the activity of various hormones and the de novo synthesis of the receptors to such hormones, as insulin and ACTH. During the process of differentiation important changes occur in the synthesis of various proteins, such as actin, tubulin, and other proteins which also make up the cellular cytoskeleton, forming part of the lipid transportation within the adipose cell. The adipocytic differentiation of 3T3 cells depends on adipogenic serum factors used in the supplementary culture medium. These adipogenic factors seem to play an important role in the development of adipose tissue. There are hormones, chemical agents and serum factors which modulate adipocytic differentiation. The clone must be susceptible to adipocytic differentiation, it must reach a quiescent state and find itself in adipogenic conditions for the 3T3 cells to differentiate into adipocytes. It must also carry out an DNA synthesis which is an expression of the new phenotype. The differentiation of 3T3 cells in terminal. The fact that these cells present an adipocytic conversion under physiologic conditions and with adipogenic hormones which exist in the whole animal has been demonstrated. All of these characteristics show that the 3T3 cells may be used as an adequate experimental system to analyze the events which occur during the differentiation and development of adipose tissue. PMID:1726451

  10. Human amniotic epithelial cell feeder layers maintain iPS cell pluripotency by inhibiting endogenous DNA methyltransferase 1

    PubMed Central

    CHEN, QING; QIU, CHAOLIN; HUANG, YONGYI; JIANG, LIZHEN; HUANG, QIN; GUO, LIHE; LIU, TE

    2013-01-01

    Maintaining induced pluripotent stem (iPS) cells in an undifferentiated, self-renewing state during long-term cultivation is, at present, a major challenge. We previously showed that human amniotic epithelial cells (HuAECs) were able to provide a good source of feeder cells for mouse and human embryonic or spermatogonial stem cells; however, the epigenetic mechanisms have not been elucidated. In the present study, mouse embryonic fibroblasts (MEFs) and HuAECs were compared as feeder layers for the long-term culture of human iPS cells. The HuAEC feeders allowed human iPS cells to maintain a high level of alkaline phosphatase (AP) activity and to express key stem cell markers during long-term subculture whereas the MEF feeders did not,. Moreover, the HuAEC feeders significantly affected the cell cycle regulation of the iPS cells, maintaining them in the resting stage and the early stage of DNA synthesis (G0/G1 stage). Furthermore, the CpG islands of the Nanog and Oct4 promoters were hypomethylated, while the Nanog- and Oct4-specific loci exhibited higher levels of histone H3 acetylation and lower levels of H3K27 trimethylation in iPS cells cultured on HuAECs compared with those cultured on MEFs. The DNA methyltransferase 1 (DNMT1) expression in iPS cells cultured on HuAECs was shown to be lower than in those cultured on MEFs. In addition, DNMT1-silenced human iPS cells were able to maintain pluripotency over long-term culture on MEFs. In combination, these results suggest that endogenous DNMT1 expression in human iPS cells may be regulated by HuAEC feeder cells and that Nanog and Oct4 are crucial components required for the maintenance of iPS cells in an undifferentiated, proliferative state, capable of self-renewal. PMID:24223636

  11. Human amniotic epithelial cell feeder layers maintain iPS cell pluripotency by inhibiting endogenous DNA methyltransferase 1.

    PubMed

    Chen, Qing; Qiu, Chaolin; Huang, Yongyi; Jiang, Lizhen; Huang, Qin; Guo, Lihe; Liu, Te

    2013-11-01

    Maintaining induced pluripotent stem (iPS) cells in an undifferentiated, self-renewing state during long-term cultivation is, at present, a major challenge. We previously showed that human amniotic epithelial cells (HuAECs) were able to provide a good source of feeder cells for mouse and human embryonic or spermatogonial stem cells; however, the epigenetic mechanisms have not been elucidated. In the present study, mouse embryonic fibroblasts (MEFs) and HuAECs were compared as feeder layers for the long-term culture of human iPS cells. The HuAEC feeders allowed human iPS cells to maintain a high level of alkaline phosphatase (AP) activity and to express key stem cell markers during long-term subculture whereas the MEF feeders did not,. Moreover, the HuAEC feeders significantly affected the cell cycle regulation of the iPS cells, maintaining them in the resting stage and the early stage of DNA synthesis (G0/G1 stage). Furthermore, the CpG islands of the Nanog and Oct4 promoters were hypomethylated, while the Nanog- and Oct4-specific loci exhibited higher levels of histone H3 acetylation and lower levels of H3K27 trimethylation in iPS cells cultured on HuAECs compared with those cultured on MEFs. The DNA methyltransferase 1 (DNMT1) expression in iPS cells cultured on HuAECs was shown to be lower than in those cultured on MEFs. In addition, DNMT1-silenced human iPS cells were able to maintain pluripotency over long-term culture on MEFs. In combination, these results suggest that endogenous DNMT1 expression in human iPS cells may be regulated by HuAEC feeder cells and that Nanog and Oct4 are crucial components required for the maintenance of iPS cells in an undifferentiated, proliferative state, capable of self-renewal. PMID:24223636

  12. Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells.

    PubMed

    Lim, Justin Wee Eng; Bodnar, Andrea

    2002-09-01

    Human embryonic stem (ES) cells are pluripotent cells with the potential to differentiate into a variety of cell types, which could be used for cell transplantation therapies as well as drug discovery studies. However, the large-scale culture of undifferentiated human ES cells is currently limited by their dependency on mouse embryonic fibroblast feeder layers. The proteomics approach was employed to characterize the environment that supports the growth of undifferentiated human ES cells and to identify factors critical for their independent growth. Conditioned medium from mouse embryonic fibroblast feeder layers, STO cell line, was concentrated and subjected to analyses by two-dimensional electrophoresis mass spectrometry. In total, 136 unique protein species were identified which included some that are known to participate in cell growth and differentiation, extracellular matrix formation and remodeling, in addition to the unexpected but interesting finding of many nominally intracellular proteins. This approach has thus revealed the complexity of the environment provided by the feeder cells and provides a useful starting point for future studies. Moreover, candidates from the initial list of identified proteins can be further investigated for their effects on the growth and differentiation of human ES cells in a defined culture environment. PMID:12362336

  13. Bird Feeder

    NSDL National Science Digital Library

    Lawrence Hall of Science

    1979-01-01

    In this outdoor activity, learners construct bird feeders and set them up at to investigate bird behavior for one or two weeks. Multiple feeder designs are suggested. Learners are encouraged to observe how different birds approach the feeder, whether any birds bully other birds, what foods visiting birds eat, how often and at what times of day different birds visit, and how birds react to models of other birds on the feeder.

  14. The use of human amniotic fluid mesenchymal stem cells as the feeder layer to establish human embryonic stem cell lines.

    PubMed

    Soong, Yung-Kwei; Huang, Shang-Yu; Yeh, Chiu-Hsiang; Wang, Tzu-Hao; Chang, Kuo-Hsuan; Cheng, Po-Jen; Shaw, S W Steven

    2013-03-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into the three germ layers and possibly all tissues of the human body. To fulfil the clinical potentials for cell-based therapy, banks of hESC lines that express different combinations of the major histocompatibility genes should be established, preferably without exposing such cells to animal cells and proteins. In this study, we tested human amniotic fluid mesenchymal stem cells (AFMSCs) as feeder cells to support the growth of hESCs. Our results indicated that mitomycin-treated AFMSCs were able to support the newly established hESC lines CGLK-1 and CGLK-2. The hESC colonies cultured on AFMSCs expressed alkaline phosphatase (ALK-P), SSEA-4, TRA-1-60, TRA-1-81, Oct-4, Nanog and Sox-2, which are markers for undifferentiated hESCs. Chromosomal analyses of both hESC lines, CGLK-1 and CGLK-2, which were cultured on AFMSC feeders for 22 and 14 passages, respectively, were confirmed to be normal karyotypes (46, XX). The ability of AFMSCs as feeder cells to maintain the undifferentiated growth and pluripotency of hESCs was confirmed by in vivo formation of teratomas derived on AFMSC hESCs in severe combined immune-compromised mice. The use of AFMSCs for feeder cells to culture hESCs has several advantages, in that AFMSCs are not tumourigenic and can be expanded extensively with a short doubling time. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23460275

  15. Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety.

    PubMed

    De Corte, Peter; Verween, Gunther; Verbeken, Gilbert; Rose, Thomas; Jennes, Serge; De Coninck, Arlette; Roseeuw, Diane; Vanderkelen, Alain; Kets, Eric; Haddow, David; Pirnay, Jean-Paul

    2012-03-01

    Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process. PMID:21394485

  16. Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

    SciTech Connect

    Jeong, Hyun Jeong [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)] [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Park, Sahng Wook [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)] [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Kim, Hojeong [Department of Anatomy, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)] [Department of Anatomy, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Park, Sang-Kyu, E-mail: 49park@kd.ac.kr [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)] [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Yoon, Dojun, E-mail: mozart@kd.ac.kr [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)] [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)

    2010-02-19

    Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor {gamma}2, CCAAT/enhancer-binding protein alpha (C/EBP{alpha}), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBP{beta} or C/EBP{delta}, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

  17. Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells.

    PubMed

    Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

    2010-02-19

    Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor gamma2, CCAAT/enhancer-binding protein alpha (C/EBPalpha), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPbeta or C/EBPdelta, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes. PMID:20096664

  18. The 3T3-L1 adipocyte glycogen proteome

    PubMed Central

    2013-01-01

    Background Glycogen is a branched polysaccharide of glucose residues, consisting of ?-1-4 glycosidic linkages with ?-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. Results Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using ?-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). Conclusions These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle. PMID:23521774

  19. Trypanosoma brucei: five commonly used trypanocides assayed in vitro with a mammalian feeder layer system for cultivation of bloodstream forms.

    PubMed

    Borowy, N K; Fink, E; Hirumi, H

    1985-12-01

    The in vitro activity of five commonly used trypanocides on bloodstream forms of Trypanosoma brucei brucei TC 221 was examined in 24-well culture plates in the presence of bovine fibroblast feeder layer cells. The minimum effective concentrations determined were as follows: Berenil 1.0 microgram/ml; Samorin 10.0 micrograms/ml; Antrycide dimethylsulfate 0.1 microgram/ml; Arsobal 0.01 microgram/ml; Naganol 1.0 microgram/ml. Contrary to values obtained with other in vitro assays, minimum effective concentrations obtained here were within the range of drug levels reached in blood, plasma, or serum of humans and animals after treatment with curative doses. The trypanocidal activity of Naganol in this assay was of particular interest, since Naganol has been hitherto considered to be inactive in vitro. PMID:4076388

  20. Culture of cells derived from the human sebaceous gland under serum-free conditions without a biological feeder layer or specific matrices

    Microsoft Academic Search

    Takeshi Fujie; Takanori Shikiji; Naoyuki Uchida; Yoshio Urano; Hiroaki Nagae; Seiji Arase

    1996-01-01

    We succeeded in serially culturing cells derived from human sebaceous gland (sebocytes) under serum-free conditions. Sebaceous\\u000a glands were isolated from dispase-treated facial skin specimens and cultured using two different methods, explant culture\\u000a and dispersed cell culture, in KGM. In both types of culture the sebocytes proliferated rapidly without a biological feeder\\u000a layer or specific matrices. It was possible to cultivate

  1. Feeder Ninja

    NSDL National Science Digital Library

    Are you looking to create beautiful and elegant RSS and social feeds? Feeder Ninja can make this happen in just a few steps. On the Features section, visitors can learn about how to create attractive feed widgets for their site, along with details about how to insert the necessary code. The Examples area contains a host of recently crafted feeds and there's a helpful FAQ area. This version is compatible with all operating systems, including Linux.

  2. Melatonin promotes differentiation of 3T3-L1 fibroblasts.

    PubMed

    González, Alicia; Alvarez-García, Virginia; Martínez-Campa, Carlos; Alonso-González, C; Cos, Samuel

    2012-01-01

    Melatonin inhibits the genesis and growth of breast cancer by interfering at different levels in the estrogen-signaling pathways. Melatonin inhibits aromatase activity and expression in human breast cancer cells, thus behaving as a selective estrogen enzyme modulator. As the adipose tissue adjacent to the tumor seems to account for most aromatase expression and enzyme activity in breast tumors and also mediates the desmoplastic reaction or accumulation of undifferentiated fibroblasts around malignant epithelial cells, in this work, we studied the effects of melatonin on the conversion of preadipocytes (3T3-L1) into adipocytes and on the capability of these cells to synthesize estrogens by regulating the expression and enzyme activity of aromatase, one of the main enzymes that participates in the synthesis of estrogens in the peritumoral adipose tissue. Thus, in both differentiating and differentiated 3T3-L1 adipocytes, high concentrations of melatonin increased intracytoplasmic triglyceride accumulation, an indicator of adipogenic differentiation. Melatonin (1 mm) significantly increased the expression of both CCAAT/enhancer-binding protein ? and peroxisome proliferator-activated receptor ?, two main regulators of terminal adipogenesis, in 3T3-L1 cells. The presence of melatonin during differentiation also induced a parallel reduction in aromatase expression and activity and expression of the cells. The effects of melatonin were reversed by luzindole, a melatonin receptor antagonist, indicating that melatonin acts through known receptor-mediated mechanisms. These findings suggest that, in human breast tumors, melatonin could stimulate the differentiation of fibroblasts and reduce the aromatase activity and expression in both fibroblasts and adipocytes, thereby reducing the number of estrogen-producing cells proximal to malignant cells. PMID:21718362

  3. Cell adhesion and cell surface topography in aggregates of 3T3 and SV40- virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy

    PubMed Central

    1978-01-01

    A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants. PMID:632324

  4. Regulated renin release from 3T3-L1 adipocytes

    PubMed Central

    Fowler, Jason D.; Johnson, Nathan D.; Haroldson, Thomas A.; Brintnall, Joy A.; Herrera, Julio E.; Katz, Stephen A.; Bernlohr, David A.

    2009-01-01

    Whereas adipose tissue possesses a local renin-angiotensin system, the synthesis and regulated release of renin has not been addressed. To that end, we utilized differentiating 3T3-L1 cells and analyzed renin expression and secretion. Renin mRNA expression and protein enzymatic activity were not detectable in preadipocytes. However, upon differentiation, renin mRNA and both intracellular and extracellular renin activity were upregulated. In differentiated adipocytes, forskolin treatment resulted in a 28-fold increase in renin mRNA, whereas TNF? treatment decreased renin mRNA fourfold. IL-6, insulin, and angiotensin (Ang) II were without effect. In contrast, forskolin and TNF? each increased renin protein secretion 12- and sevenfold, respectively. Although both forskolin and TNF? induce lipolysis in adipocytes, fatty acids, prostaglandin E2, and lipopolysaccharide had no effect on renin mRNA or secretion. To evaluate the mechanism(s) by which forskolin and/or TNF? are able to regulate renin secretion, a general lipase inhibitor (E600) and PKA inhibitor (H89) were used. Both inhibitors attenuated forskolin-induced renin release, whereas they had no effect on TNF?-regulated secretion. In contrast, E600 potentiated forskolin-stimulated renin mRNA levels, whereas H89 had no effect. Neither inhibitor had any influence on TNF? regulation of renin mRNA. Relative to lean controls, renin expression was reduced 78% in the epididymal adipose tissue of obese male C57Bl/6J mice, consistent with TNF?-mediated downregulation of renin mRNA in the culture system. In conclusion, the expression and secretion of renin are regulated under a complex series of hormonal and metabolic determinants in mature 3T3-L1 adipocytes. PMID:19293336

  5. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    SciTech Connect

    Liu, Te, E-mail: liute79@yahoo.com [School of Environmental Science and Engineering, Donghua University, Shanghai 201620 (China) [School of Environmental Science and Engineering, Donghua University, Shanghai 201620 (China); Shanghai Geriatric Institute of Chinese Medicine, Shanghai 200031 (China); Cheng, Weiwei [International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030 (China)] [International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030 (China); Huang, Yongyi [Laboratoire PROTEE, Batiment R, Universite du Sud Toulon-Var, 83957 LA GARDE Cedex (France)] [Laboratoire PROTEE, Batiment R, Universite du Sud Toulon-Var, 83957 LA GARDE Cedex (France); Huang, Qin; Jiang, Lizhen [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)] [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Guo, Lihe, E-mail: liute79@yahoo.com [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)] [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2012-02-15

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: Black-Right-Pointing-Pointer microRNA-145 inhibits Sox2 expression in human iPS cells. Black-Right-Pointing-Pointer microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. Black-Right-Pointing-Pointer HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. Black-Right-Pointing-Pointer HuAECs feeder layers maintain human iPS cells pluripotency. Black-Right-Pointing-Pointer HuAECs negatively regulates the synthesis of primary precursor miRNA in human iPS.

  6. Alteration of glycolipids in ras-transfected NIH 3T3 cells.

    PubMed Central

    Matyas, G R; Aaronson, S A; Brady, R O; Fishman, P H

    1987-01-01

    Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. We analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, we found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by 125I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1a as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, we found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. Interestingly, gangliotriaosylceramide appeared when the latter cells became confluent. These results indicated that ras oncogenes derived from human tumors are capable of inducing alterations in glycolipid composition. Images PMID:3476926

  7. Regenerated silk fibroin scaffold and infrapatellar adipose stromal vascular fraction as feeder-layer: a new product for cartilage advanced therapy.

    PubMed

    Chlapanidas, Theodora; Faragň, Silvio; Mingotto, Federica; Crovato, Francesca; Tosca, Marta Cecilia; Antonioli, Barbara; Bucco, Massimo; Lucconi, Giulia; Scalise, Alessandro; Vigo, Daniele; Faustini, Massimo; Marazzi, Mario; Torre, Maria Luisa

    2011-07-01

    Articular cartilage has limited repair and regeneration potential, and the scarcity of treatment modalities has motivated attempts to engineer cartilage tissue constructs. The use of chondrocytes in cartilage tissue engineering has been restricted by the limited availability of these cells, their intrinsic tendency to lose their phenotype during the expansion, as well as the difficulties during the first cell adhesion to the scaffold. Aim of this work was to evaluate the intra-articular adipose stromal vascular fraction attachment on silk fibroin scaffold to promote chondrocytes adhesion and proliferation. Physicochemical characterization has demonstrated that three-dimensionally organized silk fibroin scaffold is an ideal biopolymer for cartilage tissue engineering; it allows cell attachment, scaffold colonization, and physically cell holding in the area that must be repaired; the use of adipose-derived stem cells is a promising strategy to promote adhesion and proliferation of chondrocytes to the scaffold as an autologous human feeder layer. PMID:21338265

  8. Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6).

    PubMed

    Dormady, S P; Zhang, X M; Basch, R S

    2000-10-24

    Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively gamma-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be gamma-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells. PMID:11050245

  9. Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

    SciTech Connect

    Lai, Peng-Yeh [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China)] [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China); Tsai, Chong-Bin [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China) [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China); Department of Ophthalmology, Chiayi Christian Hospital, Chiayi 600, Taiwan, ROC (China); Tseng, Min-Jen, E-mail: biomjt@ccu.edu.tw [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China)] [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China)

    2013-01-18

    Highlights: ? Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ? Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ? Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ? Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBP? and PPAR? was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBP?, PPAR?, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  10. 95. VIEW OF ZINC FEEDER FROM SOUTHEAST. NOTE FEEDER CONE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    95. VIEW OF ZINC FEEDER FROM SOUTHEAST. NOTE FEEDER CONE AND PIPING FROM VACUUM RECEIVER ON LEFT. PRECIPITATE PUMP MOTOR MOUNT VISIBLE BELOW FEEDER STAIRS, PUMP AND MOTOR MISSING. SUMPS ARE LOCATED UNDER THIS FLOOR, WITH ACCESS TO HATCH TO THE RIGHT OF FEEDER STAIR. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

  11. Feederism in a woman.

    PubMed

    Terry, Lesley L; Vasey, Paul L

    2011-06-01

    Feederism is a fat fetish subculture in which individuals eroticize weight gain and feeding. Feeders are individuals who claim to become sexually aroused by feeding their partners and encouraging them to gain weight. Conversely, Feedees are individuals who claim to become sexually aroused by eating, being fed, and the idea or act of gaining weight. Very little is known about this population. This report describes a woman who self-identified as a Feedee. It is unclear, at present, whether female Feederism represents a unique paraphilia or a thematic variation of morphophilia or sexual masochism. PMID:20041284

  12. Resveratrol Stimulates the Proliferation and Differentiation of Osteoblastic MC3T3-E1 Cells

    Microsoft Academic Search

    Kenichi Mizutani; Katsumi Ikeda; Yasuhiro Kawai; Yukio Yamori

    1998-01-01

    Nutritional and pharmacological factors are needed to prevent bone loss that occurs with increasing age. The chemical compounds that act on bone metabolism as nutrients in food, however, are poorly understood. The effect of resveratrol, a natural phytoestrogen, on the proliferation and differentiation of osteoblastic MC3T3-E1 cells was studied. Resveratrol dose-dependently increased DNA synthesis (10?9?10?7M) of MC3T3-E1 cells. In addition,

  13. Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines

    Microsoft Academic Search

    Ming?Chung Jiang

    1996-01-01

    Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene?transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT?29,

  14. Inhibition by insulin of resistin gene expression in 3T3-L1 adipocytes

    Microsoft Academic Search

    Fred Haugen; Aud Jřrgensen; Christian A. Drevon; Paul Trayhurn

    2001-01-01

    Expression of the gene encoding resistin, a low molecular weight protein secreted from adipose tissue postulated to link obesity and type II diabetes, was examined in 3T3-L1 adipocytes. Resistin mRNA was detected in 3T3-L1 cells by day 3 following induction of differentiation into adipocytes; by day 4 the level of resistin mRNA peaked and remained high. The PPAR? activators, rosiglitazone

  15. Pear pomace water extract inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes

    PubMed Central

    Rhyu, Jin; Kim, Min Sook; You, Mi-Kyoung; Bang, Mi-Ae

    2014-01-01

    Obesity occurs when a person's calorie intake exceeds the amount of energy burns, which may lead to pathologic growth of adipocytes and the accumulation of fat in the tissues. In this study, the effect and mechanism of pear pomace extracts on 3T3-L1 adipocyte differentiation and apoptosis of mature adipocytes were investigated. The effects of pear pomace extract on cell viability and the anti-adipogenic and proapoptotic effects were investigated via MTT assay, Oil red O staining, western blot analysis and apoptosis assay. 3T3-L1 preadipocytes were stimulated with DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 µg/ml insulin and 1 µM dexamethasone for differentiation to adipocytes. 3T3-L1 cells were cultured with PBS or water extract of pear pomace. Water extract of pear pomace effectively inhibited lipid accumulations and expressions of PPAR-? and C/EBP? in 3T3-L1 cells. It also increased expression of p-AMPK and decreased the expression of SREBP-1c and FAS in 3T3-L1 cells. The induction of apoptosis was observed in 3T3-L1 cells treated with pear pomace. These results indicate that pear pomace water extract inhibits adipogenesis and induces apoptosis of adipocytes and thus can be used as a potential therapeutic substance as part of prevention or treatment strategy for obesity. PMID:24611103

  16. Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

    PubMed Central

    Powers, S; Fisher, P B; Pollack, R

    1984-01-01

    We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two growth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the PDGF requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by measuring the PDGF requirement at different cell densities. At high cell densities, the requirement for PDGF became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF. PMID:6092919

  17. Pear pomace water extract inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes.

    PubMed

    Rhyu, Jin; Kim, Min Sook; You, Mi-Kyoung; Bang, Mi-Ae; Kim, Hyeon-A

    2014-02-01

    Obesity occurs when a person's calorie intake exceeds the amount of energy burns, which may lead to pathologic growth of adipocytes and the accumulation of fat in the tissues. In this study, the effect and mechanism of pear pomace extracts on 3T3-L1 adipocyte differentiation and apoptosis of mature adipocytes were investigated. The effects of pear pomace extract on cell viability and the anti-adipogenic and proapoptotic effects were investigated via MTT assay, Oil red O staining, western blot analysis and apoptosis assay. 3T3-L1 preadipocytes were stimulated with DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 µg/ml insulin and 1 µM dexamethasone for differentiation to adipocytes. 3T3-L1 cells were cultured with PBS or water extract of pear pomace. Water extract of pear pomace effectively inhibited lipid accumulations and expressions of PPAR-? and C/EBP? in 3T3-L1 cells. It also increased expression of p-AMPK and decreased the expression of SREBP-1c and FAS in 3T3-L1 cells. The induction of apoptosis was observed in 3T3-L1 cells treated with pear pomace. These results indicate that pear pomace water extract inhibits adipogenesis and induces apoptosis of adipocytes and thus can be used as a potential therapeutic substance as part of prevention or treatment strategy for obesity. PMID:24611103

  18. Mitigative Effect of Erythromycin on PMMA Challenged Preosteoblastic MC3T3-E1 Cells

    PubMed Central

    Shen, Yi; Wang, Weili; Li, Xiaomiao; Markel, David C.; Ren, Weiping

    2014-01-01

    Background. Aseptic loosening (AL) is a major complication of total joint replacement. Recent approaches to limiting AL have focused on inhibiting periprosthetic inflammation and osteoclastogenesis. Questions/Purposes. The purpose of this study was to determine the effects of erythromycin (EM) on polymethylmethacrylate (PMMA) particle-challenged MC3T3 osteoblast precursor cells. Methods. MC3T3 cells were pretreated with EM (0–10??g/mL) and then stimulated with PMMA (1?mg/mL). Cell viability was evaluated by both a lactate dehydrogenase (LDH) release assay and cell counts. Cell differentiation was determined by activity of alkaline phosphatase (ALP). Gene expression was measured via real-time quantitative RT-PCR. Results. We found that exposure to PMMA particles reduced cellular viability and osteogenetic potential in MC3T3 cell line. EM treatment mitigated the effects of PMMA particles on the proliferation, viability and differentiation of MC3T3 cells. PMMA decreased the gene expression of Runx2, osterix and osteocalcin, which can be partially restored by EM treatment. Furthermore, EM suppressed PMMA- induced increase of NF-?B gene expression. Conclusions. These data demonstrate that EM mitigates the effects of PMMA on MC3T3 cell viability and differentiation, in part through downregulation of NF-?B pathway. EM appeared to represent an anabolic agent on MC3T3 cells challenged with PMMA particles. PMID:25110723

  19. Low Spillage Metabolic Feeder

    NASA Technical Reports Server (NTRS)

    Evans, JuliAnn (Inventor); Gundo, Daniel P. (Inventor); Harper, Jennifer S. (Inventor); Mulenburg, Gerald M. (Inventor); Skundberg, Thomas L. (Inventor)

    1996-01-01

    An animal feeder for use in a metabolic cage is introduced. The feeder includes a confined passageway and an adjustable notched gate proceeding a food cup. The gate is adjusted so that the entry area to the food cup approximates the cross sectional head area of the animal. Food ejected from the food cup by a caged animal is dropped through a grate into a spill tray.

  20. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    NASA Astrophysics Data System (ADS)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5?Hz for 24 and 48?h. The cultured cells were treated with a low-level diode laser using powers of 5?J and 10?J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5?J and 10?J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5?J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5?J and 10?J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  1. Isolation of adult progenitor cells with neuronal potential from rabbit corneal epithelial cells in serum- and feeder layer-free culture conditions

    PubMed Central

    Yamagami, Satoru; Uchida, Saiko; Yokoo, Seiichi; Ono, Kyoko; Usui, Tomohiko; Amano, Shiro

    2010-01-01

    Purpose To isolate progenitor cells from rabbit corneal epithelial cells (CEC) in serum- and feeder layer-free culture conditions and to compare the self-renewal capacity of corneal epithelial progenitor cells obtained from the central and limbal regions of the cornea. Methods Tissue samples of New Zealand white rabbit corneas were dissected from the limbal and central regions to obtain CEC for sphere-forming culture, in which the cells formed spheres in serum-free medium containing growth factors. The number of primary and secondary sphere colonies and the size of the primary spheres were compared between the limbal and central regions. To promote differentiation, isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/laminin. The expression of epithelial, neural, and mesenchymal mRNAs was examined in the sphere colonies and their progeny by immunocytochemistry and/or the reverse transcription–polymerase chain reaction (RT–PCR). Adherent differentiated cells from the sphere colonies were also examined morphologically. Results Primary spheres were isolated from both the limbal and central regions of the cornea. The rate of primary sphere formation by CEC from the limbal region (55.6±10.6/10,000 cells) was significantly higher than that by cells from the central cornea (43.1±7.2/10,000 cells, p=0.0028), but there was no significant difference in the size of primary spheres derived from both regions. The self-renewal capacity of cells from the limbal region was higher than that of cells from the central region, as evidenced by the significantly higher secondary sphere formation rate of limbal cells (38.7±8.5/10,000 cells) in comparison with that for central cells (31.3±5.7/10,000 cells, p=0.013). The primary sphere colonies expressed bromodeoxyuridine (BrdU), a 63-kDa protein (p63), p75 neurotrophin receptor (p75NTR), and nestin, whereas their progeny expressed cytokeratin 3, cytokeratin 12, vimentin, ?-smooth muscle actin, microtubule-associated protein 2, and neuron-specific enolase on immunocytochemical analysis. These markers were confirmed by RT–PCR. Conclusions Our findings indicate that limbal CEC contain more progenitor cells with a stronger self-renewal capacity than cells from the central region. These progenitor cells differentiate into the epithelial lineage, and can also produce neuronal protein. PMID:20806049

  2. Epac, not PKA catalytic subunit, is required for 3T3-L1 preadipocyte differentiation.

    PubMed

    Ji, Zhenyu; Mei, Fang C; Cheng, Xiaodong

    2010-01-01

    Cyclic AMP plays a critical role in adipocyte differentiation and maturation. However, it is not clear which of the two intracellular cAMP receptors, exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor or protein kinase A/cAMP-dependent protein kinase, is essential for cAMP-mediated adipocyte differentiation. In this study, we utilized a well-defined adipose differentiation model system, the murine preadipocyte line 3T3-L1, to address this issue. We showed that knocking down Epac expression in 3T3-L1 cells using lentiviral based small hairpin RNAs down-regulated peroxisome proliferator-activated receptor gamma expression and dramatically inhibited adipogenic conversion of 3T3-L1 cells while inhibiting PKA catalytic subunit activity by two mechanistically distinct inhibitors, heat stable protein kinase inhibitor and H89, had no effect on 3T3-L1 adipocyte differentiation. Moreover, cAMP analog selectively activating Epac was not able to stimulate adipogenic conversion. Our study demonstrated that while PKA catalytic activity is dispensable, activation of Epac is necessary but not sufficient for adipogenic conversion of 3T3-L1 cells. PMID:20036887

  3. Metabolism of vitamin K in Swiss 3T3 mouse fibroblasts

    E-print Network

    Johnson, Terryl Marie

    1985-01-01

    Major Subject: Nutrition METABOLISM OF VITAMIN K IN SWISS 3T3 MOUSE PIBROBLASTS A Thesis by TERRYL MARIE JOHNSON Approved as to style and content by: Dr, Louise Mo enfield-Sander (Chair of Committee) Dr Gary CD Smith (Head of Department) D ~ J... Committee: Dr. Louise M. Canfield-Sander The absorption and metabolism of vitamin K was investigated in a model system of Swiss 3T3 mouse fibroblasts ~ Uptake of vitamin K into cells followed saturation kinetics' Vitamin E (0 05-0 ' 2 mM) stimulated...

  4. PACAP up-regulates the expression of apolipoprotein D in 3T3-L1 adipocytes. DRG/3T3-L1 co-cultures study.

    PubMed

    Kosacka, Joanna; Schröder, Thomas; Bechmann, Ingo; Klöting, Nora; Nowicki, Marcin; Mittag, Anja; Gericke, Martin; Spanel-Borowski, Katharina; Blüher, Matthias

    2011-01-01

    The existence of a cross-talk between nerves and fatty tissue is increasingly recognized. Using co-cultures of dorsal root ganglion (DRG)-derived cells and 3T3-L1 adipocytes, we have previously shown that the presence of fat cells enhances neurite outgrowth and number of synapses. Vice versa, neural cells induced expression of neurotrophic adipokines apolipoprotein D and E (ApoD, ApoE) and angiopoietin-1 (Ang-1) by adipocytes. Here, we tested whether pituitary adenylate cyclase-activating peptide (PACAP), which is released by sensory fibres and causes Ca(2+) influx into fat cells, is involved in ApoD induction. Using 3T3-L1 cell cultures, we found that PACAP at a dose of 1 nM up-regulated the expression of ApoD protein and mRNA approx. 2.5 fold. This effect was driven by ERK1/2 acting upon PAC1/VPAC2 receptors. In turn, PACAP-treated 3T3-L1 adipocytes in co-cultures with DRG cells enhanced neurite ramification of neurofilament 200 (NF200)-positive neurons (measured using fluorescence microscopy) and neurofilament 68 protein levels (measured using Western blot analysis). This effect could be blocked using the PAC1/VPAC2 antagonist PACAP(6-38). Scanning cytometry revealed PACAP/ApoD induced low density lipoprotein receptors (LDLR) and ApoE receptor 2 (apoER2) in NF200-positive cells. Thus, a bidirectional loop seems to exist regulating the innervation of fatty tissues: PACAP released from sensory fibres might stimulate fat cells to synthesize neurotrophic adipokines, which, in turn, support peripheral innervation. PMID:20920539

  5. Dry piston coal feeder

    DOEpatents

    Hathaway, Thomas J. (Belle Meade, NJ); Bell, Jr., Harold S. (Madison, NJ)

    1979-01-01

    This invention provides a solids feeder for feeding dry coal to a pressurized gasifier at elevated temperatures substantially without losing gas from the gasifier by providing a lock having a double-acting piston that feeds the coals into the gasifier, traps the gas from escaping, and expels the trapped gas back into the gasifier.

  6. Bird Feeder Project

    NSDL National Science Digital Library

    2011-08-20

    In this activity, learners make a backyard bird feeder with soda bottles and other simple supplies. Learners will recycle a plastic bottle to hold birdseed, create a perch for the birds to rest, and attach it to a branch or post outside. Then, enjoy birdwatching!

  7. Effects of growth hormone antagonists on 3T3-F442A preadipocyte differentiation

    E-print Network

    Gu, Tingyue

    Effects of growth hormone antagonists on 3T3-F442A preadipocyte differentiation B C Xu, W Y Chen. lournal of Endocrinology (1995) 146, 131-1 39 Introductiun Growth hormone (GH) exerts &verse growth-term GH-inducible events were studied during adipose differ- entiation, including late marker gene

  8. Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

    NASA Astrophysics Data System (ADS)

    Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

    2014-03-01

    Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

  9. Effect of Gambisan on the Inhibition of Adipogenesis in 3T3-L1 Adipocytes

    PubMed Central

    Kang, Jung Won; Nam, Dongwoo; Kim, Kun Hyung; Huh, Jeong-Eun; Lee, Jae-Dong

    2013-01-01

    This study was conducted to explore the antiadipogenic effect and possible mechanism of Gambisan on 3T3-L1 cells. For quality control, Gambisan was standardized by HPLC and the standard compounds ephedrine, epigallocatechin-3-gallate, and caffeine were screened. Cultured 3T3-L1 cells that had been induced to differentiate were treated with various concentrations of Gambisan or its major component extracts (Ephedra intermedia Schrenk, Atractylodes lancea DC., and Thea sinensis L.) for 72 hours for MTT assay to determine cell viability or 10 days for LDH assay, triglyceride assay, DNA content measurement, Oil red O staining, RT-PCR, and western blot. Gambisan significantly inhibited adipogenesis in 3T3-L1 cells by reducing triglyceride contents and lipid accumulation in a dose-dependent manner without obvious cytotoxicity. Viability and DNA content in 3T3-L1 cells treated with Gambisan were significantly higher than cells treated with the major component extracts at every concentration. The anti-adipogenic effects of Gambisan appeared to be mediated by a significant downregulation of the expression of lipoprotein lipase mRNA and PPAR?, C/EBP?, and SREBP-1 protein apart from the expression of hormone-sensitive lipase. Gambisan could act as a possible therapeutic agent for obesity. However, further studies including in vivo assays and clinical trials are needed to confirm the efficacy, safety and mechanisms of the antiobesity effects of Gambisan. PMID:24069055

  10. Diallyl trisulfide suppresses the adipogenesis of 3T3-L1 preadipocytes through ERK activation.

    PubMed

    Lii, Chong-Kuei; Huang, Chun-Yin; Chen, Haw-Wen; Chow, Mun-Yew; Lin, Yu-Ru; Huang, Chin-Shiu; Tsai, Chia-Wen

    2012-03-01

    Garlic and its organosulfur compounds display hypolipidemic effects by inhibiting fatty acid and cholesterol synthesis. We investigated the anti-adipogenic effect of diallyl trisulfide (DATS), the second most abundant organosulfide in garlic oil. We studied the effects of DATS in 3T3-L1 preadipocytes and the mechanism involved in its action. DATS (0-75 ?M) inhibited CCAAT/enhancer-binding protein (C/EBP) ? and ? and peroxisome proliferator-activated receptor (PPAR) ? mRNA and protein levels in a dose- and time-dependent manner, leading to a decrease of fatty acid synthase (FAS) expression and lipid accumulation in 3T3-L1 cells. Insulin treatment induced a transient increase in ERK phosphorylation in 3T3-L1 preadipocytes, and maximal induction was observed at 5 min and then declined. DATS, however, sustained ERK phosphorylation up to 120 min. In the presence of PD98059, the phosphorylation of ERK and suppression of PPAR? expression and DNA binding activity of PPAR? by DATS were reversed, and lipid accumulation was restored. In conclusion, these results indicate that DATS inhibits the differentiation of 3T3-L1 preadipocytes into adipocytes. DATS is likely to act by prolonging ERK activation, which leads to the down-regulation of adipogenic transcription factor expression during adipogenesis. Our results suggest that garlic may have potential as an anti-obesity agent. PMID:22137902

  11. Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells

    SciTech Connect

    Yasumoto, S.; Burkhardt, A.L.; Doniger, J.; DiPaolo, J.A.

    1986-02-01

    A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangement. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas.

  12. Pleiotrophin transforms NIH 3T3 cells and induces tumors in nude mice.

    PubMed Central

    Chauhan, A K; Li, Y S; Deuel, T F

    1993-01-01

    The pleiotrophin (PTN) gene (Ptn) encodes an 18-kDa protein that is highly conserved among mammalian species and that functions as a weak mitogen and promotes neurite-outgrowth activity in vitro. To further investigate the role PTN plays in regulating cell growth, we overexpressed the bovine PTN cDNA and now show that PTN phenotypically transforms NIH 3T3 cells, as evidenced by increased cell number at confluence, focus formation, anchorage-independent growth, and tumor formation in the nude mouse. The results demonstrate that the Ptn gene has the potential to regulate NIH 3T3 cell growth and suggest that PTN may influence abnormal cell growth in vivo. Images PMID:8421705

  13. Expression of Nanog gene promotes NIH3T3 cell proliferation

    SciTech Connect

    Zhang Jingyu [Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 (China); Graduate School, Chinese Academy of Sciences, Beijing 100080 (China); Wang Xia [Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 (China); Chen Bing [Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 (China); Suo Guangli [Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 (China); Zhao Yanhong [Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 (China); Duan Ziyuan [Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 (China); Dai Jianwu [Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080 (China)]. E-mail: jwdai@genetics.ac.cn

    2005-12-16

    Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion.

  14. Whey Protein Stimulates the Proliferation and Differentiation of Osteoblastic MC3T3-E1 Cells

    Microsoft Academic Search

    Yukihiro Takada; Seiichiro Aoe; Masayoshi Kumegawa

    1996-01-01

    We examined the effects of whey protein on osteoblastic MC3T3-E1 cells. This protein caused dose-dependent increases in [3H]thymidine incorporation and DNA content in the cells. It also increased the total protein and hydroxyproline contents in the cells. These activities were heat resistant when the protein was heated at 75°C to 90°C for 10 min. Heat-treated whey protein was first fractionated

  15. The Mitogenic and Antiapoptotic Actions of Ghrelin in 3T3-L1 Adipocytes

    Microsoft Academic Search

    MIN SEON KIM; CHO YA YOON; PIL GEUM JANG; CHAN SOO SHIN; HYE SUN PARK; JE WON RYU; YOUNGMI KIM PAK; JOONG YEOL PARK; SEONG YEON KIM; YOUNG BUM KIM; KYONG SOO PARK

    2004-01-01

    antiapoptotic effects of ghrelin. Ghrelin also stim- ulated the insulin receptor substrate-associated phosphatidylinositol 3-kinase\\/Akt pathway in 3T3-L1 preadipocytes and adipocytes, whereas inhibition of this pathway blocked the effects of ghrelin on cell proliferation, antiapoptosis and glu- cose uptake. These findings suggest that the direct effects of ghrelin on proliferation, differentiation, and apoptosis in adipocytes may play a role in regulating

  16. Inhibitory Effects of Fucoidan in 3T3-L1 Adipocyte Differentiation

    Microsoft Academic Search

    Mi-Ja Kim; Un-Jae Chang; Jin-Sil Lee

    2009-01-01

    Fucoidan is a group of sulfated fucose-containing polysaccharides that derived from non-mammalian origin such as marine brown\\u000a algae, the jelly coat from sea urchin eggs, and the sea cucumber body wall. However, potential biological activities against\\u000a obesity from fucoidan were not reported in the literature. The objective of this study was to evaluate protective effect of\\u000a fucoidan in 3T3-L1 adipocyte

  17. Resistin expression in 3T3-L1 adipocytes is reduced by arachidonic acid

    Microsoft Academic Search

    Fred Haugen; Naeem Zahid; Knut T. Dalen; Kristin Hollung; Hilde I. Nebb; Christian A. Drevon

    2004-01-01

    The resistin gene is expressed in adipocytes and encodes a protein proposed to link obesity and type 2 dia- betes. Increased plasma FFA is associated with insulin re- sistance. We examined the effect of separate FFAs on the expression of resistin mRNA in cultured murine 3T3-L1 ad- ipocytes. The FFAs tested did not increase resistin expres- sion, whereas both arachidonic

  18. Interferon Inhibits the Conversion of 3T3-L1 Mouse Fibroblasts into Adipocytes

    Microsoft Academic Search

    Susan Keay; Sidney E. Grossberg

    1980-01-01

    Confluent Swiss mouse 3T3-L1 fibroblasts slowly differentiate functionally and morphologically into adipocytes, a conversion hastened by insulin. The cells are sensitive (although less than L929 cells) to the antiviral action of mouse fibroblast interferons but not to interferons from heterologous species (human and chicken). Cultures stimulated with insulin in the presence of partially purified or electrophoretically pure mouse interferons have

  19. Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

    Microsoft Academic Search

    Phimon Atsawasuwan; Yoshiyuki Mochida; Duenpim Parisuthiman; Mitsuo Yamauchi

    2005-01-01

    Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1–4 (LOXL1–4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1–4 in MC3T3-E1 osteoblastic cells was screened by

  20. Traditional Herbal Formula Oyaksungi-San Inhibits Adipogenesis in 3T3-L1 Adipocytes.

    PubMed

    Yoo, Sae-Rom; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

    2015-01-01

    Background. Oyaksungi-san (OYSGS) is a herbal formula that has been used for treating cardiovascular diseases in traditional Asian medicine. Here, we investigated the antiadipogenic effect of OYSGS extract in 3T3-L1 adipose cells. Methods. 3T3-L1 preadipocytes were differentiated into adipocytes with or without OYSGS. After differentiation, we measured Oil Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, leptin production, mRNA, and protein levels of adipogenesis-related factors. Results. OYSGS extract dramatically inhibited intracellular lipid accumulation in the differentiated adipocytes. It also significantly suppressed the (GPDH) activity, triglyceride (TG) content, and leptin production by reducing the expression of adipogenesis-related genes including lipoprotein lipase, fatty acid binding protein 4, CCAAT/enhancer-binding protein-alpha (C/EBP-?), and peroxisome proliferator-activated receptor gamma (PPAR-?). Furthermore, OYSGS clearly enhanced phosphorylation of AMP-activated protein kinase (AMPK) as well as its substrate acetyl CoA (ACC) carboxylase. Conclusions. Our results demonstrate that OYSGS negatively controls TG accumulation in 3T3-L1 adipocytes. We suggest antiadipogenic activity of OYSGS and its potential benefit in preventing obesity. PMID:25802547

  1. Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells

    PubMed Central

    Tsai, Chin-Yi; Peh, Meng Teng; Feng, Wei; Dymock, Brian William; Moore, Philip Keith

    2015-01-01

    The effect of hydrogen sulfide (H2S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H2S was increased after 3T3L1 differentiation. The expression of the H2S-synthesising enzymes, cystathionine ?-lyase (CSE), cystathionine ?-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H2S donor compound) and sodium hydrosulfide (NaHS, a classical H2S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (?3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H2S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H2S modulate adipogenesis and adipocyte maturation. PMID:25822632

  2. Stevioside from Stevia rebaudiana Bertoni Increases Insulin Sensitivity in 3T3-L1 Adipocytes

    PubMed Central

    Mohd-Radzman, Nabilatul Hani; Ismail, Wan Iryani Wan; Jaapar, Siti Safura; Adam, Zainah; Adam, Aishah

    2013-01-01

    Stevioside from Stevia rebaudiana has been reported to exert antihyperglycemic effects in both rat and human subjects. There have been few studies on these effects in vitro. In this paper, radioactive glucose uptake assay was implemented in order to assess improvements in insulin sensitivity in 3T3-L1 cells by elevation of glucose uptake following treatment with stevioside. Oil Red-O staining and MTT assay were utilized to confirm adipocyte differentiation and cell viability, respectively. Findings from this research showed a significant increase in absorbance values in mature adipocytes following Oil Red-O staining, confirming the differentiation process. Stevioside was noncytotoxic to 3T3-L1 cells as cell viability was reduced by a maximum of 17%, making it impossible to determine its IC50. Stevioside increased glucose uptake activities by 2.1 times (p < 0.001) in normal conditions and up to 4.4 times (p < 0.001) in insulin-resistant states. At times, this increase was higher than that seen in positive control group treated with rosiglitazone maleate, an antidiabetic agent. Expressions of pY20 and p-IRS1 which were measured via Western blot were improved by stevioside treatment. In conclusion, stevioside has direct effects on 3T3-L1 insulin sensitivity via increase in glucose uptake and enhanced expression of proteins involved in insulin-signalling pathway. PMID:24391675

  3. Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells.

    PubMed

    Tsai, Chin-Yi; Peh, Meng Teng; Feng, Wei; Dymock, Brian William; Moore, Philip Keith

    2015-01-01

    The effect of hydrogen sulfide (H2S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H2S was increased after 3T3L1 differentiation. The expression of the H2S-synthesising enzymes, cystathionine ?-lyase (CSE), cystathionine ?-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H2S donor compound) and sodium hydrosulfide (NaHS, a classical H2S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (?3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H2S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H2S modulate adipogenesis and adipocyte maturation. PMID:25822632

  4. Traditional Herbal Formula Oyaksungi-San Inhibits Adipogenesis in 3T3-L1 Adipocytes

    PubMed Central

    Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

    Background. Oyaksungi-san (OYSGS) is a herbal formula that has been used for treating cardiovascular diseases in traditional Asian medicine. Here, we investigated the antiadipogenic effect of OYSGS extract in 3T3-L1 adipose cells. Methods. 3T3-L1 preadipocytes were differentiated into adipocytes with or without OYSGS. After differentiation, we measured Oil Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, leptin production, mRNA, and protein levels of adipogenesis-related factors. Results. OYSGS extract dramatically inhibited intracellular lipid accumulation in the differentiated adipocytes. It also significantly suppressed the (GPDH) activity, triglyceride (TG) content, and leptin production by reducing the expression of adipogenesis-related genes including lipoprotein lipase, fatty acid binding protein 4, CCAAT/enhancer-binding protein-alpha (C/EBP-?), and peroxisome proliferator-activated receptor gamma (PPAR-?). Furthermore, OYSGS clearly enhanced phosphorylation of AMP-activated protein kinase (AMPK) as well as its substrate acetyl CoA (ACC) carboxylase. Conclusions. Our results demonstrate that OYSGS negatively controls TG accumulation in 3T3-L1 adipocytes. We suggest antiadipogenic activity of OYSGS and its potential benefit in preventing obesity.

  5. Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan) [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065 (Japan)] [Innovation Center, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayoshi [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan)] [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

    2013-02-01

    Highlights: ? Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ? Adipose lipin-1 expression is reduced in obesity. ? ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ? Activation of PPAR-? recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-? and interleukin-1? reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-? in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-? recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

  6. Stevioside from Stevia rebaudiana Bertoni Increases Insulin Sensitivity in 3T3-L1 Adipocytes.

    PubMed

    Mohd-Radzman, Nabilatul Hani; Ismail, Wan Iryani Wan; Jaapar, Siti Safura; Adam, Zainah; Adam, Aishah

    2013-01-01

    Stevioside from Stevia rebaudiana has been reported to exert antihyperglycemic effects in both rat and human subjects. There have been few studies on these effects in vitro. In this paper, radioactive glucose uptake assay was implemented in order to assess improvements in insulin sensitivity in 3T3-L1 cells by elevation of glucose uptake following treatment with stevioside. Oil Red-O staining and MTT assay were utilized to confirm adipocyte differentiation and cell viability, respectively. Findings from this research showed a significant increase in absorbance values in mature adipocytes following Oil Red-O staining, confirming the differentiation process. Stevioside was noncytotoxic to 3T3-L1 cells as cell viability was reduced by a maximum of 17%, making it impossible to determine its IC50. Stevioside increased glucose uptake activities by 2.1 times (p < 0.001) in normal conditions and up to 4.4 times (p < 0.001) in insulin-resistant states. At times, this increase was higher than that seen in positive control group treated with rosiglitazone maleate, an antidiabetic agent. Expressions of pY20 and p-IRS1 which were measured via Western blot were improved by stevioside treatment. In conclusion, stevioside has direct effects on 3T3-L1 insulin sensitivity via increase in glucose uptake and enhanced expression of proteins involved in insulin-signalling pathway. PMID:24391675

  7. Extract of Chaga mushroom (Inonotus obliquus) stimulates 3T3-L1 adipocyte differentiation.

    PubMed

    Joo, Jeong In; Kim, Dong Hyun; Yun, Jong Won

    2010-11-01

    Chaga mushroom (Inonotus obliquus) has long been used as a folk medicine due to its numerous biological functions such as antibacterial, antiallergic, antiinflammatory and antioxidative activities. In the present study, it was found that the I. obliquus hot water extract (IOWE) activated adipogenesis of 3T3-L1 preadipocytes. Even in the absence of adipogenic stimuli by insulin, the IOWE strongly induced adipogenesis of 3T3-L1 preadipocytes. The major constituent of IOWE was glucose-rich polysaccharides with a molecular mass of 149? kDa. IOWE enhanced the differentiation of 3T3-L1 preadipocytes, increasing TG (triacylglycerol) accumulation that is critical for acquisition of the adipocyte phenotype, in a dose-dependent manner. IOWE stimulated gene expression of C/EBP? (CCAAT/enhancer-binding protein ?) and PPAR? (peroxisome proliferator-activated receptors ?) during adipocyte differentiation, and induced the expression of PPAR? target genes such as aP2 (adipocyte protein 2), LPL (lipoprotein lipase) and CD36 (fatty acid translocase). Immunoblot analysis revealed that IOWE increased the expression of adipogenic makers such as PPAR? and GLUT4 (glucose transporter 4). The luciferase reporter assay demonstrated that IOWE did not exhibit PPAR? ligand activity. Although these results require further investigation, the ability of natural mushroom product to increase PPAR? transcriptional activities may be expected to be therapeutic targets for dyslipidemia and type 2 diabetes. PMID:21031614

  8. Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes.

    PubMed

    Pheiffer, Carmen; Dudhia, Zulfaqar; Louw, Johan; Muller, Christo; Joubert, Elizabeth

    2013-10-15

    We have previously, for the first time, demonstrated that hot water extracts of Cyclopia maculata and Cyclopia subternata, endemic South African plants that are consumed as herbal teas, inhibit adipogenesis in 3T3-L1 adipocytes. The aim of this study was to extend the anti-obesity investigations of these plants by quantifying lipolysis in mature 3T3-L1 adipocytes. Glycerol concentration in culture supernatants was used as a marker of adipocyte lipolysis. Isoproterenol, a ?-adrenergic agonist and a known lipolytic agent, was used as a positive control in our assays. Lipolysis was stimulated by all extracts, although statistical significance was noted for fermented (oxidised) C. maculata only. A concentration of 80?g/ml of C. maculata extract induced maximal lipolysis (1.8-fold, p<0.001). The increased lipolysis was accompanied by an increase in the expression of hormone sensitive lipase (1.6-fold, p<0.05) and perilipin (1.6-fold, p<0.05). The plant extracts, at the concentration range assayed (0-100?g/ml), were not cytotoxic in terms of mitochondrial dehydrogenase and adenosine-5'-triphosphate activity. These results showed that C. maculata stimulates lipolysis in mature 3T3-L1 adipocytes, providing further support for the anti-obesity effects of Cyclopia spp. PMID:23880330

  9. Geometry and emplacement of basaltic feeder dykes

    NASA Astrophysics Data System (ADS)

    Galindo, I.; Gudmundsson, A.

    2009-04-01

    Dyke intrusion is known to be a complex process that usually ends in the formation of an arrested dyke. However, when the stress conditions are favourable the dyke propagates through the crust to the surface and issues magma. The dykes that connect the magma chamber with the surface are called feeder dykes. Feeder dykes are rarely observed volcanotectonic structures since they are usually covered by their own products. For this reason, outcrops are scarce and usually restricted to cliffs, ravines, and man-made outcrops. Feeder dykes from Tenerife (Canary Islands, Spain) and Iceland have been studied in order to describe their geometric characteristics and their interaction with discontinuities in the host rock, such as tension fractures, faults, and contacts between layers. Although there have been many recent eruptions in both islands, only eleven basaltic feeder dykes have been identified: eight in Tenerife and three in Iceland. They are all well preserved and with well-exposed connections between the dyke and the associated eruptive fissure and/or deposits. Generally, the feeder dykes in Tenerife and Iceland are similar as regards geometry and emplacement mechanisms. There are, however, some differences, the main ones being that, in comparison with the feeders in Tenerife, the feeders in Iceland are longer and produce longer eruptive fissures and greater eruption rates. The rate of spreading (extension) is also higher in Iceland than in Tenerife. As regards the detailed geometry and emplacement mechanism of the feeder dykes, our field data indicate as follows: (1) the overall dyke geometry is sheet-like, although plugs and sills may formed under cinder cones at high eruptive rates; (2) all the dykes and the associated eruptive fissures are segmented; (3) in the uppermost 2-3 m below the surface, the dykes are commonly with elongated empty holes in the centre (parallel with the dyke dip) with solidified magma drops on the hole walls; (4) vesicles in the dyke rock increase in size and frequency close to the surface; (5) some dykes propagate through existing faults and, where the plane of the fault is oblique to the plane of the dyke, the dyke forms small, igneous fingers that propagate within the fault plane; and (6) when a dyke and a fault intersect, the eruptive style may be affected; in particular, at the surface, the volumetric magma flow rate may be highest at such intersections. These results, particularly items 5 and 6, suggest that for forecasting, and monitoring an actual, fissure eruption, it is important to identify all faults in volcanic areas since they may have strong effects on the feeder-dyke path and the eruptive style once the dyke has reached the surface. Modelling the mechanical interaction between feeder dykes and faults should help us understand the conditions that allow magma to flow into, and occasionally up along, fault planes. Further field and modelling studies will address the stress conditions which favour the propagation of feeder dykes and eruptive fissures close to and at the surface.

  10. Nanog transforms NIH3T3 cells and targets cell-type restricted genes Dan Piestun a,1

    E-print Network

    Domany, Eytan

    Nanog transforms NIH3T3 cells and targets cell-type restricted genes Dan Piestun a,1 , Bose S and in germ cell tumors and is important for self- renewal. To understand the relation between this and cell transformation, we expressed Nanog in NIH3T3 cells, and these cells showed an increased growth rate

  11. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    SciTech Connect

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  12. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    SciTech Connect

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)] [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Yang, Ying, E-mail: yangying_sh@yahoo.com [Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)] [Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Shen, Weili, E-mail: weili_shen@hotmail.com [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)] [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)

    2013-08-16

    Highlights: •Nebivolol may act as a partial agonist of ?3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by ?3-AR. -- Abstract: Nebivolol is a third-generation ?-adrenergic receptor (?-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-? coactivator-1? (PGC-1?), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and ?3-AR blocker SR59230A markedly attenuated PGC-1?, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of ?3-AR receptors.

  13. Changes in laser-induced fluorescence responses of 3T3 fibroblasts to repetitive thermal stress

    NASA Astrophysics Data System (ADS)

    Beuthan, J.; Dressler, C.; Zabarylo, U.; Minet, O.

    2009-04-01

    The combined experimental use of laser-induced autofluorescence of cellular metabolites and methodological fundamentals of systems biology will provide access to biological thermal stress analysis on a sub cellular level. A test setup incorporating a pulsed nitrogen laser was realized with which autofluorescence of the coenzyme NADH could be measured in living 3T3 cells. The cells were subjected to different temperature stress at repetitive time intervals. When subjected to a simple mathematical analysis, the NADH concentration change measured through autofluorescence in biological cells exhibited approximate concentration-equivalent balance curves. These results add up to the fundamental know-how about the dosimetry of thermally therapeutic methods.

  14. Tunable swelling of polyelectrolyte multilayers in cell culture media for modulating NIH-3T3 cells adhesion.

    PubMed

    Qi, Wei; Cai, Peng; Yuan, Wenjing; Wang, Hua

    2014-11-01

    For polyelectrolyte multilayers (PEMs) assembled by the layer-by-layer (LbL) assembly technique, their nanostructure and properties can be governed by many parameters during the building process. Here, it was demonstrated that the swelling of the PEMs containing poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) in cell culture media could be tuned with changing supporting salt solutions during the assembly process. Importantly, the influence of the PEMs assembled in different salt solutions on NIH-3T3 cell adhesion was observable. Specifically, the cells could possess a higher affinity for the films assembled in low salt concentration (i.e. 0.15M NaCl) or no salt, the poorly swelling films in cell culture media, which was manifested by the large cell spreading area and focal adhesions. In contrast, those were assembled in higher salt concentration, highly swelling films in cell culture media, were less attractive for the fibroblasts. As a result, the cell adhesion behaviors may be manipulated by tailoring the physicochemical properties of the films, which could be performed by changing the assembly conditions such as supporting salt concentration. Such a finding might promise a great potential in designing desired biomaterials for tissue engineering and regenerative medicine. PMID:24470104

  15. 2-Deoxyglucose and cytochalasin D modulate aldolase mobility in living 3T3 cells

    PubMed Central

    1992-01-01

    Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells is immobile as measured by FRAP. Previous studies suggest that the immobile fraction may be associated with the actin cytoskeleton (Pagliaro, L. and D. L. Taylor. 1988. J. Cell Biol. 107:981-991), and it has been proposed that the association of some glycolytic enzymes with the cytoskeleton could have functional significance, perhaps involving a fundamental relationship between glycolysis, cytoplasmic organization, and cell motility. We have tested the effect of a key glycolytic inhibitor and an actin cytoskeletal modulator on the mobility of aldolase in living cells directly, using fluorescent analog cytochemistry and FRAP. We report here that the competitive hexokinase inhibitor 2-deoxyglucose releases the bound fraction of aldolase in 3T3 cells within 10 min, and that this process is reversible upon washout of the inhibitor. A similar result is produced with the actin-binding agent, cytochalasin D. These results are consistent with models in which glycolytic enzymes are not exclusively diffusion-limited, soluble proteins, but may exist partially in the solid phase of cytoplasm. Such organization has significant implications for both the modulation of cytoplasmic structure and for cellular metabolism. PMID:1500428

  16. Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

    PubMed Central

    Sadler, Jessica B. A.; Bryant, Nia J.; Gould, Gwyn W.

    2015-01-01

    The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes. PMID:25501368

  17. Anti-adipogenic effect of mulberry leaf ethanol extract in 3T3-L1 adipocytes

    PubMed Central

    Yang, Soo Jin; Park, Na-Young

    2014-01-01

    BACKGROUND/OBJECTIVES Adipogenesis is part of the cell differentiation process in which undifferentiated fibroblasts (pre-adipocytes) become mature adipocytes with the accumulation of lipid droplets and subsequent cell morphological changes. Several transcription factors and food components have been suggested to be involved in adipogenesis. The aim of this study was to determine whether mulberry leaf ethanol extract (MLEE) affects adipogenesis in 3T3-L1 adipocytes. MATERIALS/METHODS The 3T3-L1 adipocytes were treated with different doses of MLEE for 8 days starting 2 days post-confluence. Cell viability, fat accumulation, and adipogenesis-related factors including CCAAT-enhancer-binding protein alpha (C/EBP?), peroxisome proliferator-activated receptor gamma (PPAR?), PPAR? coactivator 1 alpha (PGC-1?), fatty acid synthase (FAS), and adiponectin were analyzed. RESULTS Results showed that MLEE treatments at 10, 25, 50, and 100 µg/ml had no effect on cell morphology and viability. Without evident toxicity, all MLEE treated cells had lower fat accumulation compared with control as shown by lower absorbances of Oil Red O stain. MLEE at 50 and 100 µg/ml significantly reduced protein levels of PPAR?, PGC-1?, FAS, and adiponectin in differentiated adipocytes. Furthermore, protein level of C/EBP? was significantly decreased by the treatment of 100 µg/ml MLEE. CONCLUSION These results demonstrate that MLEE treatment has an anti-adipogenic effect in differentiated adipocytes without toxicity, suggesting its potential as an anti-obesity therapeutic. PMID:25489399

  18. p53 mediates impaired insulin signaling in 3T3-L1 adipocytes during hyperinsulinemia.

    PubMed

    Posa, Jyothi Kumari; Selvaraj, Sudhagar; Sangeetha, K N; Baskaran, Sarath Kumar; Lakshmi, B S

    2014-07-01

    Hyperinsulinemia is being implicated in the development of insulin resistance but remains poorly understood. The present study focuses on p53-mediated impaired insulin signaling by hyperinsulinemia in 3T3-L1 adipocytes. Hyperinsulinemia impairs insulin-stimulated glucose uptake and its cellular signaling in a dose- and time-dependent manner. An increased level of reactive oxygen species (ROS) and stress response signals were observed, and quenching of the ROS by an antioxidant N-acetylcysteine (NAC) did not revert impaired insulin sensitivity. The tumor suppressor p53 has emerged as a crucial factor in the metabolic adaptation of cancer cells under nutritional starvation and is being studied in the development of insulin resistance in adipocytes at physiological level. Interestingly, we observed hyperinsulinemia-enhanced p53 level in a time-dependent manner without exhibiting cytotoxicity. Transient knockdown of p53 partially improved insulin sensitivity revealing a novel link between p53 and insulin signaling in adipocytes. The findings suggest that hyperinsulinemia-induced p53 impairs insulin sensitivity in 3T3-L1 adipocytes. PMID:24604666

  19. The regulation by fibroblast growth factor of early transport changes in quiescent 3T3 cells.

    PubMed

    Quinlan, D C; Hochstadt, J

    1977-11-01

    This study involves the use of fibroblast growth factor (FGF) as a substitute for exogenous serum to examine the early transport changes which occur when quiescent 3T3 cells re-initiate active growth. FGF, in nanogram amounts, together with insulin and dexamethasone, can induce mitogenesis and mitosis in 3T3 cells GO-arrested by holding in growth medium containing 0.8% calf serum. In terms of quiescent cell transport activity enhancement, FGF is 300,000-fold more effective than fresh serum, on a protein basis. In addition, very short exposure of serum-depleted cells to FGF indicates that a distinct temporal or time sequence exists in the transport system activation process. For example, uptake of alpha-aminoisobutyric acid (AIB) and uridine are stimulated very rapidly, whereas hypoxanthine uptake does not respond until much later. Closer analysis shows that AIB uptake is maximally enhanced within zero to two minutes after FGF addition to cells. Finally, the stimulatory effect of FGF on transport system activities is specific in terms of the proliferative state of the cells to which it is added, and in terms of the uptake systems which respond to it. PMID:563406

  20. Oxidative changes and apoptosis induced by 1800-MHz electromagnetic radiation in NIH/3T3 cells.

    PubMed

    Hou, Qingxia; Wang, Minglian; Wu, Shuicai; Ma, Xuemei; An, Guangzhou; Liu, Huan; Xie, Fei

    2015-03-01

    Abstract To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5?min on/10?min off, for various durations from 0.5 to 8?h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2?W/kg. A 2',7'-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect ?H2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1?h (p?3T3 cells. PMID:24665905

  1. Capsaicin Induces “Brite” Phenotype in Differentiating 3T3-L1 Preadipocytes

    PubMed Central

    Baboota, Ritesh K.; Singh, Dhirendra P.; Sarma, Siddhartha M.; Kaur, Jaspreet; Sandhir, Rajat; Boparai, Ravneet K.; Kondepudi, Kanthi K.; Bishnoi, Mahendra

    2014-01-01

    Objective Targeting the energy storing white adipose tissue (WAT) by pharmacological and dietary means in order to promote its conversion to energy expending “brite” cell type holds promise as an anti-obesity approach. Present study was designed to investigate/revisit the effect of capsaicin on adipogenic differentiation with special reference to induction of “brite” phenotype during differentiation of 3T3-L1 preadipocytes. Methods Multiple techniques such as Ca2+ influx assay, Oil Red-O staining, nutrigenomic analysis in preadipocytes and matured adipocytes have been employed to understand the effect of capsaicin at different doses. In addition to in-vitro experiments, in-vivo studies were carried out in high-fat diet (HFD) fed rats treated with resiniferatoxin (RTX) (a TRPV1 agonist) and in mice administered capsaicin. Results TRPV1 channels are expressed in preadipocytes but not in adipocytes. In preadipocytes, both capsaicin and RTX stimulate Ca2+ influx in dose-dependent manner. This stimulation may be prevented by capsazepine, a TRPV1 antagonist. At lower doses, capsaicin inhibits lipid accumulation and stimulates TRPV1 gene expression, while at higher doses it enhances accumulation of lipids and suppresses expression of its receptor. In doses of 0.1–100 µM, capsaicin promotes expression of major pro-adipogenic factor PPAR? and some of its downstream targets. In concentrations of 1 µM, capsaicin up-regulates anti-adipogenic genes. Low-dose capsaicin treatment of 3T3-L1 preadipocytes differentiating into adipocytes results in increased expression of brown fat cell marker genes. In white adipose of mice, capsaicin administration leads to increase in browning-specific genes. Global TRPV1 ablation (i.p. by RTX administration) leads to increase in locomotor activity with no change in body weight. Conclusion Our findings suggest the dual modulatory role of capsaicin in adipogenesis. Capsaicin inhibits adipogenesis in 3T3-L1 via TRPV1 activation and induces brown-like phenotype whereas higher doses. PMID:25072597

  2. Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells

    PubMed Central

    2014-01-01

    Background Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, through increases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performed in vitro studies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reported widely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters of low-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayer cultures. Methods We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity, frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified, treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding. Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation. All data are presented as the mean ± standard error. The statistical significance was determined using Student's two-sample two-tailed t tests. Results Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2 (SATA, spatial average-temporal average), we found that approximately 75 mW/cm2 produced the greatest increase in osteoblast proliferation. Ultrasound exposures at higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting that high-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells. Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequency dependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response. Conclusion This study concludes that a short-term stimulation with optimum intensity can enhance proliferation of preosteoblast-like bone cells that plays an important role in bone formation and accelerated fracture healing, also suggesting a possible therapeutic treatment for reduced bone mass. PMID:25516803

  3. Contrasting Antioxidant and Cytotoxic Effects of Peroxiredoxin I and II in PC12 and NIH3T3 Cells

    Microsoft Academic Search

    Soheil Simzar; Raymond Ellyin; Hungyi Shau; Theodore A. Sarafian

    2000-01-01

    We examined the impact of peroxiredoxin-I (Prx-I) and peroxiredoxin-II (Prx-II) stable transduction on oxidative stress in PC12 neurons and NIH3T3 fibroblasts and found variability depending on cell type and Prx subtype. In PC12 neurons, Prx-II suppressed reactive oxygen species (ROS) generation by 36% (p < 0.01) relative to vector-infected control cells. However, in NIH3T3 fibroblasts, Prx-II overexpression resulted in a

  4. Promoting activity of 3- tert-butyl-4-hydroxyanisole (BHA) in BALB\\/3T3 cell transformation

    Microsoft Academic Search

    Ayako Sakai; Naoki Miyata; Atsushi Takahashi

    1997-01-01

    3-Tert-butyl-4-hydroxyanisole (BHA), a synthetic antioxidant, is an initiator in the transformation of BALB\\/3T3 mouse embryo cells, when cells are treated first with the chemical and then with a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. In the present study, BHA was examined for its promoting activity in BALB\\/3T3 cell transformation. BHA in a range of 5–20 ?g\\/ml enhanced transformation induced by a subthreshold dose

  5. Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes.

    PubMed

    Ohnogi, Hiromu; Kudo, Yoko; Tahara, Kenichi; Sugiyama, Katsumi; Enoki, Tatsuji; Hayami, Shoko; Sagawa, Hiroaki; Tanimura, Yuko; Aoi, Wataru; Naito, Yuji; Kato, Ikunoshin; Yoshikawa, Toshikazu

    2012-01-01

    Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome. PMID:22738967

  6. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    SciTech Connect

    Chan, C.P.; McNall, S.J.; Krebs, E.G.; Fischer, E.H. (Univ. of Washington, Seattle (USA))

    1988-09-01

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using ({sup 32}P)phosphorylase {alpha} as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at {approx}5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.

  7. Fructose promotes the differentiation of 3T3-L1 adipocytes and accelerates lipid metabolism.

    PubMed

    Legeza, Balázs; Balázs, Zoltán; Odermatt, Alex

    2014-01-31

    Excessive fructose consumption and elevated glucocorticoids contribute to metabolic syndrome. We show that fructose as the only carbohydrate source is sufficient for the differentiation of 3T3-L1 fibroblasts into adipocytes. Differentiation of cells in fructose containing medium resulted in increased 11?-hydroxysteroid dehydrogenase 1 (11?-HSD1) expression and activity. Experiments with transfected HEK-293 cells suggested more efficient NADPH generation by fructose compared with glucose in the endoplasmic reticulum (ER). Adipocytes differentiated in the presence of fructose showed increased FABP4 expression, C/EBP? to C/EBP? ratio and lipolysis. Thus, excessive fructose may cause adverse metabolic effects by enhancing 11?-HSD1 activity and increasing lipolysis in adipocytes. PMID:24374344

  8. Surface extensions of 3T3 cells towards distant infrared light sources

    PubMed Central

    1991-01-01

    Using a specially designed phase-contrast light microscope with an infrared spot illuminator we found that approximately 25% of 3T3 cells were able to extend pseudopodia towards single microscopic infrared light sources nearby. If the cells were offered a pair of such light sources next to each other, 47% of the cells extended towards them. In the latter case 30% of the responding cells extended separate pseudopodia towards each individual light source of a pair. The strongest responses were observed if the infrared light sources emitted light of wavelengths in the range of 800-900 nm intermittently at rates of 30-60 pulses per min. The temperature increases of the irradiated spots can be shown to be negligible. The results suggest that the cells are able to sense specific infrared wavelengths and to determine the direction of individual sources. PMID:1860881

  9. Uptake and binding of radiolabelled phenylarsine oxide in 3T3-L1 adipocytes.

    PubMed Central

    Frost, S C; Schwalbe, M S

    1990-01-01

    Phenylarsine oxide (PAO), a trivalent arsenical, has been shown to inhibit insulin-stimulated glucose transport in 3T3-L1 adipocytes, implicating vicinal dithiols in signal transmission [Frost & Lane (1985) J. Biol. Chem. 260, 2646-2652]. To assist in the direct identification of a PAO-binding protein which might be involved in this process, we have synthesized [3H]acetylaminophenylarsine oxide [( 3H]APAO) from the amino derivative of phenylarsine oxide (NPAO). To assess the inhibitory effect of the product, a dual-labelling experiment was performed which showed that [3H]APAO inhibited insulin-stimulated 2-deoxy[1-14C]glucose transport in 3T3-L1 adipocytes with a Ki of 21 microM, identical with that of the parent compound, NPAO. Further characterization revealed that over a wide concentration range, uptake of the labelled arsine oxide was linear. Although the dithiol reagent 2,3-dimercaptopropanol (DMP) reversed PAO-induced inhibition of transport, it had no effect on the uptake of [3H]APAO. In a simple fractionation experiment approx. 50% of the radioactivity was associated with the cytosolic fraction and 50% with the total membrane fraction. Identification of radiolabelled proteins by non-reducing SDS/PAGE revealed fraction-specific binding, although many proteins were observed. Covalent modification was time-dependent and could be reversed by addition of DMP. These data further support a role for vicinal dithiols in insulin-stimulated glucose transport. Additionally, the probe described may offer a new means with which to identify the inhibitory protein or, more globally, to investigate mechanisms of action of vicinal dithiol-containing proteins. Images Fig. 5. Fig. 6. PMID:2202293

  10. Uptake and binding of radiolabelled phenylarsine oxide in 3T3-L1 adipocytes.

    PubMed

    Frost, S C; Schwalbe, M S

    1990-08-01

    Phenylarsine oxide (PAO), a trivalent arsenical, has been shown to inhibit insulin-stimulated glucose transport in 3T3-L1 adipocytes, implicating vicinal dithiols in signal transmission [Frost & Lane (1985) J. Biol. Chem. 260, 2646-2652]. To assist in the direct identification of a PAO-binding protein which might be involved in this process, we have synthesized [3H]acetylaminophenylarsine oxide [( 3H]APAO) from the amino derivative of phenylarsine oxide (NPAO). To assess the inhibitory effect of the product, a dual-labelling experiment was performed which showed that [3H]APAO inhibited insulin-stimulated 2-deoxy[1-14C]glucose transport in 3T3-L1 adipocytes with a Ki of 21 microM, identical with that of the parent compound, NPAO. Further characterization revealed that over a wide concentration range, uptake of the labelled arsine oxide was linear. Although the dithiol reagent 2,3-dimercaptopropanol (DMP) reversed PAO-induced inhibition of transport, it had no effect on the uptake of [3H]APAO. In a simple fractionation experiment approx. 50% of the radioactivity was associated with the cytosolic fraction and 50% with the total membrane fraction. Identification of radiolabelled proteins by non-reducing SDS/PAGE revealed fraction-specific binding, although many proteins were observed. Covalent modification was time-dependent and could be reversed by addition of DMP. These data further support a role for vicinal dithiols in insulin-stimulated glucose transport. Additionally, the probe described may offer a new means with which to identify the inhibitory protein or, more globally, to investigate mechanisms of action of vicinal dithiol-containing proteins. PMID:2202293

  11. Carcinogenic potential of metal nanoparticles in BALB/3T3 cell transformation assay.

    PubMed

    Sighinolfi, G L; Artoni, E; Gatti, A M; Corsi, L

    2014-10-30

    Metal-based nanoparticles (NPs), are currently used in many application fields including consumer products, pharmaceuticals, and biomedical treatments. In spite to their wide applications, an in-depth study of their potential toxic effects is still lacking. The aim of the present research was to investigate the potential initiator or promoter-like activity of different metallic NPs such as gold, iron, cobalt, and cerium using the Balb/3T3 two-stage transformation assay. The results indicated that all the selected metallic NPs, except for cobalt, when used as initiators did not induce any transformation in Balb/3T3 cell line. Moreover, Au and Fe3 O4 NPs, when used in place of the tumor promoter treatment TPA, increased significantly the number of Foci/dish as compared to the MCA treatment alone. The number of Foci/dish was 2.6 for Au NPs and 2.13 for Fe3 O4 ones, similar to those obtained by the positive control treatment (MCA?+?TPA), whereas 1.27 for MCA treatment alone. On the contrary, CeO2 NPs did not show any difference in the number of Foci/dish, as compared to MCA alone, but it decreased the number of foci by 65% in comparison to the positive control (MCA?+?TPA). As expected, cobalt NPs showed an increased cytotoxicity and only a few surviving cells were found at the time of analysis showing a number of Foci/dish of 0.13. For the first time, our data clearly showed that Au and Fe3 O4 NPs act as promoters in the two stage transformational assay, suggesting the importance to fully investigate the NPs carcinogenic potential with different models. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014. PMID:25358123

  12. Spermidine is indispensable in differentiation of 3T3-L1 fibroblasts to adipocytes

    PubMed Central

    Vuohelainen, Susanna; Pirinen, Eija; Cerrada-Gimenez, Marc; Keinänen, Tuomo A; Uimari, Anne; Pietilä, Marko; Khomutov, Alex R; Jänne, Juhani; Alhonen, Leena

    2010-01-01

    Abstract Impaired adipogenesis has been shown to predispose to disturbed adipocyte function and development of metabolic abnormalities. Previous studies indicate that polyamines are essential in the adipogenesis in 3T3-L1 fibroblasts. However, the specific roles of individual polyamines during adipogenesis have remained ambiguous as the natural polyamines are readily interconvertible inside the cells. Here, we have defined the roles of spermidine and spermine in adipogenesis of 3T3-L1 cells by using (S’)- and (R’)- isomers of ?-methylspermidine and (S,S’)-, (R,S)- and (R,R’)-diastereomers of ?,?-bismethylspermine. Polyamine depletion caused by ?-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, prevented adipocyte differentiation by suppressing the expression of its key regulators, peroxisome proliferator-activated receptor ? and CCAAT/enhancer binding protein ?. Adipogenesis was restored by supplementation of methylspermidine isomers but not of bismethylspermine diastereomers. Although both spermidine analogues supported adipocyte differentiation only (S)-methylspermidine was able to fully support cell growth after extended treatment with ?-DFMO. The distinction between the spermidine analogues in maintaining growth was found to be in their different capability to maintain functional hypusine synthesis. However, the differential ability of spermidine analogues to support hypusine synthesis did not correlate with their ability to support differentiation. Our results show that spermidine, but not spermine, is essential for adipogenesis and that the requirement of spermidine for adipogenesis is not strictly associated with hypusine modification. The involvement of polyamines in the regulation of adipogenesis may offer a potential application for the treatment of dysfunctional adipocytes in patients with obesity and metabolic syndrome. PMID:19538475

  13. Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells.

    PubMed

    Ilavenil, Soundharrajan; Arasu, Mariadhas Valan; Lee, Jeong-Chae; Kim, Da Hye; Roh, Sang Gun; Park, Hyung Su; Choi, Gi Jun; Mayakrishnan, Vijayakumar; Choi, Ki Choon

    2014-04-15

    Trigonelline is a natural alkaloid mainly found in Trigonella Foenum Graecum (fenugreek) Fabaceae and other edible plants with a variety of medicinal applications. Therefore, we investigated the molecular mechanism of trigonelline (TG) on the inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline suppressed lipid droplet accumulation in a concentration (75 and 100 ?M) dependent manner. Treatment of adipocyte with of TG down regulates the peroxisome proliferator-activated receptor (PPAR?) and CCAAT element binding protein (C/EBP-?) mRNA expression, which leads to further down regulation of other gene such as adiponectin, adipogenin, leptin, resistin and adipocyte fatty acid binding protein (aP2) as compared with respective control cells on 5th and 10th day of differentiation. Further, addition of triognelline along with troglitazone to the adipocyte attenuated the troglitazone effects on PPAR? mediated differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline might compete against troglitazone for its binding to the PPAR?. In addition, adipocyte treated with trigonelline and isoproterenol separately. Isoproterenol, a lipolytic agent which inhibits the fatty acid synthase and GLUT-4 transporter expression via cAMP mediated pathway, we found that similar magnitude response of fatty acid synthase and GLUT-4 transporter expression in trigonelline treated adipocyte. These results suggest that the trigonelline inhibits the adipogenesis by its influences on the expression PPAR?, which leads to subsequent down regulation of PPAR-? mediated pathway during adipogenesis. Our findings provide key approach to the mechanism underlying the anti-adipogenic activity of trigonelline. PMID:24369814

  14. First Molecular Cytogenetic High Resolution Characterization of the NIH 3T3 Cell Line by Murine Multicolor Banding

    PubMed Central

    Leibiger, Christine; Kosyakova, Nadezda; Mkrtchyan, Hasmik; Glei, Michael; Trifonov, Vladimir

    2013-01-01

    Since being established in 1963, the murine fibroblast cell line NIH 3T3 has been used in thousands of studies. NIH 3T3 immortalized spontaneously and became tetraploid shortly after its establishment. Here we report the first molecular cytogenetic characterization of NIH 3T3 using fluorescence in situ hybridization based multicolor banding (mcb). Overall, a complex rearranged karyotype presenting 16 breakpoints was characterized. Also it was possible to deduce the resulting gains and losses of copy numbers in NIH 3T3. Overall, only 1.8% of the NIH 3T3 genome is disome, 26.2% tri-, 60% tetra-, 10.8% quinta-, and 1.2% hexasome. Strikingly, the cell line gained only 4 derivative chromosomes since its first cytogenetic description in 1989. An attempt to align the observed imbalances of the studied cell line with their homologous regions in humans gave the following surprising result: NIH 3T3 shows imbalances as typically seen in human solid cancers of ectodermal origin. PMID:23321776

  15. Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Alexander, M.; Curtis, G.; Avruch, J.; Goodman, H.M.

    1985-10-05

    The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, the authors focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a 9-fold increase in hybridizable mRNA levels. Within 2 h of insulin addition to 3T3 adipocytes differentiated in the absence of hormone, hybridizable GAPDH mRNA levels increased 3-fold, and within 24 h GAPDH mRNA levels increased 8-fold, and (TVS) methionine incorporation into GAPDH protein increased 5-fold. These studies demonstrate that insulin, as the sole hormonal perturbant, can increase the synthesis of certain 3T3 adipocyte proteins by altering the cellular content of a specific mRNA.

  16. Cortisol effect on heat shock proteins in the C2C12 and 3T3-L1 cells.

    PubMed

    Ravikumar, Sambandam; Muthuraman, Pandurangan

    2014-08-01

    The present study was carried out to understand the effect of cortisol on heat shock protein system (Hsps) in the C2C12 and 3T3-L1 cells under co-culture system. Cells were co-cultured by using Transwell inserts with a 0.4-?m porous membrane to separate C2C12 and 3T3-L1 cells. Each cell type was grown independently on the Transwell plates. After cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 cells transferred to 3T3-L1 plates. Ten micrograms per microliter of cortisol was added to the medium. Following 72 h of treatment, the cells in the lower wells were harvested for analysis. Heat shock proteins (Hsps) such as Hsp27, Hsp70, and Hsp90 were selected for the analysis. The qRT-PCR results showed the significant increase in the mRNA expression of as Hsp27, Hsp70, and Hsp90. In addition, confocal microscopical investigation showed the cortisol treatment increases Hsps expressions in the mono and co-cultured C2C12 and 3T3-L1 cells. From the results, we concluded that the cortisol increases Hsps expression in the co-cultured C2C12 and 3T3-L1 cells, which is differed from one-dimensional mono-cultured C2C12 and 3T3-L1 cells. PMID:24825387

  17. Automatic optimal feeder design in steel casting process

    Microsoft Academic Search

    Rohallah Tavakoli; Parviz Davami

    2008-01-01

    A method for automatic optimal feeder design in steel casting processes is presented. The initial design is the casting part (without feeders) which is placed in a suitable mold box. Design of each feeder contains the following steps: determination of the feeder-neck connection point on the casting surface, initial feeder design, feeder shape optimization and feeder topology optimization. Completing designing

  18. Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts

    PubMed Central

    1990-01-01

    Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected. PMID:2114410

  19. Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution.

    PubMed

    Rizzatti, V; Boschi, F; Pedrotti, M; Zoico, E; Sbarbati, A; Zamboni, M

    2013-01-01

    The 3T3-L1 cell line, derived from 3T3 cells, is widely used in biological research on adipose tissue. 3T3-L1 cells have a fibroblast-like morphology, but, under appropriate conditions, they differentiate into an adipocyte-like phenotype. During the differentiation process, 3T3-L1 cells increase the synthesis of triglycerides and acquire the behavior of adipose cells. In particular, triglycerides accumulate in lipid droplets (LDs) embedded in the cytoplasm. The number and the size distribution of the LDs is often correlated with obesity and many other pathologies linked with fat accumulation. The integrated optical density (IOD) of the LDs is related with the amount of triglycerides in the droplets. The aim of this study is the attempt to characterize the size distribution and the IOD of the LDs in 3T3-L1 differentiated cells. The cells were differentiated into adipocytes for 5 days with a standard procedure, stained with Oil Red O and observed with an optical microscope. The diameter, area, optical density of the LDs were measured. We found an asymmetry of the kernel density distribution of the maximum Feret's diameter of the LDs with a tail due to very large LDs. More information regarding the birth of the LDs could help in finding the best mathematical model in order to analyze fat accumulation in adipocytes. PMID:24085273

  20. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  1. Serum-induced G0/G1 transition in chemically transformed 3T3 cells

    SciTech Connect

    Gray, H.E.; Buchou, T.; Mester, J.

    1987-03-01

    Quiescent, chemically transformed (benzo-a-pyrene) BALB/c 3T3 cells (BP A31) enter the cell division cycle when exposed to complete medium containing 10% fetal calf serum (FCS); the number of cells recruited is a function of the duration of serum exposure. The recruitment of cells by short (<4 h) serum pulses is not inhibited by simultaneous exposure to cycloheximide (CH), and therefore the initial commitment does not require protein synthesis. The cells enter S phase with a constant delay following the removal of CH, even if CH exposure has been continued for as long as 20 h after the end of the serum pulse. The cell recruitment by serum pulses was inhibited by 5,6-dichloro-1-..beta..-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of cytoplasmic mRNA accumulation. These data suggest that serum exposure produces a stable memory that is necessary and sufficient for the eventual progression through G1 to S phase that occurs when protein synthesis is resumed after the removal of CH; this memory probably consists of mRNA species that are induced by serum and that are stable in the absence of protein synthesis. Unexpectedly, pretreatment of quiescent BP A31 cells with CH (8-24 h) dramatically increased the fraction of the total cell population that is recruited by a serum pulse of fixed duration.

  2. Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells

    NASA Technical Reports Server (NTRS)

    Lin, H. S.; Hughes-Fulford, M.; Kumegawa, M.; Pitts, A. C.; Snowdowne, K. W.

    1993-01-01

    We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

  3. Flavanone exhibits PPARgamma ligand activity and enhances differentiation of 3T3-L1 adipocytes.

    PubMed

    Saito, Takeshi; Abe, Daigo; Sekiya, Keizo

    2009-03-01

    Flavanones are class of polyphenolic compounds, some of which are found in foods and provide health benefits. In this study, we show that flavanone significantly enhances differentiation of 3T3-L1 preadipocytes. During adipogenesis, flavanone enhanced expression of genes and accumulation of proteins that are involved in adipocyte function. Some reports have indicated that flavanone inhibits proliferation of mammalian cells, and down-regulates expression of growth-related proteins. Such proteins include phosphorylated ERK1/2, cyclins, and Cdks that are important for an early event in adipogenesis, mitotic clonal expansion (MCE). We demonstrated that flavanone did not inhibit MCE or expression of MCE-related proteins, except for a modest inhibition of cyclin D1 expression. Using luciferase reporter assays, we found that flavanone acted as a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand in a dose-dependent manner. Together, our results suggest that flavanone enhances adipogenesis, at least in part, through its PPARgamma ligand activity. PMID:19167359

  4. Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte.

    PubMed

    Fan, Huijin; Wu, Dan; Tian, Weixi; Ma, Xiaofeng

    2013-07-01

    Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC50) of 0.14 microM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARgamma during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity. PMID:24046866

  5. Monocyte chemoattractant protein-induced protein 1 impairs adipogenesis in 3T3-L1 cells.

    PubMed

    Lipert, Barbara; Wegrzyn, Paulina; Sell, Henrike; Eckel, Juergen; Winiarski, Marek; Budzynski, Andrzej; Matlok, Maciej; Kotlinowski, Jerzy; Ramage, Lindsay; Malecki, Maciej; Wilk, Waclaw; Mitus, Jerzy; Jura, Jolanta

    2014-04-01

    Monocyte chemoattractant protein-induced protein 1 (MCPIP1) encoded by the ZC3H12a gene (also known as Regnase-1) is involved in the regulation of degradation of mRNA of inflammatory modulators and for processing of pre-miRNA. These functions depend on the presence of the PIN domain. Moreover, MCPIP1 was described as a negative regulator of NF-?B and AP-1 signaling pathways although mechanisms underlying such activity remain unknown. We aimed at determining the role of MCPIP1 in adipogenesis. Here, we present evidence that Mcpip1 transcription is transiently activated during 3T3-L1 transition from pre- to adipocytes. However Mcpip1 protein expression is also strongly decreased at day one after induction of adipogenesis. Knockdown of Mcpip1 results in an upregulation of C/EBP? and PPAR? mRNAs, whereas overexpression of MCPIP1 reduces the level of both transcription factors and impairs adipogenesis. MCPIP1-dependend modulation of C/EBP? and PPAR? levels results in a modulation of the expression of downstream controlled genes. In addition, decreased C/EBP?, but not PPAR?, depends on the activity of the MCPIP1 PIN domain, which is responsible for RNase properties of this protein. Together, these data confirm that MCPIP1 is a key regulator of adipogenesis. PMID:24418043

  6. Hierarchical polymeric scaffolds support the growth of MC3T3-E1 cells.

    PubMed

    Akbarzadeh, Rosa; Minton, Joshua A; Janney, Cara S; Smith, Tyler A; James, Paul F; Yousefi, Azizeh-Mitra

    2015-02-01

    Tissue engineering makes use of the principles of biology and engineering to sustain 3D cell growth and promote tissue repair and/or regeneration. In this study, macro/microporous scaffold architectures have been developed using a hybrid solid freeform fabrication/thermally induced phase separation (TIPS) technique. Poly(lactic-co-glycolic acid) (PLGA) dissolved in 1,4-dioxane was used to generate a microporous matrix by the TIPS method. The 3D-bioplotting technique was used to fabricate 3D macroporous constructs made of polyethylene glycol (PEG). Embedding the PEG constructs inside the PLGA solution prior to the TIPS process and subsequent extraction of PEG following solvent removal (1,4-dioaxane) resulted in a macro/microporous structure. These hierarchical scaffolds with a bimodal pore size distribution (<50 and >300 ?m) contained orthogonally interconnected macro-channels generated by the extracted PEG. The diameter of the macro-channels was varied by tuning the dispensing parameters of the 3D bioplotter. The in vitro cell culture using murine MC3T3-E1 cell line for 21 days demonstrated that these scaffolds could provide a favorable environment to support cell adhesion and growth. PMID:25665851

  7. Attachment and spreadout study of 3T3 cells onto PP track etched films

    NASA Astrophysics Data System (ADS)

    Smolko, Eduardo; Mazzei, Ruben; Tadey, Daniel; Lombardo, Daniel

    2001-12-01

    Polymer surface modifications are obtained by the application of radiation treatments and other physico-chemical methods: fission fragment (ff) irradiation and etching. The biocompatibility of the surface is then observed by cell seeding and cell adhesion experiments. Approaches to improvement of the cell adhesion are obtained by different methods: for example, in PS, cell adhesion is improved after ion implantation; in PMMA, after bombarding the polymer, the surface is reconditioned with surfactants and proteins and in PVDF, cell adhesion is assayed on nuclear tracks membranes. In this work, we obtained important cell adhesion improvements in PP films by irradiation with swift heavy ions and subsequent etching of the nuclear tracks. We use BOPP (isotactic -25 ?m thickness). Irrradiations were performed with a Cf-252 californium ff source. The source has a heavy ff and a light one, with 160-200 MeV energy divided among them corresponding to ff energies between 1 and 2 MeV/amu. A chemical etching procedure consisting of a solution of sulphuric acid and chromium three oxide at 85 °C was used. The 3T3 NIH fibroblast cell line was used for the cell adhesion experiment. Here we report for the first time, the results of a series of experiments by varying the ff fluence and the etching time showing that attachment and spreadout of cells are very much improved in this cell line according to the number of pores and the pore size.

  8. Growth, cell cycle progression, and morphology of 3T3 cells following fibroin microsphere ingestion.

    PubMed

    Go, Nam Kyung; Lee, Jin Sil; Lee, Joon Ho; Hur, Won

    2015-04-01

    Cellular uptake of microspheres may cause physiological stress and toxicity. In this report, we investigated the effect of cellular uptake of fibroin microspheres on the growth, cell cycle progression, and morphology of 3T3 cells. The microspheres were prepared by physical cross-linking of fibroin molecules without any chemical modification. Fluorescent microspheres are comprised of fluorescein isothiocyanate-dextran core and fibroin shell. More than 90% of cells were determined to be fluorescence-positive following 24-h incubation with fluorescent microspheres (0.17 mg/mL). Microsphere localization in the cytoplasm was demonstrated using confocal and transmission electron microscopy. Cellular uptake of microspheres did not influence cellular viability, but microsphere concentrations above 0.1 mg/mL resulted in decreased cell proliferation. The proliferation inhibition was attributed to G2 /M phase delay in cell cycle progression and S-phase delay at higher microsphere concentrations (0.33 mg/mL). Although flow cytometry light-scattering data raised the possibility of morphological changes, Coulter counter analysis confirmed no significant size differences between cells incubated with and without microspheres. Accordingly, fibroin microspheres can be a potential vehicle for intracytoplasmic delivery of cargos, without affecting cell viability. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 1325-1331, 2015. PMID:25044553

  9. Rubus suavissimus S. Lee extract increases early adipogenesis in 3T3-L1 preadipocytes.

    PubMed

    Ezure, Tomonobu; Amano, Satoshi

    2011-04-01

    Leaves of Rubus suavissimus S. Lee (Rosaceae) are used to prepare tiencha or sweet tea, which is helpful for body weight control by restricting calorie intake in obese patients. Obesity is a risk factor for metabolic syndrome, and a possible approach to treatment is to promote early adipogenesis in adipose tissue, thereby leading to replacement of enlarged adipocytes that secrete inflammatory factors with small adipocytes.We therefore investigated the effect of extract of tiencha leaves on early adipogenesis by using 3T3-L1 preadipocytes as a model. Tiencha extract significantly and concentration-dependently increased adipogenesis measured in terms of lipid accumulation by means of Oil Red O assay and increased the expression of adiponectin and leptin. In the early phase of adipogenesis, tiencha extract increased the mRNA expression of adipogenic transcription factors CCAAT/enhancer binding protein ? (C/EBP?) and proliferator-activated receptor ? (PPAR?). In contrast, mRNA expression of other adipogenic transcription factors, C/EBP? and C/EBP?, was unaffected. The mRNA expression levels of adipocyte-specific genes encoding adipocyte protein 2 (aP2), lipoprotein lipase (LPL), and glucose transporter 4 (Glut4), which are regulated by C/EBP? and PPAR?, were also increased. A PPAR? inhibitor, GW9662, partially inhibited the enhancing effect of tiencha extract on lipogenesis. These results suggest that tiencha extract enhances early adipogenesis by increasing the expression of adipogenic transcription factors C/EBP? and PPAR?. PMID:21082272

  10. Induction of Adipocyte Differentiation by Polybrominated Diphenyl Ethers (PBDEs) in 3T3-L1 Cells

    PubMed Central

    Tung, Emily W. Y.; Boudreau, Adčle; Wade, Michael G.; Atlas, Ella

    2014-01-01

    Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that were extensively used in commercial products. PBDEs are ubiquitous environmental contaminants that are both lipophilic and bioaccumulative. Effects of PBDEs on adipogenesis were studied in the 3T3-L1 preadipocyte cell model in the presence and absence of a known adipogenic agent, dexamethasone (DEX). A PBDE mixture designed to mimic body burden of North Americans was tested, in addition to the technical mixture DE-71 and the individual congener BDE-47. The mixture, DE-71, and BDE-47 all induced adipocyte differentiation as assessed by markers for terminal differentiation [fatty acid binding protein 4 (aP2) and perilipin] and lipid accumulation. Characterization of the differentiation process in response to PBDEs indicated that adipogenesis induced by a minimally effective dose of DEX was enhanced by these PBDEs. Moreover, C/EBP?, PPAR?, and LXR? were induced late in the differentiation process. Taken together, these data indicate that adipocyte differentiation is induced by PBDEs; they act in the absence of glucocorticoid and enhance glucocorticoid-mediated adipogenesis. PMID:24722056

  11. Mineralization initiation of MC3T3-E1 preosteoblast is suppressed under simulated microgravity condition.

    PubMed

    Hu, Li-Fang; Li, Jing-Bao; Qian, Ai-Rong; Wang, Fei; Shang, Peng

    2015-04-01

    Microgravity decreases the differentiation of osteoblast. However, as this process is multistage and complex, the mechanism by which microgravity inhibits osteoblast differentiation is still unclear. We have previously found that 24?h acute treatment of simulated microgravity (SM) with a random positioning machine (RPM) significantly inhibited the differentiation of preosteoblasts and have explored whether osteoblasts show different response to microgravity condition at other stages, such as the mineralizing-stage. Murine MC3T3-E1 preosteoblasts induced for osteogenic differentiation for seven days were cultured either under normal gravity or SM conditions for 24?h. SM treatment significantly suppressed mineralized nodule formation. Alkaline phosphatase (ALP) activity was dramatically decreased, and the expression of ALP gene was downregulated. Expression of well-known markers and regulators for osteoblasts differentiation, including osteocalcin (OC), type I collagen ?1 (Col I?1), dentin matrix protein 1 (DMP1) and runt-related transcription factor 2 (Runx2), were downregulated. Western blot analysis showed that the phosphorylated extracellular signal-regulated kinase (p-ERK) level was lower under SM condition. Thus, the initiation of osteoblast mineralization is suppressed by SM condition, and the suppression may be through the regulation of ALP activity and the osteogenic gene expression. ERK signaling might be involved in this process. These results are relevant to the decrease of osteoblast maturation and bone formation under microgravity condition. PMID:25318973

  12. Nobiletin enhances differentiation and lipolysis of 3T3-L1 adipocytes

    SciTech Connect

    Saito, Takeshi [National Agricultural Research Center for Western Region, 1-3-1 Senyu-cho, Zentsuji 765-8508 (Japan); Abe, Daigo [National Agricultural Research Center for Western Region, 1-3-1 Senyu-cho, Zentsuji 765-8508 (Japan); Sekiya, Keizo [National Agricultural Research Center for Western Region, 1-3-1 Senyu-cho, Zentsuji 765-8508 (Japan)]. E-mail: ksekiya@affrc.go.jp

    2007-06-01

    Nobiletin is a polymethoxylated flavone found in certain citrus fruits. Here we demonstrate that nobiletin enhance differentiation of 3T3-L1 preadipocytes. Nobiletin dose-dependently increased accumulation of lipid droplets in adipocytes. Quantitative RT-PCR analyses indicated that nobiletin increased the expression of genes critical for acquisition of the adipocyte phenotype. Some of them were known peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) targets and PPAR{gamma} itself, however, nobiletin did not exhibit PPAR{gamma} ligand activity. We observed the expression of CCAAT/enhancer binding protein {beta} (C/EBP{beta}), a transcription factor for PPAR{gamma}, was increased by nobiletin. The activation of cAMP-responsive element binding protein (CREB) and extracellular signal-regulated kinase (ERK), which play important roles in C/EBP{beta} expression were also potentiated by nobiletin. Furthermore, nobiletin stimulated lipolysis in differentiated adipocytes, which is known to be stimulated by cAMP pathway. These results suggested that nobiletin enhanced both differentiation and lipolysis of adipocyte through activation of signaling cascades mediated by cAMP/CREB.

  13. Entrapment of Saccharomyces cerevisiae and 3T3 fibroblast cells into blue light cured hydrogels.

    PubMed

    Mishra, Swati; Scarano, Frank J; Calvert, Paul

    2012-10-01

    Hydrogels, containing yeast cells or fibroblast cells, were fabricated using blue light-induced polymerization technique. The cell-loaded prepolymer formulation was comprised of poly(ethyleneglycol) diacrylate (more than or equal to 50% v/v), 0.5 wt % Eosin Y and 0.5 wt % triethanolamine as the base oligomer, photo-initiator, and co-initiator, respectively. The two model cell lines, Saccharomyces cerevisiae and NIH 3T3 fibroblasts maintained high viability pre- and post-processing. Several bioassays have demonstrated the unaffected intracellular and extracellular activities of the cells entrapped within the hydrogels. Scanning electron microscopy confirmed the proliferation of S. cerevisiae cells that were entrapped and cultivated for 48 h in growth media, which validated the favorable microenvironment and nutrient transport in these gels. Upon entrapment, fibroblast cells remain viable upto 12 h, however they failed to attach within the crosslinked network, thus no further proliferation was observed. The tunable properties of this hydrogel system project it as a useful matrix for specialized biohybrids. PMID:22678829

  14. Identification of pathway-based toxicity in the BALB/c 3T3 cell model.

    PubMed

    Vaccari, Monica; Mascolo, Maria Grazia; Rotondo, Francesca; Morandi, Elena; Quercioli, Daniele; Perdichizzi, Stefania; Zanzi, Cristina; Serra, Stefania; Poluzzi, Vanes; Angelini, Paola; Grilli, Sandro; Colacci, Annamaria

    2014-10-16

    The particulate matter represents one of the most complex environmental mixtures, whose effects on human health and environment vary according to particles characteristics and source of emissions. The present study describes an integrated approach, including in vitro tests and toxicogenomics, to highlight the effects of air particulate matter on toxicological relevant endpoints. Air samples (PM2.5) were collected in summer and winter at different sites, representative of different levels of air pollution. Samples organic extracts were tested in the BALB/c 3T3 CTA at a dose range 1-12m(3). The effect of the exposure to the samples at a dose of 8m(3) on the whole-genome transcriptomic profile was also assessed. All the collected samples induced dose-related toxic effects in the exposed cells. The modulated gene pathways confirmed that toxicity was related to sampling season and sampling site. The analysis of the KEGG's pathways showed modulation of several gene networks related to oxidative stress and inflammation. Even if the samples did not induce cell transformation in the treated cells, gene pathways related to the onset of cancer were modulated as a consequence of the exposure. This integrated approach could provide valuable information for predicting toxic risks in humans exposed to air pollution. PMID:25450744

  15. Identification of LRP16 as a negative regulator of insulin action and adipogenesis in 3T3-L1 adipocytes.

    PubMed

    Zang, L; Xue, B; Lu, Z; Li, X; Yang, G; Guo, Q; Ba, J; Zou, X; Dou, J; Lu, J; Pan, C; Mu, Y

    2013-05-01

    Leukemia related protein 16 (LRP16) was first cloned from acute myeloid leukemia cells in our laboratory. In the present study, we sought to investigate the role of LRP16 in insulin action and sensitivity, using LRP16-depleted and -overexpressing 3T3-L1 cells. LRP16 silencing resulted in a reduction of the expression and secretion of tumor necrosis factor-alpha (TNF-?) and a concomitant increase in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-?). Moreover, LRP16 depletion promoted insulin-induced glucose uptake and adipocyte differentiation of 3T3-L1 cells. In contrast, LRP16 overexpression increased TNF-? secretion, suppressed glucose uptake, and attenuated 3T3-L1 cell differentiation. The phosphorylation levels of insulin receptor substrate 1 (IRS-1), phosphatidylinositide 3-kinase (PI3-K), and Akt were increased in LRP16-deficient 3T3-L1 cells, and conversely, diminished in LRP16-overexpressing 3T3-L1 cells, when compared to the corresponding control cells. Additionally, LRP16 overexpression raised the phosphorylation level of mammalian target of rapamycin (mTOR). The pretreatment with rapamycin, a specific inhibitor of mTOR, prevented the TNF-? elevation and PPAR-? reduction and restored the phosphorylation of IRS-1, PI3-K, and Akt in LRP16-overexpressing cells. Our data collectively indicate that LRP16 acts as a negative regulator of insulin action and adipogenesis in 3T3-L1 adipocytes, which involves the activation of the mTOR signaling pathway. PMID:23389992

  16. Hydroxytyrosol promotes mitochondrial biogenesis and mitochondrial function in 3T3-L1 adipocytes.

    PubMed

    Hao, Jiejie; Shen, Weili; Yu, Guangli; Jia, Haiqun; Li, Xuesen; Feng, Zhihui; Wang, Ying; Weber, Peter; Wertz, Karin; Sharman, Edward; Liu, Jiankang

    2010-07-01

    Hydroxytyrosol (HT) in extra-virgin olive oil is considered one of the most important polyphenolic compounds responsible for the health benefits of the Mediterranean diet for lowering incidence of cardiovascular disease, the most common and most serious complication of diabetes. We propose that HT may prevent these diseases by a stimulation of mitochondrial biogenesis that leads to enhancement of mitochondrial function and cellular defense systems. In the present study, we investigated effects of HT that stimulate mitochondrial biogenesis and promote mitochondrial function in 3T3-L1 adipocytes. HT over the concentration range of 0.1-10 micromol/L stimulated the promoter transcriptional activation and protein expression of peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha (PPARGC1 alpha, the central factor for mitochondrial biogenesis) and its downstream targets; these included nuclear respiration factors 1 and 2 and mitochondrial transcription factor A, which leads to an increase in mitochondrial DNA (mtDNA) and in the number of mitochondria. Knockdown of Ppargc1 alpha by siRNA blocked HT's stimulating effect on Complex I expression and mtDNA copy number. The HT treatment resulted in an enhancement of mitochondrial function, including an increase in activity and protein expression of Mitochondrial Complexes I, II, III and V; increased oxygen consumption; and a decrease in free fatty acid contents in the adipocytes. The mechanistic study of the PPARGC1 alpha activation signaling pathway demonstrated that HT is an activator of 5'AMP-activated protein kinase and also up-regulates gene expression of PPAR alpha, CPT-1 and PPAR gamma. These data suggest that HT is able to promote mitochondrial function by stimulating mitochondrial biogenesis. PMID:19576748

  17. ATF3 inhibits adipocyte differentiation of 3T3-L1 cells.

    PubMed

    Jang, Min Kyung; Kim, Cho Hee; Seong, Je Kyung; Jung, Myeong Ho

    2012-04-27

    ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBP? transcript and repressed the activity of the 3.6-kb mouse C/EBP? promoter, demonstrating that ATF3 downregulates C/EBP? expression. Transfection studies using mutant constructs containing 5'-deletions in the C/EBP? promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between -1921 and -1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBP? promoter spanning from -1928 to -1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBP? mRNA and repress the promoter activity of the C/EBP? gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBP? expression. Collectively, these results demonstrate that ATF3 represses the C/EBP? gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition of adipocyte differentiation. PMID:22475484

  18. A surface-tethered spheroid model for functional evaluation of 3T3-L1 adipocytes.

    PubMed

    Turner, Paul A; Harris, Lacey M; Purser, Christine A; Baker, Rodney C; Janorkar, Amol V

    2014-01-01

    In order to effectively treat obesity, it must be better understood at the cellular level with respect to metabolic state and environmental stress. However, current two-dimensional (2D) in vitro cell culture methods do not represent the in vivo adipose tissue appropriately due to the absence of complex architecture and cellular signaling. Conversely, 3D in vitro cultures have been reported to have optimal results mimicking the adipose tissue in vivo. The main aim of this study was to examine the efficacy of a novel conjugate of a genetically engineered polymer, elastin-like polypeptide (ELP) and a synthetic polymer, polyethyleneimine (PEI), toward creating a 3D preadipocyte culture system. We then used this 3D culture model to study the preadipocyte differentiation and adipocyte maintenance processes when subjected to various dosages of nutritionally relevant free fatty acids with respect to total DNA and protein content, cell viability, and intracellular triglyceride accumulation. Our results showed that 3T3-L1 preadipocytes cultured on the ELP-PEI surface formed 3D spheroids within 72?h, whereas the cells cultured on unmodified tissue culture polystyrene surfaces remained in monolayer configuration. Significant statistical differences were discovered between the 3D spheroid and 2D monolayer culture with respect to the DNA and protein content, fatty acid consumption, and triglyceride accumulation, indicating differences in cellular response. Results indicated that the 3D culture may be a more sensitive modeling technique for in vitro adipocyte culture and provides a platform for future evaluation of 3D in vitro adipocyte function. PMID:24038000

  19. Analysis of Transcription Factor Network Underlying 3T3-L1 Adipocyte Differentiation

    PubMed Central

    Choi, KyungOh; Ghaddar, Bassel; Moya, Colby; Shi, Hai; Sridharan, Gautham V.; Lee, Kyongbum; Jayaraman, Arul

    2014-01-01

    Lipid accumulation in adipocytes reflects a balance between enzymatic pathways leading to the formation and breakdown of esterified lipids, primarily triglycerides. This balance is extremely important, as both high and low lipid levels in adipocytes can have deleterious consequences. The enzymes responsible for lipid synthesis and breakdown (lipogenesis and lipolysis, respectively) are regulated through the coordinated actions of several transcription factors (TFs). In this study, we examined the dynamics of several key transcription factors (TFs) - PPAR?, C/EBP?, CREB, NFAT, FoxO1, and SREBP-1c - during adipogenic differentiation (week 1) and ensuing lipid accumulation. The activation profiles of these TFs at different times following induction of adipogenic differentiation were quantified using 3T3-L1 reporter cell lines constructed to secrete the Gaussia luciferase enzyme upon binding of a TF to its DNA binding element. The dynamics of the TFs was also modeled using a combination of logical gates and ordinary differential equations, where the logical gates were used to explore different combinations of activating inputs for PPAR?, C/EBP?, and SREBP-1c. Comparisons of the experimental profiles and model simulations suggest that SREBP-1c could be independently activated by either insulin or PPAR?, whereas PPAR? activation required both C/EBP? as well as a putative ligand. Parameter estimation and sensitivity analysis indicate that feedback activation of SREBP-1c by PPAR? is negligible in comparison to activation of SREBP-1c by insulin. On the other hand, the production of an activating ligand could quantitatively contribute to a sustained elevation in PPAR? activity. PMID:25075860

  20. Inositol hexakisphosphate inhibits mineralization of MC3T3-E1 osteoblast cultures.

    PubMed

    Addison, William N; McKee, Marc D

    2010-04-01

    Inositol hexakisphosphate (IP6, phytic acid) is an endogenous compound present in mammalian cells and tissues. Differentially phosphorylated forms of inositol are well-documented to have important roles in signal transduction, cell proliferation and differentiation, and IP6 in particular has been suggested to inhibit soft tissue calcification (specifically renal and vascular calcification) by binding extracellularly to calcium oxalate and calcium phosphate crystals. However, the effects of IP6 on bone mineralization are largely unknown. In this study, we used MC3T3-E1 osteoblast cultures to examine the effects of exogenous IP6 on osteoblast function and matrix mineralization. IP6 at physiologic concentrations caused a dose-dependent inhibition of mineralization without affecting cell viability, proliferation or collagen deposition. Osteoblast differentiation markers, including tissue-nonspecific alkaline phosphatase activity, bone sialoprotein and osteocalcin mRNA levels, were not adversely affected by IP6 treatment. On the other hand, IP6 markedly increased protein and mRNA levels of osteopontin, a potent inhibitor of crystal growth and matrix mineralization. Inositol alone (without phosphate), as well as inositol hexakis-sulphate, a compound with a high negative charge similar to IP6, had no effect on mineralization or osteopontin induction. Binding of IP6 to mineral crystals from the osteoblast cultures, as well as to synthetic hydroxyapatite crystals, was confirmed by a colorimetric assay for IP6. In summary, IP6 inhibits mineralization of osteoblast cultures by binding to growing crystals through negatively charged phosphate groups and by induction of inhibitory osteopontin expression. These data suggest that IP6 may regulate physiologic bone mineralization by directly acting extracellularly, and by serving as a specific signal at the cellular level for the regulation of osteopontin gene expression. PMID:20079473

  1. Sphingosine kinase is induced in mouse 3T3-L1 cells and promotes adipogenesiss?

    PubMed Central

    Hashimoto, Takeshi; Igarashi, Junsuke; Kosaka, Hiroaki

    2009-01-01

    Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that exerts numerous biological activities both as a receptor ligand and as an intracellular second messenger. In the present study, we explored roles of sphingosine kinase (SphK), an S1P-producing enzyme, in adipose tissue. We utilized mouse 3T3-L1 cells as an in vitro model of adipogenesis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. Real-time quantitative PCR (qRT-PCR) assays revealed that the expression levels of transcripts encoding both isoforms of SphK-1 and SphK-2 are up-regulated during adipogenesis (37.6- and 6.6-fold vs. basal, P < 0.05, respectively). Concomitantly, SphK-1/SphK-2 protein abundance and S1P contents of these cells increased at 3 days after hormonal stimulation. Loss-of-function approaches by pharmacological inhibition of SphK activity as well as by transfection with small interfering RNA (siRNA) against SphK-1 led to significant attenuation of lipid droplet accumulation and adipocyte marker gene expression. We detected marked elevation of SphK-1 mRNA in adipose tissue derived from 13-week-old ob/ob mice with obese phenotype than their lean littermates. These results suggest that increased expression of SphK, an S1P-producing enzyme, plays a significant role during adipogenesis, potentially providing a novel point of control in adipose tissue. PMID:19020339

  2. Gene expression of MC3T3-E1 osteoblastic cells on titanium and zirconia surface

    PubMed Central

    Gong, Soon-Hyun; Lee, Heesu; Pae, Ahran; Noh, Kwantae; Shin, Yong-Moon; Lee, Jung-Haeng

    2013-01-01

    PURPOSE This study was performed to define attachment and growth behavior of osteoblast-like cells and evaluate the gene expression on zirconia compared to titanium. MATERIALS AND METHODS MC3T3-E1 cells were cultured on (1) titanium and (2) zirconia discs. The tetrazolium-based colorimetric assay (MTT test) was used for examining the attachment of cells. Cellular morphology was examined by scanning electron microscopy (SEM) and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation rate. Mann-Whitney test was used to assess the significance level of the differences between the experimental groups. cDNA microarray was used for comparing the 20215 gene expressions on titanium and zirconia. RESULTS From the MTT assay, there was no significant difference between titanium and zirconia (P>.05). From the SEM image, after 4 hours of culture, cells on both discs were triangular or elongated in shape with formation of filopodia. After 24 hours of culture, cells on both discs were more flattened and well spread compared to 4 hours of culture. From the ALP activity assay, the optical density of E1 cells on titanium was slightly higher than that of E1 cells on zirconia but there was no significant difference (P>.05). Most of the genes related to cell adhesion showed similar expression level between titanium and zirconia. CONCLUSION Zirconia showed comparable biological responses of osteoblast-like cells to titanium for a short time during cell culture period. Most of the genes related to cell adhesion and signal showed similar expression level between titanium and zirconia. PMID:24353879

  3. Positive regulations of adipogenesis by Italian ryegrass [Lolium multiflorum] in 3T3-L1 cells

    PubMed Central

    2014-01-01

    Back ground Intramuscular fat deposition in the meat animal is relatively new strategy for developing the meat quality. Fat deposition is largely depending on the adipocyte proliferation and differentiation. Therefore, we investigated the effect of chloroform extract of L. multiflorum [CELM] on cell proliferation, lipid accumulation and adipocyte differentiation in 3T3-L1 cells and body weight of mouse. Results We identified 6,9-Octadecatrienoic acid, Hexadecanoic acid, 2-hydroxypropanoic acid, butane-2,3-diol and hexane-1,2,3,4,5,6-hexaol in CELM. L. multiflorum extract increased the cell viability, lipid accumulation, cell cycle progression and key transcriptional and secretory factors like PPRA?2, C/CEBP-?, adiponectin, aP2, GLUT-4, FAS and SREBP-1 mRNA expression as compared with control cells. For in-vivo, mice administered with CELM significantly increased body weight throughout the experiment periods. Further, the identified fatty acids like 3, 6, 9-Octadecatrienoic acid and Hexadecanoic acid was docked with target protein [PPRA?2] using HEX 6.12. The least binding energy considered as high affinity with target protein. The maximum affinity with the target protein was observed in the Hexadecanoic acid followed by 3, 6, 9-Octadecatrienoic acid. The binding efficacy of Hexadecanoic acid and 3, 6, 9-Octadecatrienoic acid to the active site of PPAR-?2 may be enhanced the adipocyte differentiations. Conclusion These findings suggest that CELM stimulates adipogenesis via activating the PPAR?-mediated signaling pathway in adipocyte which could be useful for the development of meat quality in animals. PMID:24917384

  4. The Role of Uncoupling Protein 2 in the Development of Insulin Resistance in 3T3-L1 Adipocytes

    E-print Network

    Goodwin, David A

    2012-07-11

    succeeded in cloning the UCP2 gene into an inducible plasmid, pRev-TRE, inducible by the addition of Doxycycline. The development of a retroviral vector, able to integrate the pRev-TRE UCP2 plasmid into the genome of 3T3-L1 adipocytes, is currently underway...

  5. Effect of cortisol on calpains in the C2C12 and 3T3-L1 cells.

    PubMed

    Muthuraman, Pandurangan; Ravikumar, Sambandam; Muthuviveganandavel, Veerappan; Kim, Jongpil

    2014-03-01

    The present study was carried out to understand the effect of cortisol on calpain system in the C2C12 and 3T3-L1 adipocyte cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4 ?m porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates. Ten microgram per milliliter of cortisol was added to the medium. Following treatment for 3 days, the cells in the lower well were harvested for analysis. Calpains such as ?-calpain, m-calpain, and calpastatin were selected for the analysis. RT-PCR results indicated the significant increase in the mRNA expression of ?-calpain, m-calpain, and calpastatin. In addition, the confocal microscopical investigation indicated the cortisol treatment increases calpain expression in the C2C12 and 3T3-L1 cells. Taking all these together, cortisol treatment with co-culture system shows most reliable status of calpains expression in the cells, which is quite distinct from one-dimensional monocultured cells. PMID:24497045

  6. Induction of a negative autocrine loop by expression of sst2 somatostatin receptor in NIH 3T3 cells.

    PubMed Central

    Rauly, I; Saint-Laurent, N; Delesque, N; Buscail, L; Estéve, J P; Vaysse, N; Susini, C

    1996-01-01

    The somatostatin receptor subtype sst2 mediates both activation of a tyrosine phosphatase activity and inhibition of cell proliferation induced by somatostatin analogues. In the absence of exogenous ligand, expression of sst2 in NIH 3T3 cells resulted in inhibition of cell growth. Polymerase chain reaction coupled to reverse transcription demonstrated that expression of sst2 in NIH 3T3 cells stimulated the expression of preprosomatostatin mRNA accompanied by a production of immunoreactive somatostatin-like peptide which corresponded predominantly to somatostatin 14. Moreover anti-somatostatin antibodies suppressed sst2-promoted inhibition of cell proliferation. Inhibition of cell proliferation associated with increased secretion of somatostatin-like immunoreactivity was also observed after expression of sst2 in human pancreatic tumor cells BxPC3 devoid of endogenous receptors. In addition, expression of sst2 in NIH 3T3 cells was associated with constitutive activation of tyrosine phosphatase PTP1C that resulted from enhanced expression of the protein. Blocking of PTP1C tyrosine phosphatase activity with orthovanadate or that of PTP1C protein with antisense PTP1C oligonucleotides decreased the sst2-induced inhibition of cell proliferation. These results, taken together, show that expression of sst2 in NIH 3T3 cells generated a negative autocrine loop by stimulating sst2 ligand production and amplifying PTP1C sst2-transducer. Sst2/ligand may function as a determinant factor involved in the negative growth control of cells. PMID:8621771

  7. Molecular mechanism of 9-cis-retinoic acid inhibition of adipogenesis in 3T3-L1 cells

    SciTech Connect

    Sagara, Chiaki; Takahashi, Katsuhiko [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)] [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan); Kagechika, Hiroyuki [School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo 101-0062 (Japan)] [School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo 101-0062 (Japan); Takahashi, Noriko, E-mail: t-noriko@hoshi.ac.jp [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)] [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)

    2013-03-29

    Highlights: ? We examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1. ? 9-cis-RA inhibited lipid accumulation in adipogenetically-induced 3T3-L1 cells. ? A RXR pan-antagonist suppressed the inhibitory effects of 9-cis-RA on adipogenesis. ? This antagonist had no effects on RXR? and PPAR? levels in 9-cis-RA-treated cells. ? 9-cis-RA-induced decrease in both RXR? and PPAR? was independent of RXR activation. -- Abstract: Retinoic acid (RA) signaling is mediated by specific nuclear hormone receptors. Here we examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1 cells. 9-cis-RA inhibits the lipid accumulation of adipogenetically induced 3T3-L1 cells. The complex of retinoid X receptor ? (RXR?) with peroxisome proliferator-activated receptor ? (PPAR?) is a major transcription factor in the process of adipogenesis, and the levels of these molecules were decreased by 9-cis-RA treatment. A RXR pan-antagonist suppressed 9-cis-RA’s inhibitory effects on adipogenesis, but not on the intracellular levels of both RXR? and PPAR?. These results suggest that 9-cis-RA could inhibit adipogenesis by activating RXR, and decrease both RXR and PPAR?s levels in a RXR activation-independent manner.

  8. Sinensetin Enhances Adipogenesis and Lipolysis by Increasing Cyclic Adenosine Monophosphate Levels in 3T3-L1 Adipocytes.

    PubMed

    Kang, Seong-Il; Shin, Hye-Sun; Kim, Se-Jae

    2015-04-01

    Sinensetin is a rare polymethoxylated flavone (PMF) found in certain citrus fruits. In this study, we investigated the effects of sinensetin on lipid metabolism in 3T3-L1 cells. Sinensetin promoted adipogenesis in 3T3-L1 preadipocytes growing in incomplete differentiation medium, which did not contain 3-isobutyl-1-methylxanthine. Sinensetin up-regulated expression of the adipogenic transcription factors peroxisome proliferator-activated receptor ?, CCAAT/enhancer-binding protein (C/EBP) ?, and sterol regulatory element-binding protein 1c. It also potentiated expression of C/EBP? and activation of cAMP-responsive element-binding protein. Sinensetin enhanced activation of protein kinase A and increased intracellular cAMP levels in 3T3-L1 preadipocytes. In mature 3T3-L1 adipocytes, sinensetin stimulated lipolysis via a cAMP pathway. Taken together, these results suggest that sinensetin enhances adipogenesis and lipolysis by increasing cAMP levels in adipocytes. PMID:25735898

  9. Thyroid-Stimulating Hormone Inhibits Adipose Triglyceride Lipase in 3T3-L1 Adipocytes through the PKA Pathway

    PubMed Central

    Jiang, Dongqing; Ma, Shizhan; Jing, Fei; Xu, Chao; Yan, Fang; Wang, Aihong; Zhao, Jiajun

    2015-01-01

    Thyroid-stimulating hormone (TSH) has been shown to play an important role in the regulation of triglyceride (TG) metabolism in adipose tissue. Adipose triglyceride lipase (ATGL) is a rate-limiting enzyme controlling the hydrolysis of TG. Thus far, it is unclear whether TSH has a direct effect on the expression of ATGL. Because TSH function is mediated through the TSH receptor (TSHR), TSHR knockout mice (Tshr-/- mice) (supplemented with thyroxine) were used in this study to determine the effects of TSHR deletion on ATGL expression. These effects were verified in 3T3-L1 adipocytes and potential underlying mechanisms were explored. In the Tshr-/- mice, ATGL expression in epididymal adipose tissue was significantly increased compared with that in Tshr+/+ mice. ATGL expression was observed to increase with the differentiation process of 3T3-L1 preadipocytes. In mature 3T3-L1 adipocytes, TSH significantly suppressed ATGL expression at both the protein and mRNA levels in a dose-dependent manner. Forskolin, which is an activator of adenylate cyclase, suppressed the expression of ATGL in 3T3-L1 adipocytes. The inhibitory effects of TSH on ATGL expression were abolished by H89, which is a protein kinase A (PKA) inhibitor. These results indicate that TSH has an inhibitory effect on ATGL expression in mature adipocytes. The associated mechanism is related to PKA activation. PMID:25590597

  10. Antisense RNA--Induced Reduction in Murine TIMP Levels Confers Oncogenicity on Swiss 3T3 Cells

    Microsoft Academic Search

    Rama Khokha; Paul Waterhouse; Simcha Yagel; Peeyush K. Lala; Christopher M. Overall; Gill Norton; David T. Denhardt

    1989-01-01

    Mouse 3T3 cell lines capable of constitutively synthesizing an RNA complementary to the messenger RNA encoding TIMP, tissue inhibitor of metalloproteinases, were constructed by transfection with appropriate plasmid constructs. Many of the lines were down-modulated for TIMP messenger RNA levels and secreted less TIMP into the culture medium. In comparison to noninvasive, nontumorigenic controls, these cells not only were invasive

  11. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    SciTech Connect

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing, E-mail: allenylq@hotmail.com; Liao, Er-Yuan, E-mail: eyliao@21cn.com

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1 cells. • Both Akt and ERK are critical adaptor molecules to mediate the effects of ghrelin.

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

    SciTech Connect

    Boschi, Federico, E-mail: federico.boschi@univr.it [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy); Department of Computer Science, University of Verona, Strada Le Grazie 15, 37134 Verona (Italy); Rizzatti, Vanni; Zamboni, Mauro [Department of Medicine, Geriatric Section, University of Verona, Piazzale Stefani 1, 37126 Verona (Italy); Sbarbati, Andrea [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy)

    2014-02-15

    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. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the size distribution of the lipid droplets in immature and mature cells. • We used the Monte Carlo simulation approach, simulating 10 thousand of fusion events. • Four different interaction models between the lipid droplets were tested. • The best model which mimics the experimental measures was selected.

  13. Bovine Collagen Peptides Compounds Promote the Proliferation and Differentiation of MC3T3-E1 Pre-Osteoblasts

    PubMed Central

    Liu, JunLi; Zhang, Bing; Song, ShuJun; Ma, Ming; Si, ShaoYan; Wang, YiHu; Xu, BingXin; Feng, Kai; Wu, JiGong; Guo, YanChuan

    2014-01-01

    Objective Collagen peptides (CP) compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells. Methods Mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2), a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP), and ELISA for osteocalcin (OC). Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0. Results Cell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days. Conclusions Bovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis. PMID:24926875

  14. 6-gingerol prevents adipogenesis and the accumulation of cytoplasmic lipid droplets in 3T3-L1 cells.

    PubMed

    Tzeng, Thing-Fong; Liu, I-Min

    2013-04-15

    6-Gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) is one of the pungent constituents of Zingiber zerumbet (L) Smith (Zingiberaceae family). In this study, we investigated the effects of 6-gingerol on the inhibition of adipogenesis in 3T3-L1 cells. After treatment with 6-gingerol in differentiation medium for 4 or 8 days, the 3T3-L1 cells were lysed for experimental analysis. Cells were stained with Oil-Red-O to detect oil droplets in adipocytes. The 3T3-L1 cells were lysed and measured for triglyceride contents. The protein expression of adipogenesis-related transcription factor was evaluated by Western blot analysis. 6-Gingerol suppressed oil droplet accumulation and reduced the droplet size in a concentration (5-15 ?g/ml)- and time-dependent manner. Treatment of 3T3-L1 cells with 6-gingerol reduced the protein levels of peroxisome proliferator-activated receptor (PPAR)? and CCAAT/enhancer-binding protein (C/EBP)?. Additionally, the protein levels of fatty acid synthase (FAS) and adipocyte-specific fatty acid binding protein (aP2) decreased upon treatment with 6-gingerol. Meanwhile, 6-gingerol diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3? (Ser9). These results suggest that 6-gingerol effectively suppresses adipogenesis and that it exerts its role mainly through the significant down-regulation of PPAR? and C/EBP? and subsequently inhibits FAS and aP2 expression. 6-Gingerol also inhibited differentiation in 3T3-L1 cells by attenuating the Akt/GSK3? pathway. Our findings provide important insights into the mechanisms underlying the anti-adipogenic activity of 6-gingerol. PMID:23369342

  15. Effect of Ganoderma applanatum mycelium extract on the inhibition of adipogenesis in 3T3-L1 adipocytes.

    PubMed

    Kim, Ji-Eun; Park, Sung-Jin; Yu, Mi-Hee; Lee, Sam-Pin

    2014-10-01

    Ganoderma applanatum (GA) and related fungal species have been used for over 2000 years in China to prevent and treat various human diseases. However, there is no critical research evaluating the functionality of GA grown using submerged culture technology. This study aimed to evaluate the effects of submerged culture GA mycelium (GAM) and its active components (protocatechualdehyde [PCA]) on preadipocyte differentiation of 3T3-L1 cells. Mouse-derived preadipocyte 3T3-L1 cells were treated with differentiation inducers in the presence or absence of GAM extracts. We determined triglyceride accumulations, glycerol-3-phosphate dehydrogenase (GPDH) activities, and differentiation makers. PCA, the active component of GAM extract, was also used to treat 3T3-L1 cells. The MTT assay showed that the GAM extract (0.01-1?mg/mL) was not toxic to 3T3-L1 preadipocyte. Treatment of cells with GAM extracts and its active components significantly decreased the GPDH activity and lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. Western blot analysis results showed that the protein expression levels of peroxisome proliferator-activated receptor ? (PPAR?), CCAAT/enhancer-binding protein ? (C/EBP?), and sterol regulatory element-binding protein 1 (SREBP1) were inhibited by the GAM extract. In addition, adipogenic-specific genes such as perilipin, fatty acid synthase (FAS), fatty acid transport protein 1 (FATP1), and fatty acid-binding protein 4 (FABP4) decreased in a dose-dependent manner. Quantitative high-performance liquid chromatography analysis showed that the GAM extract contained 1.14?mg/g PCA. GAM extracts suppressed differentiation of 3T3-L1 preadipocytes, in part, through altered regulation of PPAR?, C/EBP?, and SREBP1. These results suggest that GAM extracts and PCA may suppress adipogenesis by inhibiting differentiation of preadipocytes. PMID:25140758

  16. FGF-2 signaling induces downregulation of TAZ protein in osteoblastic MC3T3-E1 cells

    SciTech Connect

    Eda, Homare [Department of Biochemistry, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105-8461 (Japan); Department of Orthopaedic Surgery, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105-8461 (Japan); Aoki, Katsuhiko [Department of Biochemistry, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105-8461 (Japan); Marumo, Keishi; Fujii, Katsuyuki [Department of Orthopaedic Surgery, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105-8461 (Japan); Ohkawa, Kiyoshi [Department of Biochemistry, Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105-8461 (Japan)], E-mail: pko@jikei.ac.jp

    2008-02-08

    Transcriptional coactivator with PDZ-binding motif (TAZ) protein is a coactivator of Runx2 and corepressor of PPAR{gamma}. It also induces differentiation of mesenchymal cells into osteoblasts. In this study, we found that FGF-2, which inhibits bone mineralization and stimulates cell proliferation, reduced the TAZ protein expression level in osteoblast-like cells, MC3T3-E1. This reduction was recovered by removing FGF-2 from the culture medium, which also restored the osteoblastic features of MC3T3-E1 cells. Furthermore, FGF-2-induced reduction of TAZ is blocked by a SAPK/JNK-specific inhibitor. These findings suggest that the expression of TAZ protein is involved in osteoblast proliferation and differentiation. This may help elucidate the discrepancies in the effect of FGF-2 and contribute to the understanding of FGF/FGFR-associated craniosynostosis syndrome etiology and treatment.

  17. Long-term exposure of 3T3 fibroblast cells to endocrine disruptors alters sensitivity to oxidative injury.

    PubMed

    Nishimura, Yuka; Nakai, Yasuyoshi; Tanaka, Aiko; Nagao, Tetsuji; Fukushima, Nobuyuki

    2014-07-01

    When Swiss 3T3 fibroblasts were exposed to bisphenol A (BPA) or nonylphenol (NP) within a range of 0.1-100?nM for 30-45 days, increased resistance to oxidative injury was found. Western blot analysis indicated concomitant increased expression of bcl-2 protein and reduced histone methylation levels in cells after BPA or NP exposure. Using a heterologous expression system, both chemicals could stimulate G protein-coupled receptor 30 (GPR30), a transmembrane estrogen receptor predominantly expressed in 3T3 cells, at lower concentrations, which gave increased survival. Taken together, these results suggest that BPA or NP exposure might cause alterations in cellular activity against oxidative stress, possibly through GPR30. PMID:24604882

  18. Magnetic Levitation of MC3T3 Osteoblast Cells as a Ground-Based Simulation of Microgravity

    PubMed Central

    Kidder, Louis S.; Williams, Philip C.; Xu, Wayne Wenzhong

    2009-01-01

    Diamagnetic samples placed in a strong magnetic field and a magnetic field gradient experience a magnetic force. Stable magnetic levitation occurs when the magnetic force exactly counter balances the gravitational force. Under this condition, a diamagnetic sample is in a simulated microgravity environment. The purpose of this study is to explore if MC3T3-E1 osteoblastic cells can be grown in magnetically simulated hypo-g and hyper-g environments and determine if gene expression is differentially expressed under these conditions. The murine calvarial osteoblastic cell line, MC3T3-E1, grown on Cytodex-3 beads, were subjected to a net gravitational force of 0, 1 and 2 g in a 17 T superconducting magnet for 2 days. Microarray analysis of these cells indicated that gravitational stress leads to up and down regulation of hundreds of genes. The methodology of sustaining long-term magnetic levitation of biological systems are discussed. PMID:20052306

  19. Anabolic Effects of Bee Pollen Cistus ladaniferus Extract in Osteoblastic MC3T3-E1 Cells In Vitro

    Microsoft Academic Search

    Masayoshi Yamaguchi; Satoshi Uchiyama; Taeko Nakagawa

    2007-01-01

    The effect of bee pollen extract on osteoblas- tic MC3T3-E1 cells was investigated. The water- solubilized extracts, which were obtained from the bee pollen of Cistus ladaniferus ,w aspurified using the membrane fractionation method with molecular weight (MW) less than 1000. Osteoblastic cells were cultured for 72hr in a medium containing either ve- hicle or cistus extract of less than

  20. Sp1 mediates repression of the resistin gene by PPAR? agonists in 3T3-L1 adipocytes

    Microsoft Academic Search

    S. S. Chung; H. H. Choi; Y. M. Cho; H. K. Lee; K. S.. Park

    2006-01-01

    Resistin is an adipokine related to obesity and insulin resistance. Expression of the resistin gene is repressed by the treatment of peroxisome proliferator-activated receptor ? (PPAR?) agonists, thiazolidinediones (TZDs). In this study, we investigated the mechanism by which TZDs inhibit the resistin gene expression. Resistin gene expression was decreased by TZD in fully differentiated 3T3-L1 adipocytes, which was abolished after

  1. Anti-obesity effect of sulfated glucosamine by AMPK signal pathway in 3T3-L1 adipocytes

    Microsoft Academic Search

    Chang-Suk Kong; Jung-Ae Kim; Se-Kwon Kim

    2009-01-01

    In this study, we investigated the effect of sulfated glucosamine (SGlc) on adipogenesis of 3T3-L1 adipocytes during differentiation of preadipocytes into adipocytes by measuring lipid accumulation and adipogenesis related factors. Treatment with SGlc reduced the triglyceride content in Oil-Red O staining and enhanced glycerol secretion in adipocytes in a dose-dependent manner. In addition, SGlc induced the down-regulation of adipogenesis related

  2. Bromocriptine inhibits adipogenesis and lipogenesis by agonistic action on ?2-adrenergic receptor in 3T3-L1 adipocyte cells.

    PubMed

    Mukherjee, Rajib; Yun, Jong Won

    2013-05-01

    The primary goals of the present study were to investigate the inhibitory effects of bromocriptine (BC) on adipogenesis and lipogenesis in 3T3-L1 adipocyte cells as well as to elucidate its molecular mechanism of action. Adipogenic and lipogenic capacity of BC-treated cells was evaluated by oil red-O staining, triglyceride content assay, real-time RT-PCR and immunoblotting. To determine the mechanism responsible for the anti-obesity effect of BC, we applied two methods. Firstly, we knocked down dopamine D2 receptor (D2R) up to 50% using siRNA. Secondly, we blocked the activity of ?2-adrenergic receptor (?2-AR) by yohimbine treatment and monitored its effects on adipogenic and lipogenic events in 3T3-L1 cells. BC decreased the expression levels of adipogenic activators, including Ppar?, Ppar?, and Cebp?, as well as major lipogenic target genes, including Me1, Acc1, 6Pgd, Fasn, and Prkaa1. Moreover, BC markedly reduced intracellular nitric oxide formation in a dose-dependent manner and expression of pro-inflammatory genes, Tnf? and Il6, which reflects attenuated pro-inflammatory responses. Further, upon treatment with BC, D2R-deficient cells displayed a significant decrease in lipogenic activity compared to control cells, whereas yohimbine-treated cells exhibited no reduction in lipogenic activity. BC can effectively attenuate adipogenesis and lipogenesis in 3T3-L1 cells by downregulating the expression of lipogenic genes and proteins. Our current experimental data collectively establish that the anti-obesity effects of BC are not D2R-dependent but result from the action of ?2-AR in 3T3-L1 adipocytes. PMID:23271132

  3. Anti-obesity effect of carboxymethyl chitin by AMPK and aquaporin-7 pathways in 3T3-L1 adipocytes

    Microsoft Academic Search

    Chang-Suk Kong; Jung-Ae Kim; Soon-Sun Bak; Hee-Guk Byun; Se-Kwon Kim

    2011-01-01

    The aim of this study was to investigate the anti-obesity effect of carboxymethyl-chitin (CM-chitin), a water-soluble derivative of chitin, by measuring lipid accumulation and adipogenesis related factors in 3T3-L1 adipocytes. CM-chitin was synthesized by means of carboxymethylation reaction. Its inhibitory effect on lipid accumulation was investigated by measuring triglyceride content and glycerol release level. The gene and protein levels associated

  4. [Studies on oncogene of nasopharyngeal carcinoma. I. Transforming activity of CNE-2 DNA in NIH3T3 cells].

    PubMed

    Yao, G Q

    1989-04-01

    We have obtained foci of transformed NIH3T3 cells through transfection of human DNA from CNE-2 (an undifferentiated epithelial cell line of nasopharyngeal carcinoma). The DNA from primary foci can be used in subsequent cycles of transfection, resulting in secondary foci capable of forming clones on soft agar and producing tumors 30 days after innoculation into nude mice. The tumors were later proved histologically to be fibrosarcomas. PMID:2528417

  5. Cirsium brevicaule A. GRAY leaf inhibits adipogenesis in 3T3-L1 cells and C57BL/6 mice

    PubMed Central

    2013-01-01

    Background Various flavonoids obtained from the genus Cirsium have been reported to exhibit beneficial effects on health. The present study evaluated the antiobesity effects of Cirsium brevicaule A. GRAY leaf (CL) by using 3T3-L1 cells and C57BL/6 mice that were fed a high-fat diet (HFD). Methods Dried CL powder was serially extracted with solvents of various polarities, and these extracts were tested for antiadipogenic activity using 3T3-L1 adipocytes. Mice were fed experimental HFD supplemented with dried CL powder for 4 wk. Lipid levels and mRNA levels of genes related to lipid metabolism were determined in 3T3-L1 adipocytes and the white adipose tissue (WAT) and liver of mice fed on a HFD. Results Treatment of 3T3-L1 adipocytes with a hexane extract of CL significantly reduced cellular lipid accumulation and expression of the fatty acid synthase (FASN) gene. Dietary CL reduced the serum levels of non-esterified fatty acids in HFD-fed mice. Significant decreases in subcutaneous WAT weight and associated FASN gene expression were observed in the mice fed the experimental CL diet. Dietary CL also reduced the hepatic lipid and serum levels of a hepatopathic indicator in the HFD-fed mice. A significant reduction in mRNA levels of FASN and HMG-CoA reductase were observed in the livers of the CL-diet group. Dietary CL, on the other hand, increased in the hepatic mRNA levels of genes related to ?-oxidation, namely peroxisome proliferator-activated receptor ?, calnitine palmitoyltrasferase 1A, and uncoupling protein 2. Expression of the insulin receptor gene was also significantly increased in the livers of mice-fed the CL diet. Conclusions The present study therefore demonstrated that CL suppresses lipid accumulation in the WAT and liver partly through inhibiting mRNA levels of FASN gene and enhancing the lipolysis-related gene expression. PMID:23945333

  6. R -?-Lipoic acid and acetyl- l -carnitine complementarily promote mitochondrial biogenesis in murine 3T3-L1 adipocytes

    Microsoft Academic Search

    W. Shen; K. Liu; C. Tian; L. Yang; X. Li; J. Ren; L. Packer; C. W. Cotman; J. Liu

    2008-01-01

    Aims\\/hypothesis  The aim of the study was to address the importance of mitochondrial function in insulin resistance and type 2 diabetes, and\\u000a also to identify effective agents for ameliorating insulin resistance in type 2 diabetes. We examined the effect of two mitochondrial\\u000a nutrients, R-?-lipoic acid (LA) and acetyl-l-carnitine (ALC), as well as their combined effect, on mitochondrial biogenesis in 3T3-L1 adipocytes.

  7. Dimethylfumarate Suppresses Adipogenic Differentiation in 3T3-L1 Preadipocytes through Inhibition of STAT3 Activity

    PubMed Central

    Go, Younghoon; Oh, Chang Joo; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2013-01-01

    The excessive accumulation of adipocytes contributes to the development of obesity and obesity-related diseases. The interactions of several transcription factors, such as C/EBP?, PPAR?, C/EBP?, Nrf2, and STAT3, are required for adipogenic differentiation. Dimethylfumarate (DMF), an immune modulator and antioxidant, may function as an inhibitor of STAT3 and an activator of Nrf2. This study examined whether DMF inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inhibiting STAT3 or activating Nrf2. DMF suppressed 3T3-L1 preadipocyte differentiation to mature adipocytes in a dose-dependent manner as determined by Oil Red O staining. The mRNA and protein levels of adipogenic genes, including C/EBP?, C/EBP?, PPAR?, SREBP-1c, FAS, and aP2, were significantly lower in DMF-treated 3T3-L1 preadipocytes. Suppression of adipogenic differentiation by DMF treatment resulted primarily from inhibition of the early stages of differentiation. DMF inhibits clonal expansion during adipogenic differentiation through induction of a G1 cell cycle arrest. Additionally, DMF regulates cell cycle-related proteins, such as p21, pRb, and cyclin D. DMF treatment markedly inhibited differentiation medium-induced STAT3 phosphorylation and inhibited STAT3 transcriptional activation of a reporter construct composed of four synthetic STAT3-response elements. Moreover, inhibition of endogenous Nrf2 activity using a dominant negative Nrf2 did not abolish the DMF-induced inhibition of adipogenic differentiation of 3T3-L1 preadipocytes. In summary, DMF is a negative regulator of adipogenic differentiation based on its regulation of adipogenic transcription factors and cell cycle proteins. This negative regulation by DMF is mediated by STAT3 inhibition, but is unlikely to involve Nrf2 activation. PMID:23637829

  8. Dietary Fatty Acids Up-Regulate the Expression of UCP2 in 3T3-L1 Preadipocytes

    Microsoft Academic Search

    Joanne M. Reilly; Mary P. Thompson

    2000-01-01

    States characterised by elevated plasma fatty acid levels are accompanied by increased UCP2 expression but the physiological regulation of UCP2 expression in white adipose tissue is not fully understood. We used 3T3-L1 preadipocytes to determine whether various dietary fatty acids (20:5, 18:3, 18:2, 18:1, 18:0) directly regulate UCP2 expression. Physiological concentrations of each class of polyunsaturated fatty acid and the

  9. Insulin Activates a Tyrosine-Specific Protein Kinase in Extracts of 3T3-L1 Adipocytes and Human Placenta

    Microsoft Academic Search

    Lilli M. Petruzzelli; Sabyasachi Ganguly; Charles J. Smith; Melanie H. Cobb; Charles S. Rubin; Ora M. Rosen

    1982-01-01

    Insulin activates a tyrosine-specific cAMP-independent protein kinase when added directly to detergent extracts of differentiated 3T3-L1 adipocytes and humal placental membranes. The kinase is also activated by antibody to the insulin receptor and, to a lesser extent, by proinsulin. It catalyzes the phosphorylation of the 92,000-dalton component of the insulin receptor, histone, and casein; in each case, tyrosine is the

  10. Involvement of an alternatively spliced mitochondrial oxodicarboxylate carrier in adipogenesis in 3T3-L1 cells

    Microsoft Academic Search

    Masashi Niimi; Lian Tao; Shi-Hua Lin; Jun Yin; Xiaoyun Wu; Hiroyuki Fukui; Junichi Kambayashi; Jianping Ye; Bing Sun

    2009-01-01

    BACKGROUND: Adipogenesis is a complex process that involves many genes\\/proteins at different stages of differentiation. In order to identify genes critical for adipogeneis, we took a novel approach based on phenotype change of individual cell, to search for genes with regulatory roles in adipogenesis genome-wide in 3T3-L1 cells. METHODS: Lentivirus-based inducible random homologous knockdown was used for the screening of

  11. Effects of C-reactive protein on adipokines genes expression in 3T3-L1 adipocytes

    SciTech Connect

    Yuan, Guoyue, E-mail: yuanguoyue@hotmail.com [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China)] [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China); Jia, Jue; Di, Liangliang [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China)] [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China); Zhou, Libin [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China)] [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China); Dong, Sijing; Ye, Jingjing; Wang, Dong; Yang, Ling; Wang, Jifang [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China)] [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China); Li, Lianxi [Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600, Yishan Road, Shanghai 200233 (China)] [Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600, Yishan Road, Shanghai 200233 (China); Yang, Ying [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China)] [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China); Mao, Chaoming [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China)] [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China); Chen, Mingdao, E-mail: mingdaochensh@yahoo.com [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China)] [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer CRP increases TNF-{alpha} and IL-6 genes expression in matured 3T3-L1 adipocytes. Black-Right-Pointing-Pointer CRP suppresses adiponectin, leptin and PPAR-{gamma} mRNA levels in matured 3T3-L1 cells. Black-Right-Pointing-Pointer Wortmannin reverses effects of CRP on adiponectin, TNF-{alpha} and leptin mRNA levels. Black-Right-Pointing-Pointer CRP may regulate IR, obesity and metabolic syndrome by this mechanism. -- Abstract: Adipose tissue is now recognized to be an important endocrine organ, secreting a variety of adipokines that are involved in the regulation of energy metabolism, insulin resistance and metabolic syndrome. C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. A number of studies have shown that elevation of CRP concentrations is an independent predictive parameter of type 2 diabetes mellitus, which is also strongly associated with various components of the metabolic syndrome. The aim of the present study is to investigate the effects of CRP on adipokines genes expression in 3T3-L1 adipocytes. Quantitative real-time PCR analysis revealed that CRP inhibited adiponectin, leptin and peroxisome proliferator-activated receptor-gamma (PPAR-{gamma}) genes expression and raised tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) mRNA levels in matured 3T3-L1 adipocytes in a dose and time-dependent manner. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by wortmannin partially reversed the effects of CRP on adiponectin, TNF-{alpha} and leptin genes expression. These results collectively suggest that CRP regulates adiponectin, TNF-{alpha}, leptin, IL-6 and PPAR-{gamma} genes expression, and that might represent a mechanism by which CRP regulates insulin resistance, obesity and metabolic syndrome.

  12. Kinetic Analysis of Regulatory Events in G1 Leading to Proliferation or Quiescence of Swiss 3T3 Cells

    Microsoft Academic Search

    A. Zetterberg; Olle Larsson

    1985-01-01

    Kinetic analysis of cellular response to serum deprivation or inhibition of protein synthesis was performed on Swiss 3T3 cells. Time-lapse cinematographic analysis of individual cells transiently exposed to serum-free medium (with or without the addition of purified growth factors) or cycloheximide enabled a detailed mapping of the magnitude and variability of cellular response in different parts of the cell cycle.

  13. Isoproterenol inhibits resistin gene expression through a G S-protein-coupled pathway in 3T3-L1 adipocytes

    Microsoft Academic Search

    Mathias Fasshauer; Johannes Klein; Susanne Neumann; Markus Eszlinger; Ralf Paschke

    2001-01-01

    Resistin was recently identified as a hormone secreted by adipocytes which leads to insulin resistance in vivo and in vitro and might therefore be an important link between obesity and diabetes. To clarify the regulation of resistin gene expression, 3T3-L1 adipocytes were treated with various agents known to modulate insulin sensitivity, and resistin mRNA was measured by quantitative real-time reverse

  14. Tumor Necrosis Factor ? Is a Negative Regulator of Resistin Gene Expression and Secretion in 3T3-L1 Adipocytes

    Microsoft Academic Search

    Mathias Fasshauer; Johannes Klein; Susanne Neumann; Markus Eszlinger; Ralf Paschke

    2001-01-01

    Resistin has recently been implicated as an adipocytokine leading to insulin resistance and, therefore, potentially linking obesity and diabetes. To further characterize the regulation of this fat-secreted protein by insulin sensitivity-modulating hormones, 3T3-L1 adipocytes were treated with tumor necrosis factor (TNF) ?, angiotensin (AT) 2, as well as growth hormone (GH), and resistin gene expression and protein secretion were determined

  15. Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3-L1 Adipocytes

    Microsoft Academic Search

    Ji Lin; Mary Anne Della-Fera; Clifton A. Baile

    2005-01-01

    Objective: Green tea catechins have been shown to promote loss of body fat and to inhibit growth of many cancer cell types by inducing apoptosis. The objective of this study was to determine whether epigallocatechin gallate (EGCG), the primary green tea catechin, could act directly on adipocytes to inhibit adipogenesis and induce apoptosis.Research Methods and Procedures: Mouse 3T3-L1 preadipocytes and

  16. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

    Microsoft Academic Search

    Ippei Kanazawa; Toru Yamaguchi; Shozo Yano; Mika Yamauchi; Masahiro Yamamoto; Toshitsugu Sugimoto

    2007-01-01

    BACKGROUND: Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. RESULTS: Adiponectin

  17. K+ efflux in NIH mouse 3T3 cells and transformed derivatives: dependence on extracellular Ca2+ and phorbol esters

    SciTech Connect

    Lubin, M.

    1988-07-01

    In culture medium deficient in Ca2+, NIH mouse 3T3 cells lose K+, gain Na+, and stop growing. A marked increase in the rate of K+ efflux accounts for this loss; Na+, K+-ATPase pump activity increases but does not fully compensate for enhanced K+ efflux. Phorbol esters and cycloheximide inhibit K+ loss in Ca2+-deficient medium. Phorbol esters inhibit K+ efflux from human fibroblasts as well, even at physiological levels of Ca2+. Two cell lines derived from NIH-3T3, one transformed by a simian virus 40 deletion mutant, the other by the polyoma virus oncogene encoding the middle-sized tumor antigen, retain K+ and can multiply in medium with low Ca2+. Efflux of K+ from these cells is relatively insensitive to reduced Ca2+ concentration, phorbol esters, and cycloheximide. The results suggest the following hypothesis: a channel, nonselective for K+ and Na+, opens when NIH-3T3 cells are in Ca2+-deficient medium; the channel is controlled by the receptor for phorbol ester (protein kinase C) and may also be regulated by a short-lived protein.

  18. Biomimetic calcium carbonate concentric microgrooves with tunable widths for promoting MC3T3-E1 cell functions.

    PubMed

    Wu, Xiaohui; Wang, Shanfeng

    2013-02-01

    Biomimetic, self-assembled calcium carbonate (CaCO(3) ) concentric microgrooves with groove widths of 5.0 and 10 ?m were fabricated through simply controlling incubation temperature. Mouse pre-osteoblastic MC3T3-E1 cells were cultured on flat and microgrooved substrates of CaCO(3) and their adhesion, spreading, proliferation, alkaline phosphatase activity, and calcium content were remarkably enhanced by the microgrooves, in particular, the narrower ones. Furthermore, focal adhesions and actin filaments of MC3T3-E1 cells could be aligned on both 5.0-?m and 10-?m-wide CaCO(3) grooves. Compared with the original round nuclei on the flat substrates and expanded round nuclei on the narrower microgrooves, the MC3T3-E1 cell nuclei on 10-?m-wide CaCO(3) grooves demonstrated preferred entrapment in the grooves and significant alignment with a smaller area after two-day culture. PMID:23184859

  19. Carnosic Acid Inhibits Lipid Accumulation in 3T3-L1 Adipocytes Through Attenuation of Fatty Acid Desaturation

    PubMed Central

    Park, Mi-Young; Sung, Mi-Kyung

    2015-01-01

    Background: Excess body fat accumulation contributes to the development of metabolic disorders that can cause adverse health effects. Carnosic acid (CA), a major bioactive component of rosemary (Rosemarinus officinalis), has been suggested to possess anti-adipogenic properties. The present study was conducted to elucidate the mechanism underlying the anti-adipogenic effects of CA. Methods: 3T3-L1 pre-adipocytes were treated with CA (0.1, 1, and 10 ?M) from day 0 to day 8 of differentiation. On day 8, biochemical markers of lipid accumulation and the degree of fatty acid desaturation were measured. Results: Oil Red O staining results, triglyceride (TG) accumulation, and glycerol 3-phosphate dehydrogenase activity suggested that CA significantly inhibited lipid accumulation in 3T3-L1 adipocytes. CA significantly decreased mRNA expression of peroxisome proliferator-activated receptor-?, sterol regulatory element-binding protein 1, and CCAAT/enhancer binding protein-? in a dose-dependent manner. Moreover, it decreased the ratio of both C16:1/C16:0 and C18:1/C18:0, with reduced expression of stearoyl CoA desaturase 1 mRNA and protein. Conclusions: These results suggest that CA efficiently suppressed adipogenesis in 3T3-L1 adipocytes and its action, at least in part, is associated with the downregulation of adipogenesis-related genes and the fatty acid composition of TG accumulated in adipocytes. PMID:25853102

  20. Aculeatin, a coumarin derived from Toddalia asiatica (L.) Lam., enhances differentiation and lipolysis of 3T3-L1 adipocytes.

    PubMed

    Watanabe, Akio; Kato, Tsuyoshi; Ito, Yusuke; Yoshida, Izumi; Harada, Teppei; Mishima, Takashi; Fujita, Kazuhiro; Watai, Masatoshi; Nakagawa, Kiyotaka; Miyazawa, Teruo

    2014-10-31

    Toddalia asiatica (L.) Lam. (T. asiatica) has been utilized traditionally for medicinal purposes such as the treatment of diabetes. Currently, the extract is considered to be a good source of anti-diabetic agents, but the active compounds have yet to be identified. In this study, we investigated the effects of fractionated T. asiatica extracts on the differentiation of 3T3-L1 preadipocytes and identified aculeatin as a potential active agent. When 3T3-L1 preadipocytes were treated with aculeatin isolated from T. asiatica in the presence of insulin, aculeatin increased cellular triglyceride levels and glycerol-3-phosphate dehydrogenase activity. This indicated that aculeatin could enhance the differentiation of preadipocytes into adipocytes. Further analyses using a DNA microarray and real-time quantitative reverse-transcription PCR showed an increase in the expression of peroxisome proliferator-activated receptor-? target genes (Pparg, Ap2, Cd36, Glut4 and Adipoq) by aculeatin, suggesting that aculeatin enhances the differentiation of 3T3-L1 cells by modulating the expression of genes critical for adipogenesis. Interestingly, after treatment of differentiated adipocytes with aculeatin, glucose uptake and lipolysis were enhanced. Overall, our results suggested that aculeatin is an active compound in T. asiatica for enhancing both differentiation and lipolysis of adipocytes, which are useful for the treatment of lipid abnormalities as well as diabetes. PMID:25445590

  1. ?-Mangostin Induces Apoptosis and Suppresses Differentiation of 3T3-L1 Cells via Inhibiting Fatty Acid Synthase

    PubMed Central

    Quan, Xiaofang; Wang, Yi; Ma, Xiaofeng; Liang, Yan; Tian, Weixi; Ma, Qingyun; Jiang, Hezhong; Zhao, Youxing

    2012-01-01

    ?-Mangostin, isolated from the hulls of Garcinia mangostana L., was found to have in vitro cytotoxicity against 3T3-L1 cells as well as inhibiting fatty acid synthase (FAS, EC 2.3.1.85). Our studies showed that the cytotoxicity of ?-mangostin with IC50 value of 20 µM was incomplicated in apoptotic events including increase of cell membrane permeability, nuclear chromatin condensation and mitochondrial membrane potential (??m) loss. This cytotoxicity was accompanied by the reduction of FAS activity in cells and could be rescued by 50 µM or 100 µM exogenous palmitic acids, which suggested that the apoptosis of 3T3-L1 preadipocytes induced by ?-mangostin was via inhibition of FAS. Futhermore, ?-mangostin could suppress intracellular lipid accumulation in the differentiating adipocytes and stimulated lipolysis in mature adipocytes, which was also related to its inhibition of FAS. In addition, 3T3-L1 preadipocytes were more susceptible to the cytotoxic effect of ?-mangostin than mature adipocytes. Further studies showed that ?-mangostin inhibited FAS probably by stronger action on the ketoacyl synthase domain and weaker action on the acetyl/malonyl transferase domain. These findings suggested that ?-mangostin might be useful for preventing or treating obesity. PMID:22428036

  2. Magnetic resonance detects changes in phosphocholine associated with Ras activation and inhibition in NIH 3T3 cells

    PubMed Central

    Ronen, S M; Jackson, L E; Beloueche, M; Leach, M O

    2001-01-01

    Ras is frequently mutated in cancer, and novel therapies are being developed to target Ras signalling. To identify non-invasive surrogate markers of Ras activation and inhibition, we used31P magnetic resonance spectroscopy (MRS) and investigated NIH 3T3 cells compared to a mutant ras transfected counterpart. The MR spectra indicated that phosphocholine (PC) levels increased significantly from 3 ± 2 fmol cell?1in NIH 3T3 cells to 13?±?4 ?fmol cell?1in the transfected cells. The PC/NTP ratio increased significantly from 0.3?±?0.1 to 0.7 ± 0.3. This could not be explained by either a faster proliferation rate or by alterations in cell cycle distribution. Both cell lines were treated with simvastatin, 17-AAG and R115777, agents which inhibit Ras signalling. Cell proliferation was inhibited in both cell lines. The spectrum of NIH 3T3 cells was not affected by treatment. In contrast, in the ras transfected cells growth inhibition was associated with an average 35?±?5% drop in PC levels and a comparable drop in PC/NTP. Thus the MRS visible increase in phosphocholine is associated with Ras activation, and response to treatment is associated with partial reversal of phosphocholine increase in ras transfected cells. MRS might therefore be a useful tool in detecting Ras activation and its inhibition following targeted therapies. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11237392

  3. Shikonin stimulates glucose uptake in 3T3-L1 adipocytes via an insulin-independent tyrosine kinase pathway.

    PubMed

    Kamei, Reiko; Kitagawa, Yoshinori; Kadokura, Michinori; Hattori, Fumiyuki; Hazeki, Osamu; Ebina, Yousuke; Nishihara, Tatsuro; Oikawa, Shinzo

    2002-04-01

    Type 2 diabetes is due to defects in both insulin action and secretion. In an attempt to discover small molecules that stimulate glucose uptake, similar to insulin, a cell-based glucose uptake screening assay was performed using 3T3-L1 adipocytes. Shikonin, a substance originally isolated from the root of the Chinese plant that has been used as an ointment for wound healing, was thus identified. Shikonin stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. Stimulation of glucose uptake was also observed in rat primary adipocytes and cardiomyocytes. Like insulin, shikonin-stimulated glucose uptake was inhibited by genistein, a tyrosine kinase inhibitor, and enhanced by vanadate, a tyrosine phosphatase inhibitor. However, in contrast to insulin, shikonin-stimulated glucose uptake was not strongly inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). In vitro phosphorylation analyses revealed that shikonin did not induce tyrosine phosphorylation of the insulin receptor, but significantly induced both Thr-308 and Ser-473 phosphorylation of Akt. Our results suggest that in 3T3-L1 adipocytes, shikonin action is not mediated primarily via the insulin receptor/PI3K pathway, but rather via another distinct tyrosine kinase-dependent pathway leading to glucose uptake involving Akt phosphorylation. PMID:11922615

  4. ?-Mangostin induces apoptosis and suppresses differentiation of 3T3-L1 cells via inhibiting fatty acid synthase.

    PubMed

    Quan, Xiaofang; Wang, Yi; Ma, Xiaofeng; Liang, Yan; Tian, Weixi; Ma, Qingyun; Jiang, Hezhong; Zhao, Youxing

    2012-01-01

    ?-Mangostin, isolated from the hulls of Garcinia mangostana L., was found to have in vitro cytotoxicity against 3T3-L1 cells as well as inhibiting fatty acid synthase (FAS, EC 2.3.1.85). Our studies showed that the cytotoxicity of ?-mangostin with IC(50) value of 20 µM was incomplicated in apoptotic events including increase of cell membrane permeability, nuclear chromatin condensation and mitochondrial membrane potential (??m) loss. This cytotoxicity was accompanied by the reduction of FAS activity in cells and could be rescued by 50 µM or 100 µM exogenous palmitic acids, which suggested that the apoptosis of 3T3-L1 preadipocytes induced by ?-mangostin was via inhibition of FAS. Futhermore, ?-mangostin could suppress intracellular lipid accumulation in the differentiating adipocytes and stimulated lipolysis in mature adipocytes, which was also related to its inhibition of FAS. In addition, 3T3-L1 preadipocytes were more susceptible to the cytotoxic effect of ?-mangostin than mature adipocytes. Further studies showed that ?-mangostin inhibited FAS probably by stronger action on the ketoacyl synthase domain and weaker action on the acetyl/malonyl transferase domain. These findings suggested that ?-mangostin might be useful for preventing or treating obesity. PMID:22428036

  5. Enhanced osteogenic differentiation of MC3T3-E1 on rhBMP-2-immobilized titanium via click reaction.

    PubMed

    Kim, Eun-Cheol; Kim, Tae-Hee; Jung, Jae-Hoon; Hong, Sung Ok; Lee, Deok-Won

    2014-03-15

    In the present study, we report about the efficacy of titanium surface-immobilized with bone morphogenetic protein-2 (BMP-2) via click reaction on enhanced osteogenic differentiation of MC3T3-E1 cells. The surface was characterized by static contact angles and XPS measurements, which indicated that pristine titanium (Ti-1) was successfully surface-modified via click chemistry (aminated titanium, Ti-4). By quantitative analysis of heparin immobilized on aminated titanium (Ti-4), we found that the Ti-4 can be used as a good candidate to immobilize biomolecules such as heparin. BMP-2 from titanium immobilized with BMP-2 (Ti-6) was released for a period of 28 days in a sustained manner. The highest proliferation rate of MC3T3-E1 cells was observed on Ti-6. Through in vitro tests including alkaline phosphatase (ALP) activity, calcium deposition and real-time polymerase chain reaction (real-time PCR), we found that Ti-6 can be used as a good implant to enhance the osteogenic differentiation of MC3T3-E1 cells. PMID:24528716

  6. Soy pinitol acts partly as an insulin sensitizer or insulin mediator in 3T3-L1 preadipocytes.

    PubMed

    Do, Gyeong-Min; Choi, Myung-Sook; Kim, Hye-Jin; Woo, Myung-Nam; Lee, Mi-Kyung; Jeon, Seon-Min

    2008-02-01

    The blood glucose-lowering property of pinitol is mediated via the insulin signaling pathway. This study was carried out to evaluate the effects of soy pinitol on adipogenesis in a 3T3-L1 cell line; 3T3-L1 preadipocytes were treated with pinitol (0-1 mM) together with insulin for 9 days. The regulation of lipid metabolism was assessed by oil-red-O staining of intracellular lipids and real-time PCR of adipogenesis-related factors. The inhibition of cell proliferation was estimated by MTT assay. Pinitol treatment did not inhibit lipid accumulation, nor did it affect expression of adipogenesis-related factors, including ADD1, aP2 and FAS, in a dose-dependent manner. Expression of adiponectin, GLUT4, IRS, C/EBPalpha and PPARgamma mRNAs, however, increased in cells treated with 0.5 mM and/or 1 mM pinitol. Pinitol treatment did not affect the inhibition of cell growth and proliferation in a dose-dependent manner. Accordingly, we suggest that pinitol is nontoxic to this cell line, and that it enhances adipogenesis by acting as an insulin sensitizer or insulin mediator via the upregulation of adiponectin, GLUT4, IRS, C/EBPalpha and PPARgamma in 3T3-L1 preadipocytes. PMID:18850231

  7. Inhibitory effect of apocynin on methylglyoxal-mediated glycation in osteoblastic MC3T3-E1 cells.

    PubMed

    Suh, Kwang Sik; Rhee, Sang Youl; Kim, Young Seol; Choi, Eun Mi

    2015-04-01

    Methylglyoxal (MG), a highly reactive metabolite of hyperglycemia, can enhance protein glycation, oxidative stress or inflammation. The present study investigated the effects of apocynin on the mechanisms associated with MG toxicity in osteoblastic MC3T3-E1 cells. Pretreatment of MC3T3-E1 cells with apocynin prevented the MG-induced protein glycation and formation of intracellular reactive oxygen species and mitochondrial superoxide in MC3T3-E1 cells. In addition, apocynin increased glutathione levels and restored the activity of glyoxalase I inhibited by MG. These findings suggest that apocynin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing the MG detoxification system. Apocynin treatment decreased the levels of proinflammatory cytokines such as tumor necrosis factor-? and interleukin-6 induced by MG. Additionally, the nitric oxide level reduced by MG was significantly increased by apocynin. These findings indicate that apocynin might exert its therapeutic effects via upregulation of glyoxalase system and antioxidant activity. Taken together, apocynin may prove to be an effective treatment for diabetic osteopathy. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25042521

  8. K+ Efflux in NIH Mouse 3T3 Cells and Transformed Derivatives: Dependence on Extracellular Ca2+ and Phorbol Esters

    NASA Astrophysics Data System (ADS)

    Lubin, Martin

    1988-07-01

    In culture medium deficient in Ca2+, NIH mouse 3T3 cells lose K+, gain Na+, and stop growing. A marked increase in the rate of K+ efflux accounts for this loss; Na+,K+-ATPase pump activity increases but does not fully compensate for enhanced K+ efflux. Phorbol esters and cycloheximide inhibit K+ loss in Ca2+-deficient medium. Phorbol esters inhibit K+ efflux from human fibroblasts as well, even at physiological levels of Ca2+. Two cell lines derived from NIH-3T3, one transformed by a simian virus 40 deletion mutant, the other by the polyoma virus oncogene encoding the middle-sized tumor antigen, retain K+ and can multiply in medium with low Ca2+. Efflux of K+ from these cells is relatively insensitive to reduced Ca2+ concentration, phorbol esters, and cycloheximide. The results suggest the following hypothesis: a channel, nonselective for K+ and Na+, opens when NIH-3T3 cells are in Ca2+-deficient medium; the channel is controlled by the receptor for phorbol ester (protein kinase C) and may also be regulated by a short-lived protein.

  9. Actein isolated from black cohosh promotes the function of osteoblastic MC3T3-E1 cells.

    PubMed

    Lee, Young Soon; Choi, Eun Mi

    2014-04-01

    Actein, isolated from black cohosh, was subjected to in vitro experiments to investigate its functional bioactivities in osteoblastic MC3T3-E1 cells. Actein caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, mineralization, and glutathione content in the cells, suggesting that actein has a stimulatory effect on osteoblastic bone formation or has potential activity against osteoporosis. We investigated the protective effects of actein on mitochondrial electron transport inhibitor, antimycin A induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant decrease in cell viability and mineralization. However, pretreatment with actein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, cardiolipin oxidation, ROS release, and nitrotyrosine increase, suggesting that actein may be useful for protecting mitochondria against a burst of oxidative stress. In addition, actein increased the phosphorylation of CREB (cAMP-response element-binding protein) inhibited by antimycin A and decreased the production of TNF-? induced by antimycin A. These findings suggest that actein could prevent oxidative damage to osteoblasts in osteoporotic patients. PMID:24552231

  10. Dehydrodiconiferyl alcohol isolated from Cucurbita moschata shows anti-adipogenic and anti-lipogenic effects in 3T3-L1 cells and primary mouse embryonic fibroblasts.

    PubMed

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-03-16

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor ? (Pparg), CCAAT/enhancer-binding protein ? (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBP?, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis. PMID:22262865

  11. Dehydrodiconiferyl Alcohol Isolated from Cucurbita moschata Shows Anti-adipogenic and Anti-lipogenic Effects in 3T3-L1 Cells and Primary Mouse Embryonic Fibroblasts*

    PubMed Central

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-01-01

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor ? (Pparg), CCAAT/enhancer-binding protein ? (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBP?, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis. PMID:22262865

  12. Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

    SciTech Connect

    Hashimoto, Takeshi, E-mail: thashimo@fc.ritsumei.ac.jp [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)] [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Yokokawa, Takumi; Endo, Yuriko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)] [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Iwanaka, Nobumasa [Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)] [Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Higashida, Kazuhiko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan) [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Faculty of Sport Science, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192 (Japan); Taguchi, Sadayoshi [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)] [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)

    2013-10-11

    Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O{sub 2} for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via decreased glucose uptake and lipogenic protein expression and increased basal lipolysis. Such an hypoxia-induced decrease in lipogenesis may be an attractive therapeutic target against lipid-associated metabolic diseases.

  13. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    SciTech Connect

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-08-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the ..cap alpha.. and ..beta.. subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  14. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    SciTech Connect

    He, Yonghan [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China) [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Li, Ying [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)] [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Shuocheng [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada)] [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Perry, Ben [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada) [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Zhao, Tiantian [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada) [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 (Canada); Wang, Yanwen, E-mail: yanwen.wang@nrc.ca [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada) [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Sun, Changhao, E-mail: sun2002changhao@yahoo.com [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)] [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)

    2013-06-28

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor ? (PPAR{sub ?}) and CCAAT element binding protein ? (C/EBP{sub ?}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on ?-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  15. Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells.

    PubMed

    Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A; Chock, P Boon

    2013-05-01

    Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signaling via a G?q/11/PLC?/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF G?q/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analyses. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in G?q/11 double knockout MEF cells. Although CTGF expression is regulated by TGF?, rPMT did not activate TGF? pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly, CTGF overexpression in serum-starved 3T3 cells using adenovirus led to phosphorylation of ribosomal protein S6, a downstream target of mTOR. However, despite the ability of CTGF to activate the mTOR pathway, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells. Our findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT. PMID:23415771

  16. Nano-hydroxyapatite particles induce apoptosis on MC3T3-E1 cells and tissue cells in SD rats.

    PubMed

    Wang, Liting; Zhou, Gang; Liu, Haifeng; Niu, Xufeng; Han, Jingyun; Zheng, Lisha; Fan, Yubo

    2012-04-28

    While the advantages of nanomaterials are being increasingly recognized, their potential toxicity is drawing more and more attention and concern. In this study, we explore the toxicity mechanism of 20-30 nm rod-shaped hydroxyapatite (HA) nanoparticles in vitro and in vivo. The nanoparticles were prepared by precipitation and characterized by IR, XRD and TEM. Concentrations of 0 ?g mL(-1), 10 ?g mL(-1), 100 ?g mL(-1), 1 mg mL(-1), and 10 mg mL(-1) were applied to the MC3T3-E1 cells for viability (MTT-test). Based on the characteristic differences of the two methods of cell death, the morphological features of the MC3T3-E1 cell line co-cultured with nano-hydroxyapatite (n-HA) (10 mg mL(-1)) for 24 h were also observed by TEM. Furthermore, important serum biochemical markers and histopathological examinations were used to evaluate the potential toxicological effect of n-HA on the major organs of SD rats injected intraperitoneally with n-HA (33.3 mg kg(-1) body weight). In the results, we found cell growth inhibition and apoptosis in MC3T3-E1 cells co-cultured with n-HA. Moreover, apoptosis but not necrosis was illustrated in liver and renal tissue by using histopathology slices and serum biochemical markers. It suggests that apoptosis may be the possible mechanism of n-HA toxicity and provides a better understanding of the biocompatibility of nanomaterials applied in human bone repair. PMID:22450902

  17. Nano-hydroxyapatite particles induce apoptosis on MC3T3-E1 cells and tissue cells in SD rats

    NASA Astrophysics Data System (ADS)

    Wang, Liting; Zhou, Gang; Liu, Haifeng; Niu, Xufeng; Han, Jingyun; Zheng, Lisha; Fan, Yubo

    2012-04-01

    While the advantages of nanomaterials are being increasingly recognized, their potential toxicity is drawing more and more attention and concern. In this study, we explore the toxicity mechanism of 20-30 nm rod-shaped hydroxyapatite (HA) nanoparticles in vitro and in vivo. The nanoparticles were prepared by precipitation and characterized by IR, XRD and TEM. Concentrations of 0 ?g mL-1, 10 ?g mL-1, 100 ?g mL-1, 1 mg mL-1, and 10 mg mL-1 were applied to the MC3T3-E1 cells for viability (MTT-test). Based on the characteristic differences of the two methods of cell death, the morphological features of the MC3T3-E1 cell line co-cultured with nano-hydroxyapatite (n-HA) (10 mg mL-1) for 24 h were also observed by TEM. Furthermore, important serum biochemical markers and histopathological examinations were used to evaluate the potential toxicological effect of n-HA on the major organs of SD rats injected intraperitoneally with n-HA (33.3 mg kg-1 body weight). In the results, we found cell growth inhibition and apoptosis in MC3T3-E1 cells co-cultured with n-HA. Moreover, apoptosis but not necrosis was illustrated in liver and renal tissue by using histopathology slices and serum biochemical markers. It suggests that apoptosis may be the possible mechanism of n-HA toxicity and provides a better understanding of the biocompatibility of nanomaterials applied in human bone repair.

  18. Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells

    PubMed Central

    Oubrahim, Hammou; Wong, Allison; Wilson, Brenda A.; Chock, P. Boon

    2013-01-01

    Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signalling via a G?q/11/PLC?/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF G?q/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analysis. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in G?q/11 double knockout MEF cells. Although CTGF expression is regulated by TGF?, rPMT did not activate TGF? pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly, CTGF overexpression in serum-starved 3T3 cells using adenovirus led to phosphorylation of ribosomal protein S6, a downstream target of mTOR. However, despite the ability of CTGF to activate the mTOR pathway, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells. Our findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT. PMID:23415771

  19. A novel polypeptide from Cervus nippon Temminck proliferation of epidermal cells and NIH3T3 cell line.

    PubMed

    Guan, Shu-Wen; Duan, Leng-Xin; Li, Yuan-Yuan; Wang, Ben-Xiang; Zhou, Qiu-Li

    2006-01-01

    A novel polypeptide, velvet antler polypeptide (VAPPs), having a stimulary effect on proliferation of some cell was isolated from the velvet antler of sika deer (Cervus nippon Temminck). This polypeptide consists of a single chain of 32 amino-acid residues VLSAT DKTNV LAAWG KVGGN APAFG AEALE RM. VAPPs showed marked stimulary effect on rat epidermal cells and NIH3T3 cell line (dose range from 10-40 mg x L(-1) and 5-80 mg x L(-1), respectively). PMID:16770447

  20. The role of the coating and aggregation state in the interactions between iron oxide nanoparticles and 3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Safi, Malak; Berret, Jean-François

    Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of the iron oxide sub-10 nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 gmol-1). We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. The strong uptake shown by the citrate-coated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes.

  1. Photocopy of original nap (from feeder book), New York State ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Photocopy of original nap (from feeder book), New York State Archives and Manuscripts, Albany, New York), Holnes Hutchinson, surveyor, 1834 - Glens Falls Feeder, Sluice, Along south side of Glens Falls Feeder between locks 10 & 20, Hudson Falls, Washington County, NY

  2. Structural Assessment of Small Bore Feeder Piping

    E-print Network

    BACKGROUND Structural Assessment of Small Bore Feeder Piping Kathryn Tang, Janos Mann, Skerdi. Supervisor: A. N. Sinclair CASE ONE CANDU REACTORS HAVE 380+ SMALL BORE FEEDER PIPES. THE PIPES PIPES AND VALIDATE COMPUTATIONAL METHODS COMPARED TO FULL SCALE TESTS TO FAILURE. THE CLIENT CANDU

  3. Anti-obesity effects of hispidin and Alpinia zerumbet bioactives in 3T3-L1 adipocytes.

    PubMed

    Tu, Pham Thi Be; Tawata, Shinkichi

    2014-01-01

    Obesity and its related disorders have become leading metabolic diseases. In the present study, we used 3T3-L1 adipocytes to investigate the anti-obesity activity of hispidin and two related compounds that were isolated from Alpinia zerumbet (alpinia) rhizomes. The results showed that hispidin, dihydro-5,6-dehydrokawain (DDK), and 5,6-dehydrokawain (DK) have promising anti-obesity properties. In particular, all three compounds significantly increased intracellular cyclic adenosine monophosphate (cAMP) concentrations by 81.2% ± 0.06%, 67.0% ± 1.62%, and 56.9% ± 0.19%, respectively. Hispidin also stimulated glycerol release by 276.4% ± 0.8% and inhibited lipid accumulation by 47.8% ± 0.16%. Hispidin and DDK decreased intracellular triglyceride content by 79.5% ± 1.37% and 70.2% ± 1.4%, respectively, and all three compounds inhibited glycerol-3-phosphate dehydrogenase (GPDH) and pancreatic lipase, with hispidin and DDK being the most potent inhibitors. Finally, none of the three compounds reduced 3T3-L1 adipocyte viability. These results highlight the potential for developing hispidin and its derivatives as anti-obesity compounds. PMID:25322285

  4. Gel Microstructure Regulates Proliferation and Differentiation of MC3T3-E1 Cells Encapsulated in Alginate Beads

    PubMed Central

    Lee, Baek-Hee; Li, Bing; Guelcher, Scott A.

    2012-01-01

    For cell transplantation into damaged tissues, viable cells must be delivered to the defect site in a suitable carrier. However, the hypoxic and nutrient-limited environment in the carrier can induce massive cell death. The aims of this study were to increase the viability and regulate the behavior of osteoprogenitor cells encapsulated in alginate hydrogels through control of the gel microstructure. Cell survivability in alginate beads was improved through the use of ?-MEM as the solvent for alginic acid sodium salt and CaCl2 solutions, which supplied additional nutrients for the cells compared to water or buffer. The mesh size and shear modulus of the hydrogel were hypothesized to regulate proliferation and differentiation of osteoprogenitor cells. MC3T3-E1 cells demonstrated enhanced osteoblast differentiation when encapsulated in high-density alginate with smaller mesh size and more rigid mechanical properties, as confirmed by increased alkaline phosphatase activity and osteocalcin secretion. However, MC3T3-E1 cells encapsulated in low-density alginate beads with a larger mesh size and more compliant mechanical properties exhibited increased proliferation. These results demonstrate that the microstructure of alginate hydrogels can regulate the behavior of osteoprogenitor cells, thus suggesting that the tuning the properties of the gel may be a useful approach for enhancing new bone formation. PMID:22306825

  5. Dissociation of tumour-promoter-induced effects on prostaglandin release, polyamine synthesis and cell proliferation of 3T3 cells.

    PubMed Central

    Lanz, R; Brune, K

    1981-01-01

    The phorbol ester 12-O-tetradecanoylphorbol 13-acetate induces tumour promotion, inflammation, cell proliferation and prostaglandin release. Recent reports suggest that the prostaglandins released by 12-O-tetradecanoylphorbol 13-acetate (TPA) initiate a cascade of events leading to polyamine synthesis and cell proliferation. In experiments designed to test this contention, it was found that addition of TPA (1 microM to 1 nM) to confluent mouse 3T3 fibroblasts successively caused the release of prostaglandins E2 and I2, induction of the enzyme ornithine decarboxylase (EC 4.1.1.17), stimulation of [3H]thymidine incorporation into DNA, and cell proliferation. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone (1 microM) or the non-steroidal anti-inflammatory drug indomethacin (1 microM) inhibited TPA-induced prostaglandin release. However, dexamethasone enhanced the other effects of TPA, whereas indomethacin was ineffective. Addition of prostaglandin E2 to the cultures did not induce ornithine decarboxylase activity and cell proliferation. Pretreatment of the cells with 1,3-diaminopropane (1 mM) or alpha-methylornithine (5 mM), inhibitors of polyamine synthesis, decreased TPA-induced ornithine decarboxylase activity without affecting DNA synthesis. TPA stimulated [3H]thymidine incorporation into DNA, even when the ornithine decarboxylase activity was completely blocked. These data suggest that the proliferative effect of TPA on 3T3 cells is independent of prostaglandin release and polyamine synthesis. PMID:7306036

  6. Modification of Heterotrimeric G-Proteins in Swiss 3T3 Cells Stimulated with Pasteurella multocida Toxin

    PubMed Central

    Babb, Rebecca C.; Homer, Karen A.; Robbins, Jon; Lax, Alistair J.

    2012-01-01

    Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, Gq, G12 and Gi. PMT has been shown by others to lead to the deamidation of recombinant G?i at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native G? subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the G? subunit of the Gq and Gi families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these G? subunits. Surprisingly, PMT also induced a similar modification of G?11, a member of the Gq family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of G?13 was observed following PMT treatment of cells, suggesting differential modification of this G? subunit by PMT. Gs was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated G?i, but not G?q. We also show that PMT inhibits the GTPase activity of Gq. PMID:23144805

  7. PU.1 promotes miR-191 to inhibit adipogenesis in 3T3-L1 preadipocytes.

    PubMed

    Ji, Shengyue; Li, Weili; Bao, Lijuan; Han, Peng; Yang, Weiping; Ma, Li; Meng, Fanxu; Cao, Binyun

    2014-08-22

    PU.1, an Ets family transcription factor, was previously demonstrated expressed in 3T3-L1 preadipocytes and had an negative effect on adiopogenesis. However, the underlying mechanism remains elusive. Here, miR-191 was identified as an inhibitor of adipocyte differentiation through targeting the 3' untranslated regions of C/EBP?, the initial factor in the C/EBP?/?-PPAR? terminal pathway of adipogenic differentiation. MiR-191 suppressed the lipid accumulation by Oil Red O staining and downregulated the levels of adipogenic marker genes PPAR? (P<0.01), aP2 (P<0.01) and FAS (P<0.05). Then, we found that PU.1 overexpression resulted in upregulation of miR-191 and adipogenic inhibition. Likewise, PU.1 overexpression rescued the miR-191 decrease and resisted the adipogenic promotion caused by miR-191 oligonucleotide inhibitor. Collectively, these results revealed that PU.1 promoted miR-191 to suppress adipogenesis 3T3-L1 preadipocyte and indicated a new mechanism of PU.1 inhibiting adipogenesis. PMID:25094047

  8. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes.

    PubMed

    He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

    2013-06-28

    Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT element binding protein ? (C/EBP?), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on ?-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins. PMID:23727383

  9. Overexpression of PGC-1? improves insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes.

    PubMed

    Gao, Chun-Lin; Liu, Guang-Ling; Liu, Shi; Chen, Xiao-Hui; Ji, Chen-Bo; Zhang, Chun-Mei; Xia, Zheng-Kun; Guo, Xi-Rong

    2011-07-01

    The co-transcription factor peroxisome proliferator-activated receptor ? coactivator-1? (PGC-1?) was first identified in 2002. Although the function of PGC-1? in white adipose tissue (WAT) is largely unknown, it has been studied extensively in the liver, cardiac muscle, and skeletal muscle. Herein, we investigated PGC-1? overexpression in 3T3-L1 adipocytes. The main findings were as follows: (i) 3T3-L1 adipocytes overexpressing PGC-1? showed improved insulin sensitivity and elevated insulin-stimulated glucose uptake; (ii) mitochondrial cristae became broader and more ordered, additional smaller mitochondria emerged, mitochondrial DNA increased, and fission 1 protein (Fis1) mRNA expression was greatly elevated; (iii) intracellular ATP levels increased, but no changes were observed in mitochondrial membrane potential, uncoupling protein (UCP) mRNA expression, or reactive oxygen species (ROS) production; and (iv) mitochondrial metabolism factors, namely, acetyl-coenzyme A carboxylase 2 (ACC2) and hexokinase 2 (HK2) were downregulated, while cytochrome c oxidase subunit IV (COX IV) was upregulated. In conclusion, PGC-1? affects not only insulin sensitivity but also mitochondrial biogenesis and function. We believe that the role of PGC-1? is distinct from that of PGC-1? in WAT. PMID:21499715

  10. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan) [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065 (Japan)] [Innovation Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan)] [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  11. A fraction of unripe kiwi fruit extract regulates adipocyte differentiation and function in 3T3-L1 cells.

    PubMed

    Abe, Daigo; Saito, Takeshi; Kubo, Yasutaka; Nakamura, Yoshimasa; Sekiya, Keizo

    2010-01-01

    Adipocyte dysfunction is strongly associated with the development of insulin resistance and diabetes, and regulation of adipogenesis is important in prevention of diabetes. We prepared a 100% methanol fraction of methanolic extract from unripe kiwi fruit (Actinidia deliciosa), designated KMF (kiwi fruit methanol fraction). When applied to 3T3-L1 preadipocyte cells, KMF promoted adipocyte differentiation, increased glycerol-3-phosphate dehydrogenase (GPDH) activity, and increased triglyceride (TG) content. KMF markedly increased mRNA expression of peroxisome proliferator-activated receptor gamma (PPARgamma)-the master adipogenic transcription factor-and its target genes. Moreover, KMF increased mRNA expression and protein secretion of adiponectin, whereas mRNA expression and secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were decreased. Compared with troglitazone, KMF decreased the production of reactive oxygen species (ROS) and nuclear factor-kappaB (NFkappaB) activation. Glucose uptake was stimulated by KMF in differentiated 3T3-L1 adipocytes. Taken together, these results indicate that KMF may exert beneficial effects against diabetes via its ability to regulate adipocyte differentiation and function. PMID:20087882

  12. 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes.

    PubMed

    Salt, I P; Connell, J M; Gould, G W

    2000-10-01

    Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR), a compound that activates 5'-AMP-activated protein kinase (AMPK), has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study, we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes, in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently, closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation, assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle, in which AMPK stimulation promotes glucose transport to provide ATP as a fuel, AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes, inhibiting triacylglycerol synthesis, to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may, therefore, give insight into the mechanism of insulin action. PMID:11016448

  13. ITER Magnet Feeder: Design, Manufacturing and Integration

    NASA Astrophysics Data System (ADS)

    CHEN, Yonghua; ILIN, Y.; M., SU; C., NICHOLAS; BAUER, P.; JAROMIR, F.; LU, Kun; CHENG, Yong; SONG, Yuntao; LIU, Chen; HUANG, Xiongyi; ZHOU, Tingzhi; SHEN, Guang; WANG, Zhongwei; FENG, Hansheng; SHEN, Junsong

    2015-03-01

    The International Thermonuclear Experimental Reactor (ITER) feeder procurement is now well underway. The feeder design has been improved by the feeder teams at the ITER Organization (IO) and the Institute of Plasma Physics, Chinese Academy of Sciences (ASIPP) in the last 2 years along with analyses and qualification activities. The feeder design is being progressively finalized. In addition, the preparation of qualification and manufacturing are well scheduled at ASIPP. This paper mainly presents the design, the overview of manufacturing and the status of integration on the ITER magnet feeders. supported by the National Special Support for R&D on Science and Technology for ITER (Ministry of Public Security of the People's Republic of China-MPS) (No. 2008GB102000)

  14. Insulin and isoproterenol induce phosphorylation of the particulate cyclic GMP-inhibited, low Km cyclic AMP phosphodiesterase (cGI PDE) in 3T3-L1 adipocytes.

    PubMed

    Vasta, V; Smith, C J; Calvo, J; Belfrage, P; Manganiello, V C

    1992-03-31

    The cGI PDE in particulate fractions of differentiated adipocytes (but not control 3T3-L1 fibroblasts) was cross-reactive with a polyclonal antibody raised against the bovine adipose cGI PDE. The 3T3-L1 adipocyte cGI PDE is a 135 kDa protein which is phosphorylated in 32P-labeled cells in response to beta-agonist or insulin. These results indicate that the 3T3-L1 cGI PDE is similar in structure and hormonal regulation to the analogous enzyme in the rat adipocyte. PMID:1314573

  15. Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells

    PubMed Central

    1995-01-01

    Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions. PMID:7542247

  16. A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

    PubMed Central

    Masubuchi, Yosuke; Nakagawa, Yuko; Ma, Jinhui; Sasaki, Tsutomu; Kitamura, Tadahiro; Yamamoto, Yoritsuna; Kurose, Hitoshi; Kojima, Itaru; Shibata, Hiroshi

    2013-01-01

    Background Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. Methodology/Principal Findings In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The ? subunits of Gs (G?s) and G14 (G?14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor ? (PPAR? and CCAAT/enhancer-binding protein ? (C/EBP? at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of G?s but not YM-254890, an inhibitor of G?14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of G?s had the opposite effects. Conclusions 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism. PMID:23336004

  17. Enhanced Effects of 1,25(OH)2D3 Plus Genistein on Adipogenesis and Apoptosis in 3T3-L1 Adipocytes

    Microsoft Academic Search

    Srujana Rayalam; Mary Anne Della-Fera; Suresh Ambati; Jeong-Yeh Yang; Hea Jin Park; Clifton A. Baile

    2008-01-01

    Objective:To investigate the ability of 1,25(OH)2D3 (D) and genistein (G), alone and in combination, to inhibit adipogenesis and induce apoptosis in 3T3-L1 adipocytes.Methods and Procedures:3T3-L1 preadipocytes and mature adipocytes were incubated with various concentrations of D and G, alone and in combination, for 48 h. Viability was determined using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay. Post-confluent

  18. A possible role of oxidative stress in the vanadium-induced cytotoxicity in the MC3T3E1 osteoblast and UMR106 osteosarcoma cell lines

    Microsoft Academic Search

    Ana Mar??a Cortizo; Liliana Bruzzone; Silvina Molinuevo; Susana Beatriz Etcheverry

    2000-01-01

    The cytotoxicity and free radical production induced by vanadium compounds were investigated in an osteoblast (MC3T3E1) and an osteosarcoma (UMR106) cell lines in culture. Vanadate induced cell toxicity, reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) increased in a concentration-dependent manner (0.1–10 mM) after 4 h. The concentration–response curve of vanadate-induced cytotoxicity and oxidative stress in MC3T3E1

  19. Fucoxanthin exerts differing effects on 3T3-L1 cells according to differentiation stage and inhibits glucose uptake in mature adipocytes

    SciTech Connect

    Kang, Seong-Il [Department of Biology, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of)] [Department of Biology, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of); Ko, Hee-Chul [Jeju Sasa Industry Development Agency, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of)] [Jeju Sasa Industry Development Agency, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of); Shin, Hye-Sun; Kim, Hyo-Min; Hong, Youn-Suk [Department of Biology, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of)] [Department of Biology, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of); Lee, Nam-Ho [Department of Chemistry, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of)] [Department of Chemistry, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of); Kim, Se-Jae, E-mail: sjkim@jejunu.ac.kr [Department of Biology, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of) [Department of Biology, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of); Jeju Sasa Industry Development Agency, Jeju National University, Jejusi, Jeju 690-756 (Korea, Republic of)

    2011-06-17

    Highlights: {yields} Fucoxanthin enhances 3T3-L1 adipocyte differentiation at an early stage. {yields} Fucoxanthin inhibits 3T3-L1 adipocyte differentiation at intermediate and late stages. {yields} Fucoxanthin attenuates glucose uptake by inhibiting the phosphorylation of IRS in mature 3T3-L1 adipocytes. {yields} Fucoxanthin exerts its anti-obesity effect by inhibiting the differentiation of adipocytes at both intermediate and late stages, as well as glucose uptake in mature adipocytes. -- Abstract: Progression of 3T3-L1 preadipocyte differentiation is divided into early (days 0-2, D0-D2), intermediate (days 2-4, D2-D4), and late stages (day 4 onwards, D4-). In this study, we investigated the effects of fucoxanthin, isolated from the edible brown seaweed Petalonia binghamiae, on adipogenesis during the three differentiation stages of 3T3-L1 preadipocytes. When fucoxanthin was applied during the early stage of differentiation (D0-D2), it promoted 3T3-L1 adipocyte differentiation, as evidenced by increased triglyceride accumulation. At the molecular level, fucoxanthin increased protein expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}), sterol regulatory element-binding protein 1c (SREBP1c), and aP2, and adiponectin mRNA expression, in a dose-dependent manner. However, it reduced the expression of PPAR{gamma}, C/EBP{alpha}, and SREBP1c during the intermediate (D2-D4) and late stages (D4-D7) of differentiation. It also inhibited the uptake of glucose in mature 3T3-L1 adipocytes by reducing the phosphorylation of insulin receptor substrate 1 (IRS-1). These results suggest that fucoxanthin exerts differing effects on 3T3-L1 cells of different differentiation stages and inhibits glucose uptake in mature adipocytes.

  20. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

    Microsoft Academic Search

    Xing Zhong; Ling-ling Xiu; Guo-hong Wei; Yuan-yuan Liu; Lei Su; Xiao-pei Cao; Yan-bing Li; Hai-peng Xiao

    2011-01-01

    Aim:To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.Methods:MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP,

  1. MC3T3-E1 osteoblast attachment and proliferation on porous hydroxyapatite scaffolds fabricated with nanophase powder

    PubMed Central

    Smith, Ian O; McCabe, Laura R; Baumann, Melissa J

    2006-01-01

    Porous bone tissue engineering scaffolds were fabricated using both nano hydroxyapatite (nano HA) powder (20 nm average particle size) and micro HA powder (10 ?m average particle size), resulting in sintered scaffolds of 59 vol% porosity and 8.6±1.9 ?m average grain size and 72 vol% porosity and 588±55 nm average grain size, respectively. Scanning electron microscopy was used to measure both the grain size and pore size. MC3T3-E1 osteoblast (OB) attachment and proliferation on both nano HA and micro HA porous scaffolds were quantified. As expected, OB cell number was greater on nano HA scaffolds compared with similarly processed micro HA scaffolds 5 days after seeding, while OB attachment did not appear greater on the nano HA scaffolds (p<0.05). PMID:17722535

  2. Permethrin alters adipogenesis in 3T3-L1 adipocytes and causes insulin resistance in C2C12 myotubes.

    PubMed

    Kim, Jonggun; Park, Yooheon; Yoon, Kyong Sup; Clark, J Marshall; Park, Yeonhwa

    2014-09-01

    Pyrethroids are a class of insecticides structurally derived from the naturally occurring insecticides called pyrethrins. Along with emerging evidence that exposure to insecticides is linked to altered weight gain and glucose homeostasis, exposure to pyrethroids has been linked to altered blood glucose levels in humans. Thus, the purpose of this study was to determine the role of permethrin on lipid and glucose metabolisms. Permethrin was treated to 3T3-L1 adipocytes and C2C12 myoblasts to determine its role in lipid and glucose metabolisms, respectively. Permethrin treatment resulted in increased expression of key markers of adipogenesis and lipogenesis in adipocytes. Permethrin significantly reduced insulin-stimulated glucose uptake in myotubes. This is the first report on the role of permethrin in altered lipid metabolism in adipocytes and impaired glucose homeostasis in myotubes. These results may help elucidate fundamental underlying mechanisms between insecticide exposure, particularly permethrin, and potential risk of developing obesity and its comorbidities. PMID:24911977

  3. Lactobacillus plantarum LG42 Isolated from Gajami Sik-Hae Inhibits Adipogenesis in 3T3-L1 Adipocyte

    PubMed Central

    Park, Jeong-Eun; Oh, Suk-Heung; Cha, Youn-Soo

    2013-01-01

    We investigated whether lactic acid bacteria isolated from gajami sik-hae (GLAB) are capable of reducing the intracellular lipid accumulation by downregulating the expression of adipogenesis-related genes in differentiated 3T3-L1 cells. The GLAB, Lactobacillus plantarum LG42, significantly decreased the intracellular triglyceride storage and the glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent manner. mRNA expression of transcription factors like peroxisome proliferator-activated receptor (PPAR) ? and CCAAT/enhancer-binding protein (C/EBP) ? involved in adipogenesis was markedly decreased by the GLAB treatment. Moreover, the GLAB also decreased the expression level of adipogenic markers like adipocyte fatty acid binding protein (aP2), leptin, GPDH, and fatty acid translocase (CD36) significantly. These results suggest that the GLAB inhibits lipid accumulation in the differentiated adipocyte through downregulating the expression of adipogenic transcription factors and other specific genes involved in lipid metabolism. PMID:23555088

  4. Bombesin and bombesin antagonists: studies in Swiss 3T3 cells and human small cell lung cancer.

    PubMed Central

    Woll, P. J.; Rozengurt, E.

    1988-01-01

    Bombesins are potent growth factors for murine Swiss 3T3 cells. Using these cells in chemically defined conditions we have been able to characterise the bombesin receptor and the early signals preceding DNA synthesis. We describe two substance P analogues [DArg1, DPro2, DTrp7,9, Leu11] substance P and [DArg1, DPhe5, DTrp7,9, Leu11] substance P which competitively block the binding of bombesins to their receptor and all the events leading to mitogenesis. Bombesins are secreted by human small cell lung cancers (SCLC) and may act as autocrine growth factors for these tumours, so the development of peptide bombesin antagonists could have therapeutic implications. We demonstrate that the antagonists can reversibly inhibit the growth of SCLC in vitro, with relatively little effect on other lung tumours. PMID:2841962

  5. A Prunus mume extract stimulated the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

    PubMed

    Kono, Ryohei; Okuno, Yoshiharu; Inada, Ken-ichi; Tokuda, Akihiko; Hashizume, Hiroshi; Yoshida, Munehito; Nakamura, Misa; Utsunomiya, Hirotoshi

    2011-01-01

    Osteoporosis is a serious disease caused by decreased bone mass. There is constant matrix remodeling in bones, by which bone formation is performed by osteoblastic cells, whereas bone resorption is accomplished by osteoclast cells. We investigated the effect of a Japanese apricot (Prunus mume SIBE. et ZUCC.) extract on the proliferation and osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. An alkaline phosphatase (ALP) activity assay, cell proliferation assay, alizarin red staining and expression analysis of osteoblastic genes were carried out to assess the proliferation and osteoblastic differentiation. The water-soluble fraction of Prunus mume (PWF) increased the ALP activity, cell proliferation and mineralization. The gene expression of osteopontin and bone morphogenetic protein-2, which are markers in the early period of osteoblastic differentiation, were significantly enhanced by the PWF treatment. PWF therefore stimulated the proliferation and osteoblastic differentiation of cells and may have potential to prevent osteoporosis. PMID:21979066

  6. Characterization of the respiration of 3T3 cells by laser-induced fluorescence during a cyclic heating process

    NASA Astrophysics Data System (ADS)

    Beuthan, J.; Dressler, C.; Zabarylo, U.; Minet, O.

    2010-04-01

    The use of lasers in the near infrared spectral range for laser-induced tumor therapy (LITT) demands a new understanding of the thermal responses to repetitive heat stress. The analysis of laser-induced fluorescence during vital monitoring offers an excellent opportunity to solve many of the related issues in this field. The laser-induced fluorescence of the cellular coenzyme NADH was investigated for its time and intensity behavior under heat stress conditions. Heat was applied to vital 3T3 cells (from 22°C to 50°C) according to a typical therapeutical time regime. A sharp increase in temperature resulted in non-linear time behavior when the concentration of this vital coenzyme changed. There are indications that biological systems have a delayed reaction on a cellular level. These results are therefore important for further dosimetric investigations.

  7. Sp1 mediates repression of the resistin gene by PPAR{gamma} agonists in 3T3-L1 adipocytes

    SciTech Connect

    Chung, S.S. [Genome Research Center for Diabetes and Endocrine Disease, Clinical Research Institute, Seoul National University Hospital, Seoul (Korea, Republic of); Choi, H.H. [Genome Research Center for Diabetes and Endocrine Disease, Clinical Research Institute, Seoul National University Hospital, Seoul (Korea, Republic of); Cho, Y.M. [Genome Research Center for Diabetes and Endocrine Disease, Clinical Research Institute, Seoul National University Hospital, Seoul (Korea, Republic of); Department of Internal Medicine, Seoul National University, College of Medicine, Seoul (Korea, Republic of); Lee, H.K. [Department of Internal Medicine, Seoul National University, College of Medicine, Seoul (Korea, Republic of); Park, K.S. [Genome Research Center for Diabetes and Endocrine Disease, Clinical Research Institute, Seoul National University Hospital, Seoul (Korea, Republic of) and Department of Internal Medicine, Seoul National University, College of Medicine, Seoul (Korea, Republic of)]. E-mail: kspark@snu.ac.kr

    2006-09-15

    Resistin is an adipokine related to obesity and insulin resistance. Expression of the resistin gene is repressed by the treatment of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists, thiazolidinediones (TZDs). In this study, we investigated the mechanism by which TZDs inhibit the resistin gene expression. Resistin gene expression was decreased by TZD in fully differentiated 3T3-L1 adipocytes, which was abolished after treatment of cycloheximide (a protein synthesis inhibitor). TZD could not repress the expression of the resistin gene in the presence of mithramycin A (an Sp1 binding inhibitor). Sp1 binding site of the resistin promoter (-122/-114 bp) was necessary for the repression. Further investigation of the effect of TZDs on the modification of Sp1 showed that the level of O-glycosylation of Sp1 was decreased in this process. These results suggest that PPAR{gamma} activation represses the expression of the resistin gene by modulating Sp1 activity.

  8. Cultured 3T3L1 adipocytes dispose of excess medium glucose as lactate under abundant oxygen availability

    NASA Astrophysics Data System (ADS)

    Sabater, David; Arriarán, Sofía; Romero, María Del Mar; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Mariŕ

    2014-01-01

    White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation.

  9. Nephronectin expression is regulated by SMAD signaling in osteoblast-like MC3T3-E1 cells.

    PubMed

    Tsukasaki, Masayuki; Yamada, Atsushi; Yoshimura, Kentaro; Miyazono, Agasa; Yamamoto, Matsuo; Takami, Masamichi; Miyamoto, Yoichi; Morimura, Naoko; Kamijo, Ryutaro

    2012-08-24

    Nephronectin (Npnt) is an extracellular matrix protein known to be a ligand for the integrin ?8?1. We previously demonstrated that Npnt expression was suppressed by TGF-? through ERK1/2 and JNK in osteoblasts. In this study, we found that inhibition of a TGF-? type I receptor (TGF-? R1, Alk5) by a specific inhibitor {2-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine} strongly induced Npnt expression in osteoblast-like MC3T3-E1 cells. The Alk5 inhibitor-induced increase of Npnt expression occurred in both time- and dose-dependent manners, while that expression was also induced by introduction of an siRNA for Smad2, a central intracellular mediator of TGF-? signaling. These results suggest that the expression of Npnt is regulated by the Alk5-SMAD signaling pathway in osteoblasts. PMID:22842459

  10. St. John's wort promotes adipocyte differentiation and modulates NF-?B activation in 3T3-L1 cells.

    PubMed

    Hatano, Tomoko; Sameshima, Yuka; Kawabata, Mami; Yamada, Shizuo; Shinozuka, Kazumasa; Nakabayashi, Toshikatsu; Mizuno, Hideya

    2014-01-01

    St. John's wort (SJW), or Hypericum perforatum, is a perennial herb that has been used in the treatment of depression in several countries. Though its therapeutic effect on depression has been extensively studied, its influence on metabolic syndrome is yet to be fully characterized. Therefore, we investigated the effect of SJW extract on adipocyte differentiation and its anti-inflammatory effects by using 3T3-L1 preadipocytes. Oil Red O staining indicated that SJW promotes adipocyte differentiation, while immunoblots indicated that SJW increases the expression of peroxisome proliferator activated receptor ? (PPAR?), a nuclear receptor regulating adipocyte differentiation, and adiponectin, an anti-inflammatory adipokine. Furthermore, the anti-inflammatory activity of SJW was demonstrated by its inhibition of the activation of nuclear factor-?B (NF-?B), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-? (TNF-?) decreased the expression of the NF-?B inhibitor I?B?, and increased its phosphorylation. Treatment with SJW further decreased the TNF-?-induced perturbation in I?B? expression and phosphorylation, which indicated that SJW mediated the inhibition of NF-?B activation. In addition, SJW decreased the TNF-?-induced increase in the mRNA levels of pro-inflammatory adipokines, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Collectively, our results indicate that SJW treatment could promote adipocyte differentiation probably through its anti-inflammatory activity, which in turn suggests that SJW has the potential to minimize the risk factors of metabolic syndrome. PMID:24989005

  11. Osmotic shrinkage elicits FAK- and Src phosphorylation and Src-dependent NKCC1 activation in NIH3T3 cells.

    PubMed

    Rasmussen, Line Jee Hartmann; Müller, Helene Steenkćr Holm; Jřrgensen, Bente; Pedersen, Stine Falsig; Hoffmann, Else Kay

    2015-01-15

    The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10-30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2-10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 ?M) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by ?50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by ?15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process. PMID:25377086

  12. The t(8;21) fusion protein, AML1/ETO, transforms NIH3T3 cells and activates AP-1.

    PubMed

    Frank, R C; Sun, X; Berguido, F J; Jakubowiak, A; Nimer, S D

    1999-03-01

    The 8;21 translocation is the most common cytogenetic abnormality in human acute myelogenous leukemia, joining the AML1 gene on chromosome 21, to the ETO gene on chromosome 8, forming the AML1/ETO fusion gene. The AMLI/ETO fusion protein has been shown to function mainly as a transcriptional repressor of AML1 target genes and to block AML1 function in vitro and in vivo. However, AML1/ETO can also activate the BCL-2 promoter and cause enhanced hematopoietic progenitor self-renewal in vitro, suggesting gain-of-functions unique to the fusion protein. We used NIH3T3 cells to determine the transforming capacity of AML1/ETO, and to further characterize its mechanism of action. Expression of AML1/ETO in NIH3T3 cells caused cell-type specific cell death, and cellular transformation, characterized by phenotypic changes, anchorage-independent growth, and tumor formation in nude mice. In contrast, neither expression of AML1A, AML1B or ETO altered the normal growth pattern of the cells. To investigate the mechanism of transformation by AML1/ETO, we analysed the levels of activated, phosphorylated c-Jun (ser63) and other constituents of the AP-1 complex, in the presence of various AML1/ETO related proteins. Expression of AML1/ETO increased the level of c-Jun-P (ser63), and activated AP-1 dependent transcription, which was inhibited by expression of a dominant-negative c-Jun protein. Mutational analysis revealed that the runt homology domain (RHD) and a C-terminal transcriptional repression domain in AML1/ETO are required for transformation, activation of c-Jun and increased AP-1 activity. These results establish the transforming potential of the t(8;21) fusion protein and link this gain-of-function property to modulation of AP-1 activity. PMID:10208431

  13. Overexpression of the short form of the growth hormone receptor in 3T3-L1 mouse preadipocytes

    SciTech Connect

    Bick, T.; Frick, G.P.; Leonard, D. [Univ. of Massachusetts Medical School, Worchester, MA (United States)] [and others

    1994-12-31

    In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHR{sub L}) and a smaller isoform, GHR{sub S} that consists of the extracellular domain and a unique hydrophillic carboxyl terminus. We examined the hypothesis that GHR{sub S} may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHR{sub S} was ligated into the mammalian expression vector pcDNA-I/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects. Sixteen of 24 neomycin resistant clones secreted at least twice as much GHR{sub s} in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold. The amount of GHR{sub L} in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHR{sub L}. The transfected cDNA for GHR{sub S} directs the synthesis of a 50 kDa protein. We conclude that GHR{sub S} contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHR{sub S} in 3T3-L1 cells altered cell function in the absence of GH. 20 refs., 4 figs.

  14. The Effect of Bovine Parathyroid Hormone Withdrawal on MC3T3-E1 Cell Proliferation and Phosphorus Metabolism

    PubMed Central

    Li, Sijia; Cui, Tongxia; Li, Zhonghe; Zhang, Bin; Li, Zhuo; Wu, Jianxiong; Liang, Xinling; Lin, Zheng; Shi, Wei

    2015-01-01

    Hypocalcemia and hypophosphatemia are common complications after parathyroidectomy (PTX). Sudden removal of high circulating levels of parathyroid hormone (PTH) causes decreased osteoclastic resorption resulting in a decreased bone remodeling space. These phenomena are likely due to an increased influx of calcium and phosphorus into bone. However, there are currently no data to support this hypothesis. In this study, we found that PTX significantly reduced levels of PTH, calcium and phosphate. Compared with preoperative levels, after 1 year, postoperative PTH, calcium and phosphate levels were 295.6 ± 173.7 pg/mL (P < 0.05), 86.62 ± 15.98 mg/dL (P < 0.05) and 5.56 ± 2.03 mg/dL (P < 0.05), respectively. We investigated continuous bovine PTH administration as well as withdrawal of bovine PTH stimulation in the mouse osteoblast precursor cell line MC3T3-E1. MC3T3-E1 cells were cultured with continuous bovine PTH treatment for 20 days or with transient bovine PTH treatment for 10 days. High doses of continuous bovine PTH exposure strongly reduced cell proliferation, alkaline phosphatase activity and the number of mineralized calcium nodules. However, withdrawal of bovine PTH (100 ng/mL) significantly increased the number of mineralized calcium nodules and caused a rapid decline in calcium and phosphorus content of culture medium. In conclusion, continuous exposure to bovine PTH inhibited osteoblast differentiation and reduced the formation of mineralized nodules. However, this inhibition was removed and mineralized nodule formation resumed with withdrawal of bovine PTH. According to the results of our clinical examinations and in vitro experiments, we hypothesize that the sudden removal of high levels of PTH may cause an increased influx of calcium and phosphorus into bone after PTX. PMID:25775025

  15. [Effects of isoquercitrin from Craibiodendron yunnanense on osteogenic differentiation of MC3T3-E1 cells].

    PubMed

    Duan, Ai-Zhu; Deng, Xu-Liang; Li, Rong-Tao

    2014-10-01

    Natural products especially flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. The present study is to investigate the effects of isoquercitrin from Craibiodendron yunnanense with different concentrations at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) on proliferation, differentiation and mineralization of MC3T3-E1. Cell proliferation was assessed by CCK-8 kit at 1, 3, 5 and 7 days of culture. Alkaline phosphatase (ALP) activity were performed qualitatively and quantitatively on day 7, and alizarin red S staining was employed to access the mineralization of cells on day 21. The osteogenic markers ALP, collagen type I (COL 1A1), runt-related transcription factor 2 (Runx-2) and Osterix were detected to analysis early osteogenic differentiation of cells on day 3 by RT-PCR. The results showed that isoquercitrin had a dose-dependent effect on the proliferation, osteogenic differentiation, mineralization and gene expression of MC3T3-E1 in the range from 1 x 10(-7) to 1 x 10(-5) mol x L(-1). At concentrations above 1 x 10(-4) mol x L(-1) isoquercitrin showed cytotoxicity, while 1 x 10(-6) mol x L(-1) is the optimal concentration of isoquercitrin to improve the osteoblastic activity. All these results implied that isoquercitrin might be the major composition of traditional Chinese medicine C. yunnanense to treat bone fractures. PMID:25612450

  16. The 3T3-L1 fibroblast to adipocyte conversion is accompanied by increased expression of angiopoietin-1, a ligand for tie2.

    PubMed

    Stacker, S A; Runting, A S; Caesar, C; Vitali, A; Lackmann, M; Chang, J; Ward, L; Wilks, A F

    2000-01-01

    The tie2 receptor tyrosine kinase plays a key role in angiogenesis, and the remodeling and maturation of blood vessels. In this study we have used a factor-dependent cell line (Ba/F3) expressing a chimeric receptor containing the extracellular domain of mouse tie2 and the transmembrane and cytoplasmic domain of the erythropoietin receptor to identify specific binding activity associated with an adipogenic sub-line of 3T3 fibroblasts (3T3-L1). 3T3-L1 fibroblasts are capable of undergoing differentiation to adipocytes under specific culture conditions. When compared to 3T3-L1 cells, the adipocyte differentiated cultures, which contain both pre-adipocytes and adipocytes, exhibited a significantly increased ability to support the growth of Ba/F3 cells expressing the chimeric receptor. Using probes specific for two recently described ligands for tie2, Ang-1 and Ang-2, we have shown that mRNA encoding Ang-1 is upregulated when 3T3-L1 fibroblasts are differentiated to adipocytes. These results suggest that the levels of Ang-1 protein and mRNA in 3T3-L1 cells can be regulated by cellular differentiation in adipose development. PMID:11334054

  17. Enucleation of Feeder Cells and Egg Cells with Psoralens

    PubMed Central

    McGarry, Thomas J.; Bonaguidi, Michael; Lyass, Ljuba; Kessler, John A.; Bodily, Jason M.; Doglio, Lynn

    2010-01-01

    The cell nucleus must be inactivated or destroyed in order to generate feeder layers for cultured cells or to prepare recipient egg cells for nuclear transfer. Existing enucleation techniques are either cumbersome or employ toxic chemicals. Here we report a new method to enucleate cells by treatment with a psoralen and long-wave ultraviolet light. The technique is >90% efficient and causes little cytoplasmic damage to the treated cell. We have used psoralen treatment to enucleate a wide variety of cells, including eggs, sperm, HeLa cells, and fibroblasts. Colonies of human embryonic stem cells (hESCs) and human keratinocyte precursors grown on psoralen-treated feeders are indistinguishable from those grown on gamma-irradiated or mitomycin-C treated cells. Psoralen enucleation provides a rapid, simple, and non-toxic method to generate feeder cells. The technique is also useful for nuclear transfer studies in species with large eggs whose cleavage divisions are not regulated by cell cycle checkpoints. PMID:19705441

  18. Magnetically labeled feeder system for mouse pluripotent stem cell culture.

    PubMed

    Horie, Masanobu; Ito, Akira; Maki, Takeshi; Kawabe, Yoshinori; Kamihira, Masamichi

    2015-05-01

    We report here a magnetically labeled feeder system for mouse embryonic stem/induced pluripotent stem (ES/iPS) cells. Magnetic attraction of feeder cells labeled with magnetite nanoparticles significantly increased ES/iPS colony-forming efficiency. Magnetic labeling of feeder cells also facilitated separation of ES/iPS cells from feeder cells. PMID:25468421

  19. Manually Operated Welding Wire Feeder

    NASA Technical Reports Server (NTRS)

    Rybicki, Daniel J. (Inventor)

    2001-01-01

    A manual welding wire feeder apparatus comprising a bendable elongate metal frame with a feed roller mounted at the center thereof for rotation about an axis transverse to the longitudinal axis of the frame. The frame ends are turned up as tabs and each provided with openings in alignment with each other and the mid-width center of the roller surface. The tab openings are sized to accommodate welding wire and each extends to a side edge of the tab, both opening on the same side of the frame, whereby welding wire can be side-loaded onto the frame. On the side of the frame, opposite the roller a lock ring handle is attached tangentially and is rotatable about the attachment point and an axis perpendicular to the frame. The device is grasped in the hand normally used to hold the wire. A finger is placed through the loop ring and the frame positioned across the palm and lower fingers. The thumb is positioned atop the wire so it can be moved from the back of the frame across the roller, and towards the front. In doing so, the wire is advanced at a steady rate in axial alignment with the tab openings and roller. To accommodate different wire diameters the frame is bendable about its center in the plane of the frame axis and wire so as to keep the wire in sufficient tension against the roller and to keep the wire fixed when the frame is tilted and thumb pressure released.

  20. Fluid Shear-Induced ATP Secretion Mediates Prostaglandin Release in MC3T3-E1 Osteoblasts

    PubMed Central

    Genetos, Damian C.; Geist, Derik J.; Dawei, Liu; Donahue, Henry J.; Duncan, Randall L.

    2010-01-01

    ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear-induced ATP release was mediated through vesicular fusion and was dependent on Ca2+ entry into the cell via L-type voltage-sensitive Ca2+ channels. Degradation of secreted ATP by apyrase prevented shear-induced PGE2 release. Introduction Fluid shear induces a rapid rise in intracellular calcium ([Ca2+]i) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca2+]i is the activation of purinergic (P2) receptors resulting from shear-induced extracellular release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the possible mechanisms associated with this release. Methods MC3T3-E1 preosteoblasts were plated on type I collagen, allowed to proliferate to 90% confluency, then subjected to 12 dynes/cm2 laminar fluid flow using a parallel plate flow chamber. ATP release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap junctional intercellular communication (GJIC) and vesicular formation were added prior to shear and maintained in the flow medium for the duration of the experiment. Results and Conclusions Fluid shear produced a transient increase in ATP release compared to static MC3T3-E1 cells (59.8±15.7nM vs. 6.2±1.8nM, respectively), peaking within 1 min of onset. Inhibition of calcium entry through the L-type voltage-sensitive Ca2+ channel (L-VSCC) with nifedipine or verapamil significantly attenuated shear-induced ATP release. Channel inhibition had no effect on basal ATP release in static cells. Ca2+ -dependent ATP release in response to shear appeared to result from vesicular release, and not through gap hemichannels, since vesicle disruption with N-ethylmaleimide, brefeldin A, or monensin prevented increases in flow-induced ATP release, whereas inhibition of gap hemichannels with either 18?-glycyrrhetinic acid or 18?-glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shear-induced increases in PGE2 release. These data suggest a time line of mechanotransduction wherein fluid shear activates L-VSCC's to promote Ca2+ entry that, in turn, stimulates vesicular ATP release. Further, these data suggest that P2 receptor activation by secreted ATP mediates flow-induced prostaglandin release. PMID:15619668

  1. Inhibitory effects of Capsicum annuum L. water extracts on lipoprotein lipase activity in 3T3-L1 cells.

    PubMed

    Baek, Jongmi; Lee, Jaesung; Kim, Kyoungkon; Kim, Taewoo; Kim, Daejung; Kim, Cheonan; Tsutomu, Kanazawa; Ochir, Sarangowa; Lee, Kooyeon; Park, Cheol Ho; Lee, Yong-Jik; Choe, Myeon

    2013-04-01

    Obesity, an intractable metabolic disease, currently has no medical treatment without side effects, so studies have been actively carried out to find natural compounds that have anti-obesity activity with minimum side effects. In this study, the anti-obesity effects of water extracts of seven Capsicum annuum L. varieties being Putgochu (Pca), Oyee gochu (Oca), Kwari putgochu (Kca), Green pepper (Gca), Yellow paprika (Yca), Red paprika (Rca) and Cheongyang gochu (Cca), were examined through the evaluation of lipoprotein lipase (LPL) mRNA expression level in 3T3-L1 cells (mouse pre-adipocytes). After capsaicin elimination by chloroform defatting, freeze-dried powder of Cca was treated to 3T3-L1 cells and anti-obesity effects were examined by determining the LPL mRNA level using the RT-PCR method. Of the primary fractions, only proven fractions underwent secondary and tertiary refractionating to determine anti-obesity effects. From seven different Capsicum annuum L., there was a significant decrease of the LPL mRNA expression level of 50.9% in Cca treatment compared to the control group. A significant decrease of the LPL mRNA expression level was shown in primary fractions (Fr) 5 (36.2% decrease) and 6 (30.5% decrease) of the Cca water extracts. Due to the impurities checked by UPLC chromatography, Fr 5 and 6 were refractionated to determine the LPL mRNA expression level. Treatment of Fr 6-6 (35.8% decrease) and Fr 5-6 (35.3% decrease) showed a significant decrease in the LPL mRNA expression level. When analyzed using UPLC, major compounds of Fr 6-6 and Fr 5-6 were very similar. Subsequently, we refractionated Fr 6-6 and Fr 5-6 to isolate the major peak for structure elucidation. Treatment of Fr 5-6-1 (26.6% decrease) and Fr 6-6-1 (29.7% decrease) showed a significant decrease in the LPL mRNA expression level. Consequently, the fractions may have a possibility to ameliorate obesity through the decrease of the LPL mRNA expression level. PMID:23610601

  2. Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

    PubMed Central

    Koenig, B B; Cook, J S; Wolsing, D H; Ting, J; Tiesman, J P; Correa, P E; Olson, C A; Pecquet, A L; Ventura, F; Grant, R A

    1994-01-01

    The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs. Images PMID:8065329

  3. Mutational spectrum of myeloid malignancies with inv(3)/t(3;3) reveals a predominant involvement of RAS/RTK signaling pathways

    PubMed Central

    Gröschel, Stefan; Sanders, Mathijs A.; Hoogenboezem, Remco; Zeilemaker, Annelieke; Havermans, Marije; Erpelinck, Claudia; Bindels, Eric M. J.; Beverloo, H. Berna; Döhner, Hartmut; Löwenberg, Bob; Döhner, Konstanze; Delwel, Ruud

    2015-01-01

    Myeloid malignancies bearing chromosomal inv(3)/t(3;3) abnormalities are among the most therapy-resistant leukemias. Deregulated expression of EVI1 is the molecular hallmark of this disease; however, the genome-wide spectrum of cooperating mutations in this disease subset has not been systematically elucidated. Here, we show that 98% of inv(3)/t(3;3) myeloid malignancies harbor mutations in genes activating RAS/receptor tyrosine kinase (RTK) signaling pathways. In addition, hemizygous mutations in GATA2, as well as heterozygous alterations in RUNX1, SF3B1, and genes encoding epigenetic modifiers, frequently co-occur with the inv(3)/t(3;3) aberration. Notably, neither mutational patterns nor gene expression profiles differ across inv(3)/t(3;3) acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome cases, suggesting recognition of inv(3)/t(3;3) myeloid malignancies as a single disease entity irrespective of blast count. The high incidence of activating RAS/RTK signaling mutations may provide a target for a rational treatment strategy in this high-risk patient group. PMID:25381062

  4. Mutational spectrum of myeloid malignancies with inv(3)/t(3;3) reveals a predominant involvement of RAS/RTK signaling pathways.

    PubMed

    Gröschel, Stefan; Sanders, Mathijs A; Hoogenboezem, Remco; Zeilemaker, Annelieke; Havermans, Marije; Erpelinck, Claudia; Bindels, Eric M J; Beverloo, H Berna; Döhner, Hartmut; Löwenberg, Bob; Döhner, Konstanze; Delwel, Ruud; Valk, Peter J M

    2015-01-01

    Myeloid malignancies bearing chromosomal inv(3)/t(3;3) abnormalities are among the most therapy-resistant leukemias. Deregulated expression of EVI1 is the molecular hallmark of this disease; however, the genome-wide spectrum of cooperating mutations in this disease subset has not been systematically elucidated. Here, we show that 98% of inv(3)/t(3;3) myeloid malignancies harbor mutations in genes activating RAS/receptor tyrosine kinase (RTK) signaling pathways. In addition, hemizygous mutations in GATA2, as well as heterozygous alterations in RUNX1, SF3B1, and genes encoding epigenetic modifiers, frequently co-occur with the inv(3)/t(3;3) aberration. Notably, neither mutational patterns nor gene expression profiles differ across inv(3)/t(3;3) acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome cases, suggesting recognition of inv(3)/t(3;3) myeloid malignancies as a single disease entity irrespective of blast count. The high incidence of activating RAS/RTK signaling mutations may provide a target for a rational treatment strategy in this high-risk patient group. PMID:25381062

  5. Genistein inhibits the proliferation and differentiation of MCF-7 and 3T3-L1 cells via the regulation of ER? expression and induction of apoptosis

    PubMed Central

    CHOI, EUN JEONG; JUNG, JAE YEON; KIM, GUN-HEE

    2014-01-01

    The present study investigated the effect of the phytochemical genistein on the proliferation and differentiation of MCF-7 and 3T3-L1 cells via the regulation of estrogen receptor-? (ER?) expression and the induction of apoptosis. When MCF-7 human breast cancer cells were treated with 50, 100, 150 and 200 ?M genistein for 24, 48 or 72 h, cell growth was significantly decreased in a concentration-dependent manner. Notably, the patterns of ER? expression and proliferation in MCF-7 cells treated with genistein were similar. Furthermore, ER? expression in differentiating 3T3-L1 cells was significantly inhibited by 48 h treatment with 50 ?M genistein, which was selected based on the results of cytotoxicity assays on 3T3-L1 preadipocytes [lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays]. Under the same conditions, genistein-induced apoptotic features were observed in MCF-7 and differentiating 3T3-L1 cells. This observation is supported by the finding that B-cell lymphoma 2 (Bcl-2) expression was reduced while that of Bcl-2-associated X protein (Bax) was induced by genistein. The results of the present study suggest that an ER?-related pathway and the induction of apoptosis are involved in the proliferation of MCF-7 cells and the differentiation of 3T3-L1 cells. PMID:25009600

  6. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

    NASA Technical Reports Server (NTRS)

    Pavalko, F. M.; Chen, N. X.; Turner, C. H.; Burr, D. B.; Atkinson, S.; Hsieh, Y. F.; Qiu, J.; Duncan, R. L.

    1998-01-01

    Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

  7. Protective effect of quercitrin against hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells.

    PubMed

    Choi, Eun Mi

    2012-03-01

    The protective effect of quercitrin on the response of osteoblastic MC3T3-E1 cells to oxidative stress was evaluated. Osteoblasts were incubated with H(2)O(2) and/or quercitrin, and markers of osteoblast function and oxidative damage were examined. Quercitrin treatment reversed the cytotoxic effect of H(2)O(2) significantly (P<0.05). This effect was blocked by ICI182780 and LY294002, suggesting that quercitrin's effect might be involved in estrogen action and results from PI3K mediated signaling pathway. Pretreatment of quercitrin increased collagen content, alkaline phosphatase (ALP) activity, and calcium deposition of osteoblasts compared with H(2)O(2) treated cells and these effects were blocked by ERKs and p38 mitogen-activated protein kinases (MAPKs) inhibitors such as PD98059 and SB203580, respectively. These suggest that quercitrin-induced protective effect against osteoblast dysfunction by oxidative stress is associated with increased activation of ERKs and p38 MAPK. Pretreatment with quercitrin also reduced the increase in bone-resorbing factor, receptor activator of nuclear factor-kB ligand (RANKL) and oxidative damage markers (malondialdehyde, protein carbonyl, and nitrotyrosine) induced by H(2)O(2). These results suggest that quercitrin may be protective against H(2)O(2)-induced dysfunction in osteoblasts. PMID:20822887

  8. Mechanically induced c-fos expression is mediated by cAMP in MC3T3-E1 osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1999-01-01

    In serum-deprived MC3T3-E1 osteoblasts, mechanical stimulation caused by mild (287 x g) centrifugation induced a 10-fold increase in mRNA levels of the proto-oncogene, c-fos. Induction of c-fos was abolished by the cAMP-dependent protein kinase inhibitor H-89, suggesting that the transient c-fos mRNA increase is mediated by cAMP. Down-regulation of protein kinase C (PKC) activity by chronic TPA treatment failed to significantly reduce c-fos induction, suggesting that TPA-sensitive isoforms of PKC are not responsible for c-fos up-regulation. In addition, 287 x g centrifugation increased intracellular prostaglandin E2 (PGE2) levels 2.8-fold (P<0. 005). Since we have previously shown that prostaglandin E2 (PGE2) can induce c-fos expression via a cAMP-mediated mechanism, we asked whether the increase in c-fos mRNA was due to centrifugation-induced PGE2 release. Pretreatment with the cyclooxygenase inhibitors indomethacin and flurbiprofen did not hinder the early induction of c-fos by mechanical stimulation. We conclude that c-fos expression induced by mild mechanical loading is dependent primarily on cAMP, not PKC, and initial induction of c-fos is not necessarily dependent on the action of newly synthesized PGE2.

  9. Effects of sulfonylurea drugs on adiponectin production from 3T3-L1 adipocytes: implication of different mechanism from pioglitazone.

    PubMed

    Kanda, Yukiko; Matsuda, Masafumi; Tawaramoto, Kazuhito; Kawasaki, Fumiko; Hashiramoto, Mitsuru; Matsuki, Michihiro; Kaku, Kohei

    2008-07-01

    Adiponectin is a fat-derived cytokine with anti-diabetic and anti-atherogenic properties. In this study, effects of sulfonylureas (SUs) on adiponectin production and the action mechanism were evaluated using 3T3-L1 adipocytes. The cells were incubated with glimepiride, glibenclamide, gliclazide, pioglitazone, metformin and the medium only as the control. In the control, the adiponectin level evaluated as the production rate per 24 h was not changed, while pioglitazone significantly increased the adiponectin level. SUs also increased the adiponectin level, but metformin failed to show any increase in adiponectin production. SUs induced adiponectin gene expression as well as pioglitazone. Pioglitazone significantly increased adipogenesis, but glimepiride did not. The aP2 gene expression was increased by pioglitazone, but not by glimepiride. Forskolin, a protein kinase A stimulator, reduced the adiponectin production stimulated by glimepiride but not by pioglitazone. These observations strongly suggest that SUs stimulate the adiponectin production through a different mechanism from pioglitazone, namely an interaction with protein kinase A activity. The significance of the extrapancreatic action of SUs observed in this study should be further evaluated in the clinical field. PMID:18455831

  10. 1?-Hydroxy-2-oxopomolic acid isolated from Agrimonia pilosa extract inhibits adipogenesis in 3T3-L1 cells.

    PubMed

    Ahn, Eun-Kyung; Lee, Jung A; Seo, Dong-Wan; Hong, Seong Su; Oh, Joa Sub

    2012-01-01

    In order to determine anti-adipogenic effect, this study investigated 1?-hydroxy-2-oxopomolic acid (HOA) isolated from Agrimonia pilosa inhibits adipocyte differentiation and expression of adipogenic marker genes, such as peroxisome proliferator activated receptor ? (PPAR?), CCAAT-enhancer-binding protein ? (C/EBP?), glucose transporter 4 (GLUT4), adiponectin, adipocyte fatty acid-binding protein 2 (aP2), adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c (ADD1/SREBP1c), resistin, and fatty acid synthase (Fas) in 3T3-L1 preadipocyte. We demonstrated that HOA induced a significant decrease in lipid accumulation and expression of adipogenic marker genes in a dose-dependent manner. In addition, HOA reduced the transcripitional activity of PPAR? induced by troglitazone, a potent diabetes agent; it also suppressed expression of PPAR? and C/EBP? protein levels. Our data suggest that HOA isolated from Agrimonia pilosa inhibits adipocyte differentiation through downregulation of various adipocytokines by blocking PPAR? and C/EBP? expression. PMID:22687396

  11. IGF 2 expression in 3T3 adipocytes in response to serum from hypophysectomized or diabetic swine

    SciTech Connect

    Srinivas, V.; White, M.E.; Ramsay, T.G. (Ohio State Univ., Columbus (United States))

    1990-02-26

    Expression of IGF-2 and changes in its expression in response to systemic endocrine alterations have not been demonstrated for adipocytes. Adipocytes were induced to develop within cultures of 3T3-L1 cells using a medium containing 0.5mM isobutylmethylxanthine, 1uM insulin and 100ng hydrocortisone/ml for 48 hours of exposure. Cultures containing developing adipocytes were incubated with 10% pig serum and 1 uM insulin for several days. The resultant adipocyte cultures were then treated with either 10% pig serum, diabetic pig serum or hypophysectomized pig serum in DMEM for 48 hours. Adipocytes within the cultures were separated from undifferentiated cells using percoll density gradient centrifugation. Total RNA was isolated from adipocytes and dot blotted. Blots were probed with a {sup 32}P-cDNA probe for rat IGF-2. IGF-2 was expressed by the adipocytes and the pattern of expression showed specific differences between serum treatments; IGF-2 expression was highest in cells exposed to normal pig serum, less expressed in cells exposed to diabetic serum and with minimal expression in adipocytes incubated with hypophysectomized pig serum. These data suggest that adipocyte expression of IGF-2 is influenced by endocrine factors present in pig serum.

  12. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    PubMed

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed. PMID:25246127

  13. Influence of zinc deficiency on cell-membrane fluidity in Jurkat, 3T3 and IMR-32 cells.

    PubMed Central

    Verstraeten, Sandra V; Zago, M Paola; MacKenzie, Gerardo G; Keen, Carl L; Oteiza, Patricia I

    2004-01-01

    We investigated whether zinc deficiency can affect plasma membrane rheology. Three cell lines, human leukaemia T-cells (Jurkat), rat fibroblasts (3T3) and human neuroblastoma cells (IMR-32), were cultured for 48 h in control medium, in zinc-deficient medium (1.5 microM zinc; 1.5 Zn), or in the zinc-deficient medium supplemented with 15 microM zinc (15 Zn). The number of viable cells was lower in the 1.5 Zn group than in the control and 15 Zn groups. The frequency of apoptosis was higher in the 1.5 Zn group than in the control and 15 Zn groups. Membrane fluidity was evaluated using the 6-(9-anthroyloxy)stearic acid and 16-(9-anthroyloxy)palmitic acid probes. Membrane fluidity was higher in 1.5 Zn cells than in the control cells; no differences were observed between control cells and 15 Zn cells. The effect of zinc deficiency on membrane fluidity at the water/lipid interface was associated with a higher phosphatidylserine externalization. The higher membrane fluidity in the hydrophobic region of the bilayer was correlated with a lower content of arachidonic acid. We suggest that the increased fluidity of the membrane secondary to zinc deficiency is in part due to a decrease in arachidonic acid content and the apoptosis-related changes in phosphatidylserine distribution. PMID:14629198

  14. Electrical Stimulation of NIH-3T3 Cells with Platinum-PEDOT-Electrodes Integrated in a Bioreactor

    PubMed Central

    Blume, Grit; Müller-Wichards, Wiebke; Goepfert, Christiane; Pörtner, Ralf; Müller, Jörg

    2013-01-01

    The objective of this work involves the development and integration of electrodes for the electrical stimulation of cells within a bioreactor. Electrodes need to fit properties such as biocompatibility, large reversible charge transfer and high flexibility in view of their future application as implants on the tympanic membrane. Flexible thin-film platinum-poly(3,4-ethylene-dioxythiophene)-electrodes on a poly(ethylene terephthalate)-foil manufactured using microsystems technology were integrated into a bioreactor based on the design of a 24 well plate. The murine fibroblast cell line NIH-3T3 was cultured on the foil electrodes and the cells were stimulated with direct voltage and unipolar pulsed voltage. The amplitude, the pulse length and the ratio of pulse to pause were varied. The stimulated cells were stained in order to determine the angle between the cell cleavage plane of the dividing cells and the vector of the electric field. These angles were subsequently used to calculate the polarization index, which is a measure of the orientation of the metaphase plane of dividing cells that occurs for example during wound healing or embryonic morphogenesis. PMID:24358059

  15. Inhibition of Adipogenesis by Oligonol through Akt-mTOR Inhibition in 3T3-L1 Adipocytes

    PubMed Central

    Park, Jae-Yeo; Kim, Younghwa; Im, Jee Ae; You, Seungkwon

    2014-01-01

    Polyphenols have recently become an important focus of study in obesity research. Oligonol is an oligomerized polyphenol, typically comprised of catechin-type polyphenols from a variety of fruits, which has been found to exhibit better bioavailability and bioreactivity than natural polyphenol compounds. Here, we demonstrated that Oligonol inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression. During adipogenesis, Oligonol downregulated the mRNA levels of peroxisome proliferator-activated receptor ? (PPAR?), CCAAT/enhancer binding proteins ? (C/EBP?), and ? (C/EBP?) in a dose-dependent manner and the expression of genes involved in lipid biosynthesis. The antiadipogenic effect of Oligonol appears to originate from its ability to inhibit the Akt and mammalian target of rapamycin (mTOR) signaling pathway by diminishing the phosphorylation of ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR and forkhead box protein O1 (Foxo1). These results suggest that Oligonol may be a potent regulator of obesity by repressing major adipogenic genes through inhibition of the Akt signaling pathway, which induces the inhibition of lipid accumulation, ultimately inhibiting adipogenesis. PMID:25295069

  16. Effects of yerba maté, a plant extract formulation ("YGD") and resveratrol in 3T3-L1 adipogenesis.

    PubMed

    Santos, Juliana C; Gotardo, Erica M F; Brianti, Mitsue T; Piraee, Mahmood; Gambero, Alessandra; Ribeiro, Marcelo L

    2014-01-01

    We aimed to evaluate the in vitro effects of yerba maté, YGD (a herbal preparation containing yerba maté, guarana and damiana), and resveratrol on adipogenesis. The anti-adipogenic effects of yerba mate, YGD, resveratrol and YGD + resveratrol and yerba mate + resveratrol combinations were evaluated in 3T3-L1 cells by Oil Red staining, cellular triglyceride content, and PCR quantitative array. The results demonstrated that all of the tested compounds inhibited adipogenesis. Yerba maté extract significantly down-regulated the expression of genes that play an important role in regulating adipogenesis, such as Adig, Axin, Cebpa, Fgf10, Lep, Lpl, and Ppar?2. In addition, these genes, YGD also repressed Bmp2, Ccnd1, Fasn, and Srebf1. Resveratrol also modulated the expression of Adig, Bmp2, Ccnd1, C/EBP?, Fasn, Fgf10, Lep, Lpl, and Ppar?2. Moreover, resveratrol repressed Cebpb, Cdk4, Fgf2, and Klf15. The yerba maté extract and YGD up-regulated the expression of genes involved in inhibiting adipogenesis, such as Dlk-1, Klf2, and Ucp1. Resveratrol also induced the expression of Klf2 and Ucp1. In addition resveratrol modulated the Ddit3, Foxo1, Sirt1, and Sirt2. The combined effects of these compounds on gene expression showed similar results observed from individual treatments. Our data indicates that the synergy between the compounds favors the inhibition of adipogenesis. PMID:25338179

  17. Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

    SciTech Connect

    Shimada, Koichi, E-mail: shimada-ki@dent.nihon-u.ac.jp [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan) [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo (Japan); Ikeda, Kyoko [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan)] [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Ito, Koichi [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan) [Department of Periodontology, Nihon University School of Dentistry, Tokyo (Japan); Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo (Japan)

    2009-12-18

    Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-{alpha} stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-{alpha}-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-{alpha} and BMP signaling pathways.

  18. Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts.

    PubMed

    Tajima, R; Kawaguchi, N; Horino, Y; Takahashi, Y; Toriyama, K; Inou, K; Torii, S; Kitagawa, Y

    2001-09-26

    Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat. PMID:11583813

  19. Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty [Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina 27402 (United States); Patel, Yashomati M. [Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina 27402 (United States)], E-mail: ympatel@uncg.edu

    2008-10-15

    Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.

  20. Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

    PubMed Central

    Chowdhury, Helena H.; Kreft, Marko; Jensen, Jřrgen; Zorec, Robert

    2014-01-01

    Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway. PMID:25279585

  1. Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

    PubMed

    Chowdhury, Helena H; Kreft, Marko; Jensen, Jřrgen; Zorec, Robert

    2014-01-01

    Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway. PMID:25279585

  2. Novel effect of helenalin on Akt signaling and Skp2 expression in 3T3-L1 preadipocytes

    SciTech Connect

    Auld, Corinth A. [Department of Nutrition, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States); Hopkins, Robin G. [Department of Nutrition, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States); Fernandes, Karishma M. [Department of Nutrition, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States); Morrison, Ron F. [Department of Nutrition, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States)]. E-mail: ron_morrison@uncg.edu

    2006-07-21

    We have previously shown that the F-box protein, Skp2, is highly regulated during preadipocyte proliferation and plays a mechanistic role in p27 degradation during cell cycle progression. Data presented here demonstrate that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 protein accumulation during early phases of 3T3-L1 adipocyte differentiation. Furthermore, helenalin was shown to completely block p27 degradation, cyclin A accumulation, and G{sub 1}/S transition resulting in G{sub 1} arrest. Helenalin was also shown to block Skp2 mRNA accumulation in a concentration-dependent manner and to completely suppress hormonally induced Skp2 promoter activity suggesting transcriptional mechanisms were involved. Examination of signaling events previously determined to be important for Skp2 upregulation during adipogenesis revealed impaired Akt phosphorylation immediately preceding the inhibitory effect of helenalin on Skp2 mRNA accumulation. These studies demonstrate a novel effect of helenalin on Skp2 regulation and growth factor receptor signaling during early stages of adipocyte differentiation.

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

    PubMed

    Dave, Sandeep; Kaur, Naval Jit; Nanduri, Ravikanth; Dkhar, H Kitdorlang; Kumar, Ashwani; Gupta, Pawan

    2012-01-01

    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/EBP? and PPAR? independent of C/EBP? 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 PPAR? expression seems to stem from its ability to inhibit Akt and augment the TNF? pathway. The Akt-TSC2-mTORC1 pathway has recently been described for PPAR? expression in adipocytes. In our experiments, TNF? 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)?(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 PPAR?-regulated adipogenesis pathway at all stages and by augmenting TNF?-induced lipolysis and apoptosis in mature adipocytes. PMID:22292054

  4. Repression of GLUT4 expression by the endoplasmic reticulum stress response in 3T3-L1 adipocytes

    PubMed Central

    Miller, Ryan S.; Diaczok, Daniel; Cooke, David W.

    2007-01-01

    Expression of GLUT4 is decreased in adipocytes in obesity and type 2 diabetes, contributing to the insulin resistance of these states. Recent investigations suggest a role for activation of the ER stress response in the pathophysiology of type 2 diabetes. We investigated activation of the ER stress response in 3T3-L1 adipocytes. We show that activation of the ER stress response decreased GLUT4 expression at the level of gene transcription. Activation of the ER stress response also increased the expression of CHOP10, an inhibitor of the activity and expression of C/EBP?. As expected, activation of the ER stress response decreased expression of C/EBP?, an activator of GLUT4 expression, providing a mechanism to account for the repression of GLUT4 by ER stress activation. Our studies identify repression of GLUT4 expression as another potential mechanism for obesity-induced activation of the ER stress response to contribute to the insulin resistance of obesity. PMID:17698029

  5. Anti-adipogenic activity of compounds isolated from Idesia polycarpa on 3T3-L1 cells.

    PubMed

    Lee, Mina; Lee, Hyang Hwa; Lee, Jin-Ku; Ye, Sang-Kyu; Kim, Seung Hyun; Sung, Sang Hyun

    2013-06-01

    Recently, obesity is a complex multifactorial chronic disease increasing the risk for type 2 diabetes, coronary heart disease and hypertension, and has become a major worldwide health problem. In the course of screening natural products employing 3T3-L1 cells as an in vitro system, the methanol extract of Idesia polycarpa Maxim. Fruits (Flacourtiaceae) significantly inhibited adipocyte differentiation by measuring lipid contents using oil red O staining. One new compound, 6-(oxymethyl)-2-hydroxyphenyl-O-?-D-glucopyranosyl-(1?6)-?-D-glucopyranoside (8), was isolated along with nine known compounds (1-7 and 9-10) from CHCl3 and n-BuOH fractions of the methanol extract of I. polycarpa fruits. Among them, idescarpin (1) with 1-hydroxy-6-oxo-2-cyclohexenecarboxylate moiety showed the most potent inhibitory activity on adipocyte differentiation with IC50 values of 23.2 ?M. Idescarpin (1) dramatically suppressed the induction of C/EBP? expression, whereas it significantly increased the induction of PPAR? expression, supported by quantitative real time PCR and Western blot analysis. The down-regulation in mRNA levels of SREBP1c, SCD-1, and FAS by idescarpin (1) during adipocyte differentiation revealed that the inhibition of adipocyte differentiation was mediated by the regulation of lipogenesis. Taken together, we suggest that idescarpin (1) shows a great potential against obesity and diabetes though the anti-adipogenic activity and the up-regulation of PPAR?. PMID:23628332

  6. Intranuclear distribution of galectin-3 in mouse 3T3 fibroblasts: comparative analyses by immunofluorescence and immunoelectron microscopy.

    PubMed

    Hubert, M; Wang, S Y; Wang, J L; Sčve, A P; Hubert, J

    1995-10-01

    The intracellular distribution of carbohydrate binding protein 35 (CBP35), recently named galectin-3, was studied in mouse 3T3 fibroblasts, using immunofluorescence at the light microscope level and immunogold labeling at the ultrastructural level. In general, serum-stimulated, proliferating cells showed higher levels of labeling than quiescent cultures of the same cells. In the proliferating cells, the labeling intensity was higher in the nucleus than in the cytoplasm. Treatment of permeabilized cells or thin sections with ribonuclease A decreased the immunolabeling intensity, whereas parallel control treatments with deoxyribonuclease I failed to yield the same effect. While there appears to be general agreement between the immunofluorescence and the ultrastructural results regarding the level of CBP35 and its association with nuclear ribonucleoprotein complexes, there was one striking difference in terms of labeling of specific subnuclear structures. Immunofluorescence results indicate diffuse distribution of CBP35 within the nucleus, but the label appears to be excluded from certain "black holes," which most probably correspond to nucleoli. On the other hand, immunogold particles were observed in electron microscopy, mainly in interchromatin spaces, except for interchromatin granule clusters, at the border of condensed chromatin, on the dense fibrillar component, and at the periphery of the fibrillar centers of nucleoli. PMID:7556449

  7. The Small GTPase Rho A is Crucial for MC3T3-E1 Osteoblastic Cell Survival

    PubMed Central

    Yoshida, Tomohiko; Clark, Mary F.; Stern, Paula H.

    2009-01-01

    Prolongation of cell survival through prevention of apoptosis is considered to be a significant factor leading to anabolic responses in bone. The current studies were carried out to determine the role of the small GTPase, RhoA, in osteoblast apoptosis, since RhoA has been found to be critical for cell survival in other tissues. We investigated the effects of inhibitors and activators of RhoA signaling on osteoblast apoptosis. In addition, we assessed the relationship of this pathway to parathyroid hormone (PTH) effects on apoptotic signaling and cell survival. Rho A is activated by geranylgeranylation, which promotes its membrane anchoring. In serum-starved MC3T3-E1 osteoblastic cells, inhibition of geranylgeranylation with geranylgeranyl transferase I inhibitors increased activity of caspase-3, a component step in the apoptosis cascade, and increased cell death. Dominant negative RhoA and Y27632, an inhibitor of the RhoA effector Rho kinase, also increased caspase-3 activity. A geranylgeranyl group donor, geranylgeraniol, antagonized the effect of the geranylgeranyl tranferase I inhibitor GGTI-2166, but could not overcome the effect of the Rho kinase inhibitor. PTH 1–34, a potent antiapoptotic agent, completely antagonized the stimulatory effects of GGTI-2166, dominant negative RhoA, and Y27632, on caspase-3 activity. The results suggest that RhoA signaling is essential for osteoblastic cell survival but that the survival effects of PTH 1–34 are independent of this pathway. PMID:19184980

  8. Chlamydia Induces Anchorage Independence in 3T3 Cells and Detrimental Cytological Defects in an Infection Model

    PubMed Central

    Knowlton, Andrea E.; Fowler, Larry J.; Patel, Rahul K.; Wallet, Shannon M.; Grieshaber, Scott S.

    2013-01-01

    Chlamydia are Gram negative, obligate intracellular bacterial organisms with different species causing a multitude of infections in both humans and animals. Chlamydia trachomatis is the causative agent of the sexually transmitted infection (STI) Chlamydia, the most commonly acquired bacterial STI in the United States. Chlamydial infections have also been epidemiologically linked to cervical cancer in women co-infected with the human papillomavirus (HPV). We have previously shown chlamydial infection results in centrosome amplification and multipolar spindle formation leading to chromosomal instability. Many studies indicate that centrosome abnormalities, spindle defects, and chromosome segregation errors can lead to cell transformation. We hypothesize that the presence of these defects within infected dividing cells identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation. Here we demonstrate that infection with Chlamydia trachomatis is able to transform 3T3 cells in soft agar resulting in anchorage independence and increased colony formation. Additionally, we show for the first time Chlamydia infects actively replicating cells in vivo. Infection of mice with Chlamydia results in significantly increased cell proliferation within the cervix, and in evidence of cervical dysplasia. Confocal examination of these infected tissues also revealed elements of chlamydial induced chromosome instability. These results contribute to a growing body of data implicating a role for Chlamydia in cervical cancer development and suggest a possible molecular mechanism for this effect. PMID:23308295

  9. The small GTPase RhoA is crucial for MC3T3-E1 osteoblastic cell survival.

    PubMed

    Yoshida, Tomohiko; Clark, Mary F; Stern, Paula H

    2009-04-01

    Prolongation of cell survival through prevention of apoptosis is considered to be a significant factor leading to anabolic responses in bone. The current studies were carried out to determine the role of the small GTPase, RhoA, in osteoblast apoptosis, since RhoA has been found to be critical for cell survival in other tissues. We investigated the effects of inhibitors and activators of RhoA signaling on osteoblast apoptosis. In addition, we assessed the relationship of this pathway to parathyroid hormone (PTH) effects on apoptotic signaling and cell survival. RhoA is activated by geranylgeranylation, which promotes its membrane anchoring. In serum-starved MC3T3-E1 osteoblastic cells, inhibition of geranylgeranylation with geranylgeranyl transferase I inhibitors increased activity of caspase-3, a component step in the apoptosis cascade, and increased cell death. Dominant negative RhoA and Y27632, an inhibitor of the RhoA effector Rho kinase, also increased caspase-3 activity. A geranylgeranyl group donor, geranylgeraniol, antagonized the effect of the geranylgeranyl transferase I inhibitor GGTI-2166, but could not overcome the effect of the Rho kinase inhibitor. PTH 1-34, a potent anti-apoptotic agent, completely antagonized the stimulatory effects of GGTI-2166, dominant negative RhoA, and Y27632, on caspase-3 activity. The results suggest that RhoA signaling is essential for osteoblastic cell survival but that the survival effects of PTH 1-34 are independent of this pathway. PMID:19184980

  10. Low-level expression of purine bases in BALB/3T3 cells monitored by ultrasensitive graphene-based glass carbon electrode.

    PubMed

    Gao, Guang-Gang; Xu, Guo-Biao; Li, Jin-Lian; Cui, Ji-Wen; Wu, Dong-Mei; Qiu, Hong-Bin

    2014-12-15

    Using an ultrasensitive chemically reduced graphene oxide and ionic liquid modified glass carbon electrode (RGI-GCE), separated electrochemical signals of adenine and hypoxanthine in both human breast cancer (MCF-7) and mouse embryonic fibroblast (BALB/3T3) cells were observed. For the first time, low-level expression of purine bases in noncancerous BALB/3T3 cells can be electrochemically monitored. The metabolism of purine bases in carcinogen agent-contaminated BALB/3T3 cells was also investigated through the change of electrochemical signals ascribed to different purine bases, which opens a new electrochemical approach to the exploration of a low-level purine mechanism in noncancerous cells. PMID:25205651

  11. Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.

    PubMed Central

    Stathopoulos, V M; Coco-Maroney, A; Wei, C W; Goth, M; Zaricznyj, C; Macara, I G

    1990-01-01

    Two major species of diacylglycerol kinase (type I and type II) were separated from brain cytosol and from NIH-3T3 or ras-transformed 3T3 cells by heparin-agarose chromatography. Multiple species of diacylglycerol kinase were also detected by non-denaturing isoelectric focusing. The two peaks of activity were of similar size, both co-eluted at approximately 95 kDa from a Superose f.p.l.c. column. Type II enzyme (pI 8.0) was more active when substrate was presented in a deoxycholate/phosphatidylserine undefined environment, as opposed to an octyl glucoside/phosphatidylserine micellar environment. Type II activity was also enhanced by the presence of phosphatidylcholine as cofactor. Type I enzyme (pI 4.0) was more active in the presence of either phosphatidylserine or phosphatidylinositol. Type I and II enzymes had different ATP affinities. Both enzymes showed a preference for diacylglycerol substrates with saturated acyl chains of 10-12 carbon atoms. The cytosolic enzyme activity was able to bind to diacylglycerol-enriched membranes in NIH-3T3 fibroblasts, and this translocation was unaffected in ras-transformed 3T3 cells. These results demonstrate the presence of multiple diacylglycerol kinases in brain cytosol and NIH-3T3 and ras-transformed 3T3 cells. The enzymes differ in cofactor, ATP and substrate requirements. These results can explain some of the contradictions between previous studies of cytosolic diacylglycerol kinase activity, and suggest the presence of a family of such kinases that are differentially regulated by phospholipid cofactors. PMID:2176468

  12. Heterologous expression of C. elegans fat-1 decreases the n-6/n-3 fatty acid ratio and inhibits adipogenesis in 3T3-L1 cells

    SciTech Connect

    An, Lei, E-mail: anleim@yahoo.com.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China)] [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Pang, Yun-Wei, E-mail: yunweipang@126.com [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China)] [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Gao, Hong-Mei, E-mail: Gaohongmei_123@yahoo.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China) [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Research Unit for Animal Life Sciences, Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki-Iwama 319-0206 (Japan); Tao, Li, E-mail: Eunice8023@yahoo.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China) [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin 130118 (China); Miao, Kai, E-mail: miaokai7@163.com [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China)] [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Wu, Zhong-Hong, E-mail: wuzhh@cau.edu.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China)] [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); and others

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer Expression of C. elegans fat-1 reduces the n-6/n-3 PUFA ratio in 3T3-L1 cells. Black-Right-Pointing-Pointer fat-1 inhibits the proliferation and differentiation of 3T3-L1 preadipocytes. Black-Right-Pointing-Pointer fat-1 reduces lipid deposition in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer The lower n-6/n-3 ratio induces apoptosis in 3T3-L1 adipocytes. -- Abstract: In general, a diet enriched in polyunsaturated fatty acids (PUFAs) inhibits the development of obesity and decreases adipose tissue. The specific impacts of n-3 and n-6 PUFAs on adipogenesis, however, have not been definitively determined. Traditional in vivo and in vitro supplementation studies have yielded inconsistent or even contradictory results, which likely reflect insufficiently controlled experimental systems. Caenorhabditiselegans fat-1 gene encodes an n-3 fatty acid desaturase, and its heterologous expression represents an effective method both for altering the n-6/n-3 PUFA ratio and for evaluating the biological effects of n-3 and n-6 PUFAs. We sought to determine whether a reduced n-6/n-3 ratio could influence adipogenesis in 3T3-L1 cells. Lentivirus-mediated introduction of the fat-1 gene into 3T3-L1 preadipocytes significantly reduced the n-6/n-3 ratio and inhibited preadipocyte proliferation and differentiation. In mature adipocytes, fat-1 expression reduced lipid deposition, as measured by Oil Red O staining, and induced apoptosis. Our results indicate that a reduced n-6/n-3 ratio inhibits adipogenesis through several mechanisms and that n-3 PUFAs more effectively inhibit adipogenesis (but not lipogenesis) than do n-6 PUFAs.

  13. Effect of cortisol on caspases in the co-cultured C2C12 and 3 T3-L1 cells.

    PubMed

    Muthuraman, Pandurangan

    2014-06-01

    The present study was carried out to understand the effect of cortisol on caspase expression in the C2C12 and 3 T3-L1 cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4-?m porous membrane to separate C2C12 and 3 T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3 T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 cells were transferred to 3 T3-L1 plates. A total of 10 ?g/?l of cortisol was added to the medium. Following treatment of cortisol for 3 days, the cells in the lower well were harvested for analysis. Caspases such as caspase 3, caspase 7, and caspase 9 were selected for the analysis. qRT-PCR results indicated the significant increase in the mRNA expression of caspase 3, caspase 7, and caspase 9. Caspase 3, 7, and 9 activities were also increased in the mono- and co-cultured C2C12 and 3 T3-L1 cells. In addition, confocal microscopical investigation indicated that cortisol increases caspase expressions in the mono- and co-cultured C2C12 and 3 T3-L1 cells. Taking all these together, we concluded that the co-culture system reflects the exact effect of cortisol on caspase expression, which is quite distinct from one dimensional mono-cultured experiments. PMID:24752940

  14. Eucommia ulmoides Oliv. antagonizes H 2 O 2 -induced rat osteoblastic MC3T3-E1 apoptosis by inhibiting expressions of caspases 3, 6, 7, and 9

    Microsoft Academic Search

    Jun Lin; Yi-jing Fan; Christian Mehl; Jia-jun Zhu; Hong Chen; Ling-yan Jin; Jing-hong Xu; Hui-ming Wang

    2011-01-01

    Eucommia ulmoides Oliv. (EuO), also known as Duzhong, native to China, has been reported to have antioxidative function, but its cellular mechanism\\u000a is not fully examined yet. We investigated inhibitory effects of EuO leaf ethanol extracts on H2O2-induced apoptosis in rat osteoblastic MC3T3-E1 cells and underlying mechanisms. Locally-grown Duzhong leaves were extracted\\u000a with ethanol. MC3T3-E1 cells were treated with EuO

  15. Apelin stimulates glucose uptake through the PI3K\\/Akt pathway and improves insulin resistance in 3T3-L1 adipocytes

    Microsoft Academic Search

    Shunming ZhuFei; Fei Sun; Weijie Li; Yanjie Cao; Chen Wang; Yabin Wang; Dong Liang; Rongqing Zhang; Shenwei Zhang; Haichang Wang; Feng Cao

    2011-01-01

    Apelin, a cytokine mainly secreted by adipocytes, is closely related with insulin resistance. The underlying molecular mechanisms\\u000a of how apelin affects insulin resistance, however, are poorly understood. This study aimed to investigate the effect of apelin\\u000a on glucose metabolism and insulin resistance in 3T3-L1 adipocytes. After 10 ng\\/ml TNF-? treatment for 24 h, insulin-stimulated\\u000a glucose uptake was reduced by 47% in 3T3-L1

  16. PTH regulates ?2-adrenergic receptor expression in osteoblast-like MC3T3-E1 cells.

    PubMed

    Moriya, Shuichi; Hayata, Tadayoshi; Notomi, Takuya; Aryal, Smriti; Nakamaoto, Testuya; Izu, Yayoi; Kawasaki, Makiri; Yamada, Takayuki; Shirakawa, Jumpei; Kaneko, Kazuo; Ezura, Yoichi; Noda, Masaki

    2015-01-01

    As the aged population is soaring, prevalence of osteoporosis is increasing. However, the molecular basis underlying the regulation of bone mass is still incompletely understood. Sympathetic tone acts via beta2 adrenergic receptors in bone and regulates the mass of bone which is the target organ of parathyroid hormone (PTH). However, whether beta2 adrenergic receptor is regulated by PTH in bone cells is not known. We therefore investigated the effects of PTH on beta2 adrenergic receptor gene expression in osteoblast-like MC3T3-E1 cells. PTH treatment immediately suppressed the expression levels of beta2 adrenergic receptor mRNA. This PTH effect was dose-dependent starting as low as 1?nM. PTH action on beta2 adrenergic receptor gene expression was inhibited by a transcriptional inhibitor, DRB, but not by a protein synthesis inhibitor, cycloheximide suggesting direct transcription control. Knockdown of beta2 adrenergic receptor promoted PTH-induced expression of c-fos, an immediate early response gene. With respect to molecular basis for this phenomenon, knockdown of beta2 adrenergic receptor enhanced PTH-induced transcriptional activity of cyclic AMP response element-luciferase construct in osteoblasts. Knockdown of beta2 adrenergic receptors also enhanced forskolin-induced luciferase expression, revealing that adenylate cyclase activity is influenced by beta2 adrenergic receptor. As for phosphorylation of transcription factor, knockdown of beta2 adrenergic receptor enhanced PTH-induced phosphorylation of cyclic AMP response element binding protein (CREB). These data reveal that beta2 adrenergic receptor is one of the targets of PTH and acts as a suppressor of PTH action in osteoblasts. PMID:25164990

  17. Effects of silicon on osteoblast activity and bone mineralization of MC3T3-E1 cells.

    PubMed

    Kim, Eun-Jin; Bu, So-Young; Sung, Mi-Kyung; Choi, Mi-Kyeong

    2013-04-01

    Previous studies have reported that dietary silicon (Si) intake is positively associated with bone health including bone mineral density. Although the amount of Si intake is high among trace elements in humans, how dietary Si affects bone formation at the cellular level is not well addressed. The purpose of this study was to investigate the role of Si in osteoblast activity and bone mineralization. MC3T3-E1 was cultured as mature osteoblasts and treated with sodium metasilicate (0, 1, 5, 10, 25, 50, and 100 ?M) as a source of Si. After 7 days of treatment, 5 and 10 ?M of sodium metasilicate significantly increased intracellular alkaline phosphatase activity (p < 0.05) when compared to the control. Additionally, all doses of sodium metasilicate (1, 5, 10, 25, 50, and 100 ?M) increased mineralized nodule formation at 14 days of differentiation as evidenced by increased Alizarin Red S staining. In the analysis of gene expression, 50 ?M of sodium metasilicate upregulated type I collagen (COL-I) compared to the control group. However, the increase of COL-I gene expression as a result of treatment with 1, 10, 25, and 100 ?M of sodium metasilicate did not reach statistical significance. mRNA expression of insulin-like growth factor-I and receptor activator of NF-?B ligand was not significantly changed at any dose of sodium metasilicate (0, 1, 5, 10, 25, 50, and 100 ?M). In light of the results, we conclude that Si has a positive effect on bone metabolism by enhancing osteoblast mineralization activity. PMID:23306944

  18. Differentially Expressed Proteins in Nitric Oxide-Stimulated NIH/3T3 Fibroblasts: Implications for Inhibiting Cancer Development

    PubMed Central

    Shim, Dong Hwi

    2015-01-01

    Purpose Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. Materials and Methods The cells were treated with 300 µM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Results Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (?1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (? subunit) NG-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and NG-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (? subunit) are unknown in relation to carcinogenesis. Conclusion Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development. PMID:25684010

  19. Expression of pref-1/dlk-1 is regulated by microRNA-143 in 3T3-L1 cells.

    PubMed

    Kim, Yoon-Jin; Min, Tae Sun; Seo, Kang-Seok; Kim, Sang Hoon

    2015-03-01

    Preadipocyte factor 1 (Pref-1), also known as a delta-like 1 protein, is a transmembrane and secreted protein containing the epidermal growth factor-like repeat. Pref-1 inhibits adipocyte differentiation by activating the ERK1/2 pathway. MicroRNAs, a new class of small noncoding RNAs of 20-24 nucleotides, act as negative regulators of gene expression and result in mRNA degradation or translational repression. MicroRNA-143 (miR-143) is known to induce adipocyte differentiation; however, miR-143 targets in the regulation of adipocyte differentiation remain unknown. In this study, we investigated whether pref-1 is a miR-143 target to regulate adipogenesis. After the induction of adipocyte differentiation the level of miR-143 was increased, whereas the expression of pref-1 mRNA was decreased. The pref-1 protein level was also down-regulated in preadipocytes ectopically expressing miR-143, and recovered by miR-143 inhibitor. The binding region for miR-143 was predicted to be located between positions 247 and 252 in the 3'-UTR of pref-1. The luciferase activity of the vector containing the wild-type 3'-UTR of pref-1 was decreased by 65 % in cells transfected with miR-143 mimic compared to that of the corresponding control. In contrast, the activity of the pref-1 mutant cells was not affected by the treatment with miR-143 mimic. The ectopic expression of miR-143 mimic suppressed the phosphorylation of ERK1/2 induced by pref-1 in 3T3-L1 cells. However, the suppressed phosphorylation was restored by miR-143 inhibitor. Taken together, these data suggest that miR-143 promotes adipogenesis by directly modulating the pref-1 expression in adipocytes. PMID:25366176

  20. 3T3 cell motility and morphology before, during, and after exposure to extremely-low-frequency magnetic fields.

    PubMed

    Spadinger, I; Agnew, D; Palcic, B

    1995-01-01

    Automated image cytometry techniques were used to measure motility and morphology in 3T3 fibroblasts exposed to extremely-low-frequency (ELF) magnetic fields. Cell motility and morphology were measured as a function of time before, during, and after 3-4 hour exposures to vertically oriented, 100 microTRMS sinusoidal magnetic fields at various frequencies in the 10-63 Hz range. Sham exposures were also carried out. No static DC fields were applied, but the geomagnetic field was almost vertical and, therefore, had a large component (28.3 microT) parallel to the applied AC field. The morphology and motile behavior of the cells were characterized by mathematically defined descriptors, which were calculated and averaged for the exposure period as well as for control periods that preceded and followed the exposure period. Each experiment involved the tracking of 100 cells that were subjected to one of the test frequencies (unless a sham exposure was being conducted). Statistical analysis of the results showed that even small changes of 10-20% could be significant at the P < .05 level. Changes on this order were measured in a significant proportion of the experiments. However, because such results were seen for both the sham-exposed and the ELF-exposed cells, and because the range of values that was obtained for the sham exposures was the same as that obtained for the ELF exposures, we concluded that there was no evidence to show that any of the measured changes were attributable to the applied ELF magnetic field. PMID:7677794

  1. 3T3 cell motility and morphology before, during, and after exposure to extremely-low-frequency magnetic fields

    SciTech Connect

    Spadinger, I.; Palcic, B. [British Columbia Cancer Research Centre, Vancouver, British Columbia (Canada). Cancer Imaging; Agnew, D. [Ontario Hydro, Whitby, Ontario (Canada). Health and Safety Div.

    1995-08-01

    Automated image cytometry techniques were used to measure motility and morphology in 3T3 fibro-blasts exposed to extremely-low-frequency (ELF) magnetic fields. Cell motility and morphology were measured as a function of time before, during, and after 3--4 hour exposures to vertically oriented, 100 {mu}T{sub RMS} sinusoidal magnetic fields at various frequencies in the 10--63 Hz range. Sham exposures were also carried out. No static DC fields were applied, but the geomagnetic field was almost vertical and, therefore, had a large component (28.3 {mu}T) parallel to the applied AC field. The morphology and motile behavior of the cells were characterized by mathematically defined descriptors, which were calculated and averaged for the exposure period as well as for control periods that preceded and followed the exposure period. Each experiment involved the tracking of 100 cells that were subjected to one of the test frequencies (unless a sham exposure was being conducted). Statistical analysis of the results showed that even small changes of 10--20% could be significant at the P < .05 level. Changes on this order were measured in a significant proportion of the experiments. However, because such results were seen for both the sham-exposed and the ELF-exposed cells, and because the range of values that was obtained for the sham exposures was the same as that obtained for the ELF exposures, the authors concluded that there was no evidence to show that any of the measured changes were attributable to the applied ELF magnetic field.

  2. Controlled release of simvastatin from in situ forming hydrogel triggers bone formation in MC3T3-E1 cells.

    PubMed

    Park, Yoon Shin; David, Allan E; Park, Kyung Min; Lin, Chia-Ying; Than, Khoi D; Lee, Kyuri; Park, Jun Beom; Jo, Inho; Park, Ki Dong; Yang, Victor C

    2013-04-01

    Simvastatin (SIM), a drug commonly administered for the treatment of hypercholesterolemia, has been recently reported to induce bone regeneration/formation. In this study, we investigated the properties of hydrogel composed of gelatin-poly(ethylene glycol)-tyramine (GPT) as an efficient SIM delivery vehicle that can trigger osteogenic differentiation. Sustained delivery of SIM was achieved through its encapsulation in an injectable, biodegradable GPT-hydrogel. Cross-linking of the gelatin-based GPT-hydrogel was induced by the reaction of horse radish peroxidase and H(2)O(2). GPT-hydrogels of three different matrix stiffness, 1,800 (GPT-hydrogel1), 5,800 (GPT-hydrogel2), and 8,400 Pa (GPT-hydrogel3) were used. The gelation/degradation time and SIM release profiles of hydrogels loaded with two different concentrations of SIM, 1 and 3 mg/ml, were also evaluated. Maximum swelling times of GPT-hydrogel1, GPT-hydrogel2, and GPT-hydrogel3 were observed to be 6, 12, and 20 days, respectively. All GPT-hydrogels showed complete degradation within 55 days. The in vitro SIM release profiles, investigated in PBS buffer (pH 7.4) at 37°C, exhibited typical biphasic release patterns with the initial burst being more rapid with GPT-hydrogel1 compared with GPT-hydrogel3. Substantial increase in matrix metalloproteinase-13, osteocalcin expression levels, and mineralization were seen in osteogenic differentiation system using MC3T3-E1 cells cultured with GPT-hydrogels loaded with SIM in a dose-dependent manner. This study demonstrated that controlled release of SIM from a biodegradable, injectable GPT-hydrogel had a promising role for long-term treatment of chronic degenerative diseases such as disc degenerative disease. PMID:23250670

  3. Reduced hormone-stimulated adenylate cyclase activity in NIH-3T3 cells expressing the EJ human bladder ras oncogene.

    PubMed Central

    Tarpley, W G; Hopkins, N K; Gorman, R R

    1986-01-01

    Recent studies have shown that the 21-kilodalton protein (p21) Ha-ras gene product shares sequence homology with and may exhibit biochemical properties similar to the mammalian guanine nucleotide-binding proteins. These data suggested that one of the biochemical functions of p21 in the vertebrate cell may be to regulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. We determined both in intact NIH-3T3 murine cells and in membranes isolated from these cells that the hormone-stimulated adenylate cyclase activity of cells expressing the EJ human bladder carcinoma oncogene (EJ-ras) is significantly reduced compared with control cells. Thus, the levels of cAMP measured in the EJ-ras-transformed cells by radioimmunoassay are reduced 78% and 93% after prostaglandin and isoproterenol stimulation, respectively, compared with the levels in control cells. Treatment of the EJ-ras-transformed cells with pertussis toxin or cholera toxin did not correct the alterations in adenylate cyclase activity. Cells expressing the normal human Ha-ras gene displayed intermediate levels of adenylate cyclase hormone sensitivity; these levels of adenylate cyclase activity were greater than those in the EJ-ras-transformed cells but lower than in control cells. Hormone-stimulated adenylate cyclase activities in cells transfected with Rous sarcoma virus DNA were similar to those in control cells. These data support the hypothesis that both the normal and mutated Ha-ras p21s are related to guanine nucleotide-binding proteins. Images PMID:3012529

  4. NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.

    PubMed Central

    Johansen, T; Bjřrkřy, G; Overvatn, A; Diaz-Meco, M T; Traavik, T; Moscat, J

    1994-01-01

    In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells. Images PMID:8264633

  5. Structural Basis for Recognition of H3T3ph and Smac/DIABLO N-terminal Peptides by Human Survivin

    SciTech Connect

    Du, Jiamu; Kelly, Alexander E.; Funabiki, Hironori; Patel, Dinshaw J. (MSKCC); (Rockefeller)

    2012-03-02

    Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.

  6. Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes.

    PubMed

    Kuonen, R; Weissenstein, U; Urech, K; Kunz, M; Hostanska, K; Estko, M; Heusser, P; Baumgartner, S

    2013-01-01

    Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10??g/mL VALE and 1??g/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo. PMID:24379890

  7. Flavonol acylglycosides from flower of Albizia julibrissin and their inhibitory effects on lipid accumulation in 3T3-L1 cells.

    PubMed

    Yahagi, Tadahiro; Daikonya, Akihiro; Kitanaka, Susumu

    2012-01-01

    Obesity is a serious health problem worldwide. We investigated the anti-obesity effect of the flower of Albizia julibrissin DURAZZ. (Leguminosae). A 90% EtOH extract of the flower inhibited adipogenesis in 3T3-L1 preadipocytes, as well as the activity of glycerol-3-phosphate dehydrogenase (GPDH) activity. New flavonol acylglycosides (1-4) and eighteen known compounds (5-22) were isolated by bioassay-directed fractionation. These new glycosides were elucidated to be 3?-(E)-p-coumaroylquercitrin (1), 3?-(E)-feruloylquercitrin (2), 3?-(E)-cinnamoylquercitrin (3), and 2?-(E)-cinnamoylquercitrin (4) on the basis of spectroscopic and chemical analysis. These compounds inhibited adipogenesis in 3T3-L1 preadipocytes. In particular, 2 exhibited potent inhibitory effects on triglyceride accumulation. Furthermore, GPDH activity was inhibited by 2. Additionally, 2 inhibited glucose uptake in 3T3-L1 adipocytes. These results indicate that the 90% EtOH extract and compounds isolated from the flower of A. julibrissin inhibit adipogenesis in 3T3-L1 preadipocytes and may have anti-obesity effect through the inhibition of preadipocyte differentiation. PMID:22223384

  8. Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes

    PubMed Central

    Kuonen, R.; Weissenstein, U.; Urech, K.; Kunz, M.; Hostanska, K.; Estko, M.; Heusser, P.; Baumgartner, S.

    2013-01-01

    Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10??g/mL VALE and 1??g/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo. PMID:24379890

  9. ?-Tocotrienol induced cell cycle arrest and apoptosis via activating the Bax-mediated mitochondrial and AMPK signaling pathways in 3T3-L1 adipocytes.

    PubMed

    Wu, Shu-Jing; Huang, Guang-Yu; Ng, Lean-Teik

    2013-09-01

    This study aimed to examine the anti-proliferative effects of ?-, ?- and ?-tocotrienols (?T3, ?T3 and ?T3), and ?-tocopherol on 3T3-L1 adipocytes. Results showed that compared with other vitamin E analogues, ?T3 demonstrated the most potent anti-proliferative effect on 3T3-L1 cells. It significantly caused a reduction in mitochondrial membrane potential (??m) and an increase in ROS formation, as well as inducing cell apoptosis and cell cycle arrest at S phase. Further studies showed that it down-regulated Bcl-2 and PPAR-? expression, suppressed Akt and ERK activation and phosphorylation, and caused cytochrome c release from mitochondria to cytosol, whereas it up-regulated CD95 (APO-1/CD95) and Bax expression, and caused caspase-3 and JNK activation, PARP cleavage and AMPK phosphorylation. Pretreatments with caspase-3 (z-DEVD-fmk) and AMPK (CC) inhibitors significantly suppressed the ?T3-induced ROS production and cell death. Caspase-3 inhibitor also efficiently blocked CD95 (APO-1/CD95) and Bax expression, caspase-3 activation and PARP cleavage, whereas antioxidant N-acetyl-l-cysteine, AMPK inhibitor and AMPK siRNA effectively blocked the AMPK phosphorylation. Taken together, these results conclude that the potent anti-proliferative and anti-adipogenic effects of ?T3 on 3T3-L1 adipocytes could be through the Bax-mediated mitochondrial and AMPK signaling pathways. PMID:23816832

  10. Dynamic profile and adipogenic role of growth differentiation factor 5 (GDF5) in the differentiation of 3T3-L1 preadipocytes.

    PubMed

    Pei, Zhou; Yang, Yi; Kiess, Wieland; Sun, Chengjun; Luo, Feihong

    2014-10-15

    Adipocyte differentiation is key to determining the number of adipocytes during the development of obesity. Recent studies have shown that growth differentiation factor-5 (GDF5) promotes brown adipogenesis, however its role in white adipogenesis is still uncertain. The aim of the present study was to investigate the effect of GDF5 on white adipogenesis using 3T3-L1 preadipocyte model. In the present study, GDF5 was found to be differentially regulated during adipocyte differentiation. GDF5 protein increased the differentiation of 3T3-L1 preadipocytes, especially when these cells were exposed to hormone cocktails without insulin. During adipogenesis, GDF5 enhanced the expression of genes related to adipocyte differentiation and caused cells to enter the S phase. Short-hairpin-RNA knockdown of GDF5 in 3T3-L1 cells was found to prevent adipogenesis induced by a standard hormone cocktail and to downregulate the expression of adipocyte genes and proteins, this impairment could be partly rescued by GDF5 protein. Collectively, these results suggest that GDF5 can promote progression of the cell-cycle and increase numbers of cells in S phase, GDF5 might play a critical role in 3T3-L1 preadipocyte differentiation. PMID:25078108

  11. Soyasaponins Aa and Ab exert an anti-obesity effect in 3T3-L1 adipocytes through downregulation of PPAR?.

    PubMed

    Yang, Seung Hwan; Ahn, Eun-Kyung; Lee, Jung A; Shin, Tai-Sun; Tsukamoto, Chigen; Suh, Joo-won; Mei, Itabashi; Chung, Gyuhwa

    2015-02-01

    Saponins are a diverse group of biologically functional products in plants. Soyasaponins are usually glycosylated, which give rise to a wide diversity of structures and functions. In this study, we investigated the effects and molecular mechanism of soyasaponins Aa and Ab in regulating adipocyte differentiation and expression of adipogenic marker genes in 3T3-L1 adipocytes. Soyasaponins Aa and Ab dose-dependently inhibited the accumulation of lipids and the expression of adiponectin, adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c, adipocyte fatty acid-binding protein 2, fatty acid synthase, and resistin in 3T3-L1 adipocytes. In addition, soyasaponins Aa and Ab suppressed the transcriptional activity of peroxisome proliferator-activated receptor ? (PPAR?) in HEK 293T cells. Furthermore, we confirmed that the expression of PPAR? and of CCAAT-enhancer-binding protein ? (C/EBP?) was suppressed at both the mRNA and protein levels in 3T3-L1 adipocytes by treatment with soyasaponins Aa and Ab. Taken together, these findings indicate that soyasaponin Aa and Ab markedly inhibit adipocyte differentiation and expression of various adipogenic marker genes through the downregulation of the adipogenesis-related transcription factors PPAR? and C/EBP? in 3T3-L1 adipocytes. PMID:25366162

  12. Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells.

    PubMed

    Kim, Hyejin; Tabata, Atsushi; Tomoyasu, Toshifumi; Ueno, Tomomi; Uchiyama, Shigeto; Yuasa, Keizo; Tsuji, Akihiko; Nagamune, Hideaki

    2015-01-01

    Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equol was tenfold more effective than daidzein. These differentiation-promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression. PMID:24557631

  13. Coal gasification: New challenge for the Beaumont rotary feeder

    NASA Technical Reports Server (NTRS)

    Stelian, J.

    1977-01-01

    The use of rotary feeders in the coal gasification process is described with emphasis on the efficient conversion of coal to clean gaseous fuels. Commercial applications of the rotary feeder system are summarized.

  14. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...bulkheads forming fire zones, at least one emergency lighting feeder must supply only the emergency lights between two adjacent main vertical fire zone bulkheads. The emergency lighting feeder must be separated...

  15. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...bulkheads forming fire zones, at least one emergency lighting feeder must supply only the emergency lights between two adjacent main vertical fire zone bulkheads. The emergency lighting feeder must be separated...

  16. NIH3T3 cells expressing the deleted in colorectal cancer tumor suppressor gene product stimulate neurite outgrowth in rat PC12 pheochromocytoma cells

    PubMed Central

    1994-01-01

    The Deleted in Colorectal Cancer (DCC) gene is a candidate tumor suppressor gene that is predicted to encode a transmembrane polypeptide with strong similarity to the neural cell adhesion molecule (N-CAM) family. Previous studies have suggested that several different N-CAMs, when expressed in non-neuronal cell types can stimulate neurite outgrowth from PC12 rat pheochromocytoma cells. Based on the predicted structural similarity of DCC to N-CAMs, we sought to determine whether NIH3T3 cells expressing DCC could stimulate neurite outgrowth in PC12 cells. We found that NIH3T3 cell lines expressing DCC could stimulate PC12 cells to extend neurites. Supernatants from DCC-transfected NIH3T3 cells did not induce neurite outgrowth above background levels, suggesting that cell-cell interaction was required. NIH3T3 cells expressing a truncated form of DCC, lacking the majority of the cytoplasmic domain sequences, also failed to induce neurite outgrowth above the levels seen with control NIH3T3 cells, suggesting that the cytoplasmic domain of DCC was necessary for its neurite-promoting function. In contrast to NGF-mediated neurite outgrowth, the DCC- mediated response was inhibited by treatment with pertussis toxin or the combination of N- and L-type calcium channel blockers, and was unaffected by the transcriptional inhibitor cordycepin. The data suggest that the DCC protein can function in a fashion analogous to other N-CAMs to alter PC12 cell phenotype through intracellular pathways distinct from those involved in NGF signaling. PMID:8132705

  17. Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-Cbl in 3T3-L1 adipocytes.

    PubMed Central

    Ribon, V; Saltiel, A R

    1997-01-01

    We report here that the product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl reaches a maximum within 1-2 min after stimulation by insulin and gradually declines thereafter. The tyrosine phosphorylation of c-Cbl was also observed after treatment of 3T3-L1 adipocytes with epidermal growth factor, whereas platelet-derived growth factor had no effect. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with fusion proteins containing the Src homology 2 (SH2) domains of Crk and the Fyn tyrosine kinase, but not with fusion proteins containing the SH2 domains of either the p85 subunit of phosphatidylinositol 3'-kinase or the tyrosine phosphatase SHPTP2/Syp. Furthermore insulin stimulates the association of c-Cbl with endogenous c-Crk and Fyn in intact 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl is regulated during adipocyte differentiation. Neither insulin-like growth factor 1 nor insulin stimulated the tyrosine phosphorylation of c-Cbl in 3T3-L1 fibroblasts. Moreover, c-Cbl is not tyrosine phosphorylated in response to insulin in cells expressing high levels of the human insulin receptor, or in hepatocytes, despite comparable levels of c-Cbl expression. These results suggest that c-Cbl might have a novel function in the regulation of insulin receptor intracellular signalling in 3T3-L1 adipocytes. PMID:9210408

  18. Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay.

    PubMed

    Muramatsu, Dai; Sasaki, Kiyoshi; Kuroda, Sachiko; Hayashi, Kumiko; Tanaka, Noriho; Sakai, Ayako

    2009-04-30

    A short-term cell transformation assay has recently been developed, using Bhas 42 cells which were established from BALB/c 3T3 cells transfected by v-Ha-ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay has been reported to be capable of detecting initiating and promoting activities of chemical carcinogens, according to the different protocols, initiation assay and promotion assay, respectively. The assay is superior to classical transformation assays in cost and labor performance. The present study was carried out to compare its sensitivity with that of a classical BALB/c 3T3 cell system. We performed the Bhas 42 cell transformation assay with inorganic arsenic compounds which are potent environmental carcinogens in human but not mutagens in bacteria or weak mutagens in mammalian cells in vitro. Sodium arsenite, disodium arsenate, and their metabolites, monomethylarsonic acid and dimethylarsinic acid (DMAA) were included in the study. Sodium arsenite was positive in the initiation assay and all compounds except for DMAA were positive in the promotion assay. These results were compared with reported data in a two-stage BALB/c 3T3 cell transformation assay. The sensitivity of Bhas 42 cell transformation assay was found to be similar to that of the conventional BALB/c 3T3 cell transformation assay for the detection of initiating activities of arsenic compounds. For the detection of promoting activities, its sensitivity was equivalent to that of the two-stage BALB/c 3T3 cell transformation assay where the target cells were initiated with sub-threshold dose of 3-methylcholanthrene, confirming that Bhas 42 cells behave as initiated cells in the transformation assay. PMID:19386250

  19. Using Bird Feeders for Wintertime Ecological Instruction.

    ERIC Educational Resources Information Center

    Gannon, John E.; Weber, Peter G.

    1985-01-01

    Describes studies at winter bird feeders which enhance instruction of ecology at the University of Michigan biological station. Background material is provided for field activities in which black-capped chickadees are observed and for home range determination and population estimate activities. (DH)

  20. Pythium Root Rot (and Feeder Root Necrosis)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pythium species cause a number of diseases on corn. Among the Pythium diseases, root rot presents the least conspicuous aboveground symptoms. Broadly defined, root rot also includes feeder root necrosis. At least 16 species of Pythium are known to cause root rot of corn. These include P. acanthicu...

  1. TEXAS FEEDER SCHOOLS SUMMER/FALL 2014

    E-print Network

    John, Lizy Kurian

    THE Austin 1 2440336 HILL COUNTRY CHRISTIAN SCHOOL Austin 2 2440338 AUSTIN WALDORF SCHOOL Austin 1 2440339TEXAS FEEDER SCHOOLS SUMMER/FALL 2014 September 23, 2014 HS Code HS Name HS City Total 2440010 ABILENE HIGH SCHOOL Abilene 2 2440012 COOPER HIGH SCHOOL Abilene 3 2440025 WYLIE HIGH SCHOOL Abilene 3

  2. TEXAS FEEDER SCHOOLS SUMMER/FALL 2013

    E-print Network

    Johnston, Daniel

    GRIFFIN SCHOOL THE Austin 1 2440338 AUSTIN WALDORF SCHOOL Austin 1 2440339 GARZA INDEPENDENCE HIGH SCHOOLTEXAS FEEDER SCHOOLS SUMMER/FALL 2013 October 3, 2013 HS Code HS Name TX HS City Total 2440005 ABERNATHY HIGH SCHOOL Abernathy 1 2440010 ABILENE HIGH SCHOOL Abilene 5 2440012 COOPER HIGH SCHOOL Abilene 4

  3. New resource axes for deposit feeders?

    Microsoft Academic Search

    Robert F; L. Self; Peter A. Jumars

    Recent work on selectivity in deposit feeders has focused on the importance of particle size . In field experiments with exotic sediments of known characteristics (glass beads), we demon- strate that selective ingestion in a multitentaculate, surface deposit feeding ampharetid poly- chaete depends upon particle specific gravities and surface textures . The degree of selectivity for specific gravity is shown

  4. The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.

    PubMed

    Park, Yong; Lee, Seung Jin; Choi, In Young; Lee, Se Ryeon; Sung, Hwa Jung; Kim, Jong Hoon; Yoo, Young Do; Geum, Dong Ho; Kim, Sun Haeng; Kim, Byung Soo

    2010-06-01

    The use of a mouse embryonic fibroblast (MEF) feeder for culture of embryonic stem cells (ESCs) is a widely accepted method, regardless of the ESCs' origin and type. In this study, we performed the undifferentiated propagation of human ES cell lines (hESCs, H1, and HSF6) and mouse ES cell lines (mESCs, D3, and CE3), which were previously maintained on an MEF feeder, using human placenta-derived fibroblast-like cell (HPC) feeders originated from chorionic villi of women who had undergone therapeutic abortion due to known maternal disease that is aggravated by pregnancy. Moreover, we tried to introduce the HPC feeder for the establishment of inducible pluripotent stem cells (iPSCs) from human placental mesenchymal stem cells (MSCs). On the HPC feeder we were able to propagate ESCs and iPSCs colonies as an undifferentiated state up to the 50th passage and 20th passage, respectively. Maintenance of undifferentiated ESCs was identified by the expression of ALP, SSEA-1, SSEA-4, TRA-81, TRA-60, Oct-4, Nanog, or Rex-1. Also, addition of leukemia inhibitory factor was not required for undifferentiated propagation of mESCs on the HPC feeder. The efficiency and expression of three germ layer markers of embryoid bodies (EBs) from ESCs were satisfactory in both the MEF and HPC group. EBs formed from iPSCs were scant, and differentiation to the three germ layers was identifiable by reverst transcription-polymerase chain reactio (RT-PCR) only in the HPC group. In conclusion, the HPC feeder can efficiently support the undifferentiated propagation of hESCs, mESCs, and iPSCs, suggesting that human placenta may be a useful source of universal feeder cells for hESC, mESC, and iPSC culture. PMID:20698773

  5. Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles

    SciTech Connect

    St?pnik, Maciej, E-mail: mstep@imp.lodz.pl [Nofer Institute of Occupational Medicine, ?ód? (Poland)] [Nofer Institute of Occupational Medicine, ?ód? (Poland); Arkusz, Joanna; Smok-Pieni??ek, Anna [Nofer Institute of Occupational Medicine, ?ód? (Poland)] [Nofer Institute of Occupational Medicine, ?ód? (Poland); Bratek-Skicki, Anna; Salvati, Anna; Lynch, Iseult; Dawson, Kenneth A. [Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4 (Ireland)] [Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4 (Ireland); Gromadzi?ska, Jolanta [Nofer Institute of Occupational Medicine, ?ód? (Poland)] [Nofer Institute of Occupational Medicine, ?ód? (Poland); De Jong, Wim H. [National Institute for Public Health and the Environment, Antonie van Leeuwenhoeklaan 9 NL?3720, Bilthoven (Netherlands)] [National Institute for Public Health and the Environment, Antonie van Leeuwenhoeklaan 9 NL?3720, Bilthoven (Netherlands); Rydzy?ski, Konrad [Nofer Institute of Occupational Medicine, ?ód? (Poland)] [Nofer Institute of Occupational Medicine, ?ód? (Poland)

    2012-08-15

    The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but not to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (?-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on cells. -- Highlights: ? Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ? Ludox CL-X silica NPs are cytotoxic to both cell lines. ? In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ? Cell cycle analysis shows alterations in both cell lines with both silica NP tested. ? Buthionine sulfoximine enhances cytotoxicity of Ludox CL-X in 3T3-L1 cells.

  6. Epidermal growth factor receptor-mediated expression of NF-?B transcription factor in osteoblastic MC3T3-E1 cells cultured under a low-calcium environment

    Microsoft Academic Search

    A. Matsumoto; Y. Deyama; A. Deyama; M. Okitsu; Y. Yoshimura; K. Suzuki

    1998-01-01

    We examined the effect of a low calcium environment on DNA-protein binding reaction activity of the transcription factor, NF-?B in osteoblastic MC3T3-E1 cells, using an electrophoretic mobility shift assay. Binding sites for the NF-?B sequence in DNA in nuclear protein in MC3T3-E1 cells are present. This DNA-protein binding reaction activity increased in MC3T3-E1 cells with EGF treatment, compared with those

  7. Maximum Photovoltaic Penetration Levels on Typical Distribution Feeders: Preprint

    SciTech Connect

    Hoke, A.; Butler, R.; Hambrick, J.; Kroposki, B.

    2012-07-01

    This paper presents simulation results for a taxonomy of typical distribution feeders with various levels of photovoltaic (PV) penetration. For each of the 16 feeders simulated, the maximum PV penetration that did not result in steady-state voltage or current violation is presented for several PV location scenarios: clustered near the feeder source, clustered near the midpoint of the feeder, clustered near the end of the feeder, randomly located, and evenly distributed. In addition, the maximum level of PV is presented for single, large PV systems at each location. Maximum PV penetration was determined by requiring that feeder voltages stay within ANSI Range A and that feeder currents stay within the ranges determined by overcurrent protection devices. Simulations were run in GridLAB-D using hourly time steps over a year with randomized load profiles based on utility data and typical meteorological year weather data. For 86% of the cases simulated, maximum PV penetration was at least 30% of peak load.

  8. A human endothelial cell feeder system that efficiently supports the undifferentiated growth of mouse embryonic stem cells.

    PubMed

    Zhou, Haisheng; Yong, Jun; Sun, Xiaomeng; Wang, Chengyan; Yang, Weifeng; Zhang, Pengbo; Zhu, Jingliang; Shi, Cheng; Ding, Mingxiao; Deng, Hongkui

    2008-11-01

    Feeder cells are commonly used to culture embryonic stem cells to maintain their undifferentiated and pluripotent status. Conventionally, mouse embryonic fibroblasts (MEFs), supplemented with leukemia inhibitory factor (LIF), are used as feeder cells to support the growth of mouse embryonic stem cells (mESCs) in culture. To prepare for fresh MEF feeder or for MEF-conditioned medium, sacrifice of mouse fetuses repeatedly is unavoidable in these tedious culture systems. Here we report the discovery of a human endothelial cell line (ECV-304 cell line) that efficiently supports growth of mESCs LIF-free conditions. mESCs that were successfully cultured for eight to 20 passages on ECV-304 feeders showed morphological characteristics similar to cells cultured in traditional feeder cell systems. These cells expressed the stem cell markers Oct3/4, Nanog, Sox2, and SSEA-1. Furthermore, cells cultured on the ECV-304 cell line were able to differentiate into three germ layers and were able to generate chimeric mice. Compared with traditional culture systems, there is no requirement for mouse fetuses and exogenous LIF does not need to be added to the culture system. As a stable cell line, the ECV-304 cell line efficiently replaces MEFs as an effective feeder system and allows the efficient expansion of mESCs. PMID:18557766

  9. Induction of acyl-CoA-binding protein and its mRNA in 3T3-L1 cells by insulin during preadipocyte-to-adipocyte differentiation.

    PubMed Central

    Hansen, H O; Andreasen, P H; Mandrup, S; Kristiansen, K; Knudsen, J

    1991-01-01

    The induction of acyl-CoA-binding protein (ACBP) and ACBP mRNA was investigated in 3T3-L1 cells during growth and insulin-induced differentiation. The level of ACBP relative to both total soluble protein and DNA increased during insulin-stimulated conversion of 3T3-L1 cells from preadipocytes into fully developed adipocytes. So did the total rate of lipogenesis, as measured by incorporation of [1-14C]acetate. A similar increase in ACBP mRNA relative to total RNA was observed. These results therefore suggest that ACBP plays a specific role in the lipogenic process. However, this role might be indirect, as the increase in lipogenesis preceded the increase in ACBP. The significance of this finding is discussed. PMID:1859362

  10. Differential gene expression in ?-irradiated BALB\\/3T3 fibroblasts under the influence of 3-aminobenzamide, an inhibitior of parp enzyme

    Microsoft Academic Search

    R. S. Cardoso; A. R. Espanhol; G. A. S. Passos; E. T. Sakamoto-Hojo

    2002-01-01

    3-Aminobenzamide (3AB) is an inhibitor of poly (ADP-ribose) polymerase (PARP), an enzyme implicated in the maintenance of genomic integrity, which is activated in response to radiation-induced DNA strand breaks. cDNA macroarray membranes containing 1536 clones were used to characterize the gene expression profiles displayed by mouse BALB\\/3T3 fibroblasts (A31 cell line) in response to ionizing irradiation alone or in combination

  11. Effects of aqueous extracts of raw pu-erh tea and ripened pu-erh tea on proliferation and differentiation of 3T3-L1 preadipocytes.

    PubMed

    Cao, Zhen-Hui; Yang, Hui; He, Zhan-Long; Luo, Cheng; Xu, Zhi-Qiang; Gu, Da-Hai; Jia, Jun-Jing; Ge, Chang-Rong; Lin, Qiu-Ye

    2013-08-01

    Pu-erh tea has shown anti-obesity effects but little is known about its effect on proliferation and differentiation of preadipocytes. This study investigated the effects of the aqueous extracts of raw pu-erh tea and ripened pu-erh tea on proliferation and differentiation of murine 3T3-L1 preadiopocytes. We examined dose and time effects of both aqueous extracts on proliferation of 3T3-L1 preadipocytes. The contents of triglycerides in cytoplasm and the mRNA expression of critical transcriptional factors involved in differentiation were determined. Cytotoxicity and apoptosis rate of preadipocytes by pu-erh tea extracts treatment were test for toxic and pro-apoptotic effects. Both aqueous extracts of pu-erh tea inhibited the proliferation of 3T3-L1 preadipocytes at the selected time points. At lower concentration of raw pu-erh tea extracts (less than 300 µg/ml) and ripened pu-erh tea extracts (less than 350 µg/ml), no significant cytotoxic and pro-apoptotic were observed. Ripened pu-erh tea was more effective with lower IC50 than raw pu-erh tea. Both extracts suppressed the differentiation and down-regulated the gene expression of peroxisome proliferator-activated receptor-? and CCAAT/enhancer binding proteins-?. Therefore, these results indicate that both aqueous extracts of pu-erh tea can inhibit proliferation and differentiation with ripened pu-erh tea more potent. Polyphenol rich in both extracts may play a role in the inhibition of proliferation and differentiation of 3T3-L1 preadipocytes. PMID:23027678

  12. Effects of modified Shu-Gan-Liang-Xue decoction combined with anastrozole on osteoblastic proliferation and differentiation of MC3T3-E1 cells.

    PubMed

    Zhou, Fei; Han, Shuyan; Zhou, Ning; Zheng, Wenxian; Li, Pingping

    2015-03-01

    Aromatase inhibitors (AIs) are widely used in the treatment of hormone?dependent breast cancer and as a result, aromatase inhibitor?associated bone loss (AIBL) has become a major concern amongst patients receiving AI treatment. Modified Shu?Gan?Liang?Xue decoction (mSGLXD), a clinical prescription, has been used for ameliorating AIBL in patients with breast cancer for decades and has achieved good clinical efficacy. However, the mechanism underlying how mSGLXD influences bone homeostasis and alleviates AIBL has remained elusive. In the present study, mSGLXD was supplemented with Rhizoma Drynariae containing phytoestrogens, and the safety of mSGLXD was evaluated. mSGLXD did not possess estrogenic activity and significantly inhibited the proliferation of estrogen receptor?positive breast cancer cell line MCF?7, which suggested that mSGLXD was safe for postmenopausal patients with breast cancer. Subsequently, the effects of mSGLXD alone or in combination with anastrozole on osteoblastic MC3T3?E1 cell proliferation and differentiation were investigated. Cell counting kit?8, reverse transcription?polymerase chain reaction and biochemical methods, such as ELISA and alizarin red S staining, were used in the present study. It was revealed that mSGLXD not only stimulated MC3T3?E1 cell proliferation, but also upregulated alkaline phosphatase and osteocalcin gene and protein expression levels. High concentrations of anastrozole (10 or 100 µmol/l) markedly inhibited MC3T3?E1 cell proliferation, but this inhibitory effect was attenuated by mSGLXD. Furthermore, mSGLXD increased MC3T3?E1 cell mineralization following ??glycerophosphate and ascorbic acid induction. Therefore, the results of the present study suggested that mSGLXD may be a promising adjuvant therapy, with high safety and efficacy, for the prevention and treatment of AIBL in patients with breast cancer who receive AI treatment. PMID:25405542

  13. Lysophosphatidic Acid-induced ERK Activation and Chemotaxis in MC3T3-E1 Preosteoblasts are Independent of EGF Receptor Transactivation

    SciTech Connect

    Karagiosis, Sue A.; Chrisler, William B.; Bollinger, Nikki; Karin, Norman J.

    2009-06-01

    Growing evidence indicates that bone-forming osteoblasts and their progenitors are target cells for the lipid growth factor lysophosphatidic acid (LPA) which is produced by degranulating platelets at sites of injury. LPA is a potent inducer of bone cell migration, proliferation and survival in vitro and an attractive candidate to facilitate preosteoblast chemotaxis during skeletal regeneration in vivo, but the intracellular signaling pathways mediating the effects of this lipid on bone cells are not defined. In this study we measured the ability of LPA to stimulate extracellular signal-related kinase (ERK1/2) in MC3T3-E1 preosteoblastic cells and determined the contribution of this pathway to LPA-stimulated chemotaxis. LPA-treated cells exhibited a bimodal activation of ERK1/2 with maximal phosphorylation at 5 and 60 minutes. The kinetics of ERK1/2 phosphorylation were not coupled to Ras activation or LPA-induced elevations in cytosolic Ca2+. While LPA is coupled to the transactivation of the EGF receptor in many cell types, LPA-stimulated ERK1/2 activation in MC3T3-E1 cells was unaffected by inhibition of EGF receptor function. ERK isoforms rapidly accumulated at nuclear sites in LPA-treated cells, a process that was blocked if ERK1/2 phosphorylation was prevented with the MEK1 inhibitor U0126. Blocking ERK1/2 phosphorylation with U0126 also diminished MC3T3-E1 cell migration and altered the normal disassembly of LPA-induced stress fibers, while the inhibition of EGF receptor function had no effect on LPA-coupled preosteoblast motility. Our results identify ERK1/2 activation as a mediatora mediator of LPA-stimulated MC3T3-E1 cell migration that may be relevant to preosteoblast motility during bone repair in vivo.

  14. Effects of modified Shu-Gan-Liang-Xue decoction combined with anastrozole on osteoblastic proliferation and differentiation of MC3T3-E1 cells

    PubMed Central

    ZHOU, FEI; HAN, SHUYAN; ZHOU, NING; ZHENG, WENXIAN; LI, PINGPING

    2015-01-01

    Aromatase inhibitors (AIs) are widely used in the treatment of hormone-dependent breast cancer and as a result, aromatase inhibitor-associated bone loss (AIBL) has become a major concern amongst patients receiving AI treatment. Modified Shu-Gan-Liang-Xue decoction (mSGLXD), a clinical prescription, has been used for ameliorating AIBL in patients with breast cancer for decades and has achieved good clinical efficacy. However, the mechanism underlying how mSGLXD influences bone homeostasis and alleviates AIBL has remained elusive. In the present study, mSGLXD was supplemented with Rhizoma Drynariae containing phytoestrogens, and the safety of mSGLXD was evaluated. mSGLXD did not possess estrogenic activity and significantly inhibited the proliferation of estrogen receptor-positive breast cancer cell line MCF-7, which suggested that mSGLXD was safe for postmenopausal patients with breast cancer. Subsequently, the effects of mSGLXD alone or in combination with anastrozole on osteoblastic MC3T3-E1 cell proliferation and differentiation were investigated. Cell counting kit-8, reverse transcription-polymerase chain reaction and biochemical methods, such as ELISA and alizarin red S staining, were used in the present study. It was revealed that mSGLXD not only stimulated MC3T3-E1 cell proliferation, but also upregulated alkaline phosphatase and osteocalcin gene and protein expression levels. High concentrations of anastrozole (10 or 100 ?mol/l) markedly inhibited MC3T3-E1 cell proliferation, but this inhibitory effect was attenuated by mSGLXD. Furthermore, mSGLXD increased MC3T3-E1 cell mineralization following ?-glycerophosphate and ascorbic acid induction. Therefore, the results of the present study suggested that mSGLXD may be a promising adjuvant therapy, with high safety and efficacy, for the prevention and treatment of AIBL in patients with breast cancer who receive AI treatment. PMID:25405542

  15. Effect of Low and High-Intensity Pulsed Ultrasound on Collagen Posttranslational Modifications in MC3T3-E1 Osteoblasts

    Microsoft Academic Search

    M. Saito; K. Fujii; T. Tanaka; S. Soshi

    2004-01-01

    Different intensities of pulsed ultrasound have distinct biological effects on bone mineralization in the process of bone fracture repair, even across a narrow range (e.g., 30–120 mW\\/cm 2). The aim of our study was to elucidate the effect of low-intensity (30 mW\\/cm 2) and high-intensity (120 mW\\/cm 2) pulsed ultrasound on collagen metabolism by using MC3T3-E1 osteoblasts. Of special interest

  16. Activation of AMP-Activated Protein Kinase Attenuates Tumor Necrosis Factor-?-Induced Lipolysis via Protection of Perilipin in 3T3-L1 Adipocytes

    PubMed Central

    Hong, Seok-Woo; Lee, Jinmi; Park, Se Eun; Rhee, Eun-Jung; Park, Cheol-Young; Oh, Ki-Won; Park, Sung-Woo

    2014-01-01

    Background Tumor necrosis factor (TNF)-? and AMP-activated protein kinase (AMPK) are known to stimulate and repress lipolysis in adipocytes, respectively; however, the mechanisms regulating these processes have not been completely elucidated. Methods The key factors and mechanism of action of TNF-? and AMPK in lipolysis were investigated by evaluating perilipin expression and activity of protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2 ? (eIF2?) by Western blot and an immunofluorescence assay in 24-hour TNF-?-treated 3T3-L1 adipocytes with artificial manipulation of AMPK activation. Results Enhancement of AMPK activity by the addition of activator minoimidazole carboxamide ribonucleotide (AICAR) suppressed TNF-?-induced lipolysis, whereas the addition of compound C, an inhibitor of AMPK phosphorylation, enhanced lipolysis. Perilipin, a lipid droplet-associated protein, was decreased by TNF-? and recovered following treatment with AICAR, showing a correlation with the antilipolytic effect of AICAR. Significant activation of PERK/eIF2?, a component of the unfolded protein response signaling pathway, was observed in TNF-? or vesicle-treated 3T3-L1 adipocytes. The antilipolytic effect and recovery of perilipin expression by AICAR in TNF-?-treated 3T3-L1 adipocytes were significantly diminished by treatment with 2-aminopurine, a specific inhibitor of eIF2?. Conclusion These data indicated that AICAR-induced AMPK activation attenuates TNF-?-induced lipolysis via preservation of perilipin in 3T3-L1 adipocytes. In addition, PERK/eIF2? activity is a novel mechanism of the anti-lipolytic effect of AICAR. PMID:25325265

  17. Human transforming growth factor type. cap alpha. coding sequence is not a directed-acting oncogene when overexpressed in NIH 3T3 cells

    SciTech Connect

    Finzi, E.; Fleming, T.; Segatto, O.; Pennington, C.Y.; Bringman, T.S.; Derynck, R.; Aaronson, S.A.

    1987-06-01

    A peptide secreted by some tumor cells in vitro imparts anchorage-independent growth to normal rat kidney (NRK) cells and has been termed transforming growth factor type ..cap alpha.. (TGF-..cap alpha..). To directly investigate the transforming properties of this factor, the human sequence coding for TGF-..cap alpha.. was placed under the control of either a metallothionein promoter or a retroviral long terminal repeat. These constructs failed to induce morphological transformation upon transfection of NIH 3T3 cells, whereas viral oncogenes encoding a truncated form of its cognate receptor, the EGF receptor, or another growth factor, sis/platelet-derived growth factor 2, efficiently induced transformed foci. Binding assays were done using (/sup 125/I)-EGF. When NIH 3T3 clonal sublines were selected by transfection of TGF-..cap alpha.. expression vectors in the presence of a dominant selectable market, they were shown to secrete large amounts of TGF-..cap alpha.. into the medium, to have downregulated EGF receptors, and to be inhibited in growth by TGF-..cap alpha.. monoclonal antibody. These results indicated that secreted TGF-..cap alpha.. interacts with its receptor at a cell surface location. Single cell-derived TGF-..cap alpha..-expressing sublines grew to high saturation density in culture. These and other results imply that TGF-..cap alpha.. exerts a growth-promoting effect on the entire NIH 3T3 cell population after secretion into the medium but little, if any, effect on the individual cell synthesizing this factor. It is concluded that the normal coding sequence for TGF-..cap alpha.. is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.

  18. Actions of ?-apo-carotenoids in differentiating cells: Differential effects in P19 cells and 3T3-L1 adipocytes.

    PubMed

    Wang, Cynthia X; Jiang, Hongfeng; Yuen, Jason J; Lee, Seung-Ah; Narayanasamy, Sureshbabu; Curley, Robert W; Harrison, Earl H; Blaner, William S

    2015-04-15

    ?-Apo-carotenoids, including ?-apo-13-carotenone and ?-apo-14'-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of ?-apo-13-carotenone and ?-apo-14'-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these ?-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of ?-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two ?-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either ?-apo-13-carotenone or ?-apo-10'-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some ?-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation. PMID:25602703

  19. Effect of Achyranthes bidentata Blume on 3T3-L1 Adipogenesis and Rats Fed with a High-Fat Diet

    PubMed Central

    Oh, Sang Deog; Kim, Mihyun; Min, Byung-Il; Choi, Gi Soon; Kim, Sun-Kwang; Bae, Hyunsu; Kang, Chulhun; Kim, Deok-Gon; Kim, Chang Keun

    2014-01-01

    The present study investigated the antiobesity effect of Achyranthes bidentata Blume root water extract in a 3T3-L1 adipocyte differentiation model and rats fed with a high-fat diet. To investigate the effect of Achyranthes bidentata Blume on adipogenesis in vitro, differentiating 3T3-L1 cells in adipocyte-induction media were treated every two days with Achyranthes bidentata Blume at various concentrations (1 to 25??g/mL) for eight days. We found that Achyranthes bidentata Blume root inhibited 3T3-L1 adipocyte differentiation without affecting cell viability, and Western blot analysis revealed that phospho-Akt expression was markedly decreased, whereas there was no significant change in perilipin expression. Furthermore, administration of Achyranthes bidentata Blume root (0.5?g/kg body weight for six weeks) to rats fed with a high-fat diet significantly reduced body weight gain without affecting food intake, and the level of triglyceride was significantly decreased when compared to those in rats fed with only a high-fat diet. These results suggest that Achyranthes bidentata Blume root water extract could have a beneficial effect on inhibition of adipogenesis and controlling body weight in rats fed with a high-fat diet. PMID:24963319

  20. ?? Adrenergic Receptor Activation Suppresses Bone Morphogenetic Protein (BMP)-Induced Alkaline Phosphatase Expression in Osteoblast-Like MC3T3E1 Cells.

    PubMed

    Yamada, Takayuki; Ezura, Yoichi; Hayata, Tadayoshi; Moriya, Shuichi; Shirakawa, Jumpei; Notomi, Takuya; Arayal, Smriti; Kawasaki, Makiri; Izu, Yayoi; Harada, Kiyoshi; Noda, Masaki

    2015-06-01

    ? adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of ?2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells. J. Cell. Biochem. 116: 1144-1152, 2015. © 2014 Wiley Periodicals, Inc. PMID:25536656

  1. Activation of AMPK participates hydrogen sulfide-induced cyto-protective effect against dexamethasone in osteoblastic MC3T3-E1 cells.

    PubMed

    Yang, Ming; Huang, Yue; Chen, Jia; Chen, Yi-lei; Ma, Jian-jun; Shi, Pei-hua

    2014-11-01

    Long-time glucocorticoids (GCs) usage causes osteoporosis. In the present study, we explored the potential role of hydrogen sulfide (H2S) against dexamethasone (Dex)-induced osteoblast cell damage, and focused on the underlying mechanisms. We showed that two H2S-producing enzymes, cystathionine ?-synthase (CBS) and cystathionine ?-lyase (CSE), were significantly downregulated in human osteonecrosis tissues as well as in Dex-treated osteoblastic MC3T3-E1 cells. H2S donor NaHS as well as the CBS activator S-adenosyl-l-methionine (SAM) inhibited Dex-induced viability reduction, death and apoptosis in MC3T3-E1 cells. NaHS activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling, which participated its cyto-protective activity. AMPK inhibition by its inhibitor (compound C) or reduction by targeted-shRNA suppressed its pro-survival activity against Dex in MC3T3-E1 cells. Further, we found that NaHS inhibited Dex-mediated reactive oxygen species (ROS) production and ATP depletion. Such effects by NaHS were again inhibited by compound C and AMPK?1-shRNA. In summary, we show that H2S inhibits Dex-induced osteoblast damage through activation of AMPK signaling. H2S signaling might be further investigated as a novel target for anti-osteoporosis treatment. PMID:25445596

  2. Expression of a novel insulin-activated amino acid transporter gene during differentiation of 3T3-L1 preadipocytes into adipocytes.

    PubMed

    Liao, K; Lane, M D

    1995-03-28

    A cDNA encoding a novel insulin-activated adipocyte amino acid transporter (designated AAAT) was cloned from a mouse 3T3-L1 adipocyte library. The deduced amino acid sequence of the cDNA corresponds to a protein of 553 amino acids that possesses 56% amino acid sequence identity to the human neutral amino acid transporter and 42% identity to the rat brain glutamate transporter. Transient transfection of 3T3-L1 preadipocytes with an AAAT expression vector led to insulin-dependent uptake of L-serine and to a lesser extent, uptake of L-alanine and L-glutamate. Expression of the AAAT message is tissue-specific, with the highest level occurring in mouse adipose tissue and a lower level in lung. Unlike other sodium-dependent amino acid transporter mRNAs, the AAAT message is not expressed in brain, kidney, liver or heart and only traces are detected in spleen, thymus and skeletal muscle. Consistent with its high level in adipose tissue, expression of the AAAT message is markedly increased when 3T3-L1 preadipocytes are induced to differentiate into adipocytes. PMID:7702599

  3. The ?-SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1

    PubMed Central

    Xie, Weili; Xie, Qi; Jin, Meishan; Huang, Xiaoxiao; Zhang, Xiaodong; Shao, Zhengkai; Wen, Guangwu

    2014-01-01

    Silicon carbide (SiC), a compound of silicon and carbon, with chemical formula SiC, the beta modification (?-SiC), with a zinc blende crystal structure (similar to diamond), is formed at temperature below 1700°C. ?-SiC will be the most suitable ceramic material for the future hard tissue replacement, such as bone and tooth. The in vitro cytotoxicity of ?-SiC nanowires was investigated for the first time. Our results indicated that 100?nm long SiC nanowires could significantly induce the apoptosis in MC3T3-E1 cells, compared with 100??m long SiC nanowires. And 100?nm long SiC nanowires increased oxidative stress in MC3T3-E1 cells, as determined by the concentrations of MDA (as a marker of lipid peroxidation) and 8-OHdG (indicator of oxidative DNA damage). Moreover, transmission electron microscopy (TEM) was performed to evaluate the morphological changes of MC3T3-E1 cells. After treatment with 100?nm long SiC nanowires, the mitochondria were swelled and disintegrated, and the production of ATP and the total oxygen uptake were also decreased significantly. Therefore, ?-SiC nanowires may have limitations as medical material. PMID:24967352

  4. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPARgamma activation.

    PubMed

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-Il; Uemura, Taku; Hirai, Shizuka; Kawada, Teruo

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPARgamma by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPARgamma target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPARgamma and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome. PMID:19891958

  5. Quercetin reversed lipopolysaccharide-induced inhibition of osteoblast differentiation through the mitogen?activated protein kinase pathway in MC3T3-E1 cells.

    PubMed

    Wang, Xin-Chun; Zhao, Nzhi-Jun; Guo, Chun; Chen, Jing-Tao; Song, Jin-Ling; Gao, Li

    2014-12-01

    Quercetin, a flavonoid found in onions and other vegetables, has potential inhibitory effects on bone resorption in vivo and in vitro. In our previous study it was identified that quercetin triggered the apoptosis of lipopolysaccharide (LPS)?induced osteoclasts and inhibited bone resorption. Currently, little information is available detailing the effect of quercetin on osteoblast differentiation and bone formation in bacteria?induced inflammatory diseases. The present study aimed to investigate the effect of quercetin on osteoblast differentiation in MC3T3?E1 osteoblasts stimulated with LPS. LPS significantly downregulated the mRNA expression of osteoblast?related genes in the MC3T3?E1 cells. By contrast, quercetin significantly restored the LPS?suppressed mRNA expression of osteoblast?related genes in a dose?dependent manner. Quercetin also restored the protein expression of Osterix in MC3T3?E1 cells suppressed by LPS. Furthermore, quercetin selectively triggered the activation of the mitogen?activated protein kinase (MAPK) pathway by enhancing the expression of extracellular signal-regulated kinase and reducing the expression of c?Jun N?terminal kinase. These data suggest that quercetin reversed the inhibition of osteoblast differentiation induced by LPS through MAPK signaling. These findings suggest that quercetin may be of potential use as a therapeutic agent to restore osteoblast function in bacteria?induced bone diseases. PMID:25323558

  6. Pneumatic solids feeder for coal gasification reactor

    SciTech Connect

    Notestein, J.E.; Halow, J.S.

    1991-12-31

    This invention is comprised of a pneumatic feeder system for a coal gasification reactor which includes one or more feeder tubes entering the reactor above the level of the particle bed inside the reactor. The tubes are inclined downward at their outer ends so that coal particles introduced into the tubes through an aperture at the top of the tubes slides downward away from the reactor and does not fall directly into the reactor. Pressurized gas introduced into, or resulting from ignition of recycled combustible gas in a chamber adjacent to the tube ends, propels the coal from the tube into the reactor volume and onto the particle bed. Leveling of the top of the bed is carried out by a bladed rotor mounted on the reactor stirring shaft. Coal is introduced into the tubes from containers above the tubes by means of rotary valves placed across supply conduits. This system avoids placement of feeder hardware in the plenum above the particle bed and keeps the coal from being excessively heated prior to reaching the particle bed.

  7. Real time detection and correction of distribution feeder operational problems

    SciTech Connect

    Subramanian, A.K.; Huang, J.C.

    1995-12-31

    The paper presents a new technique that detects and corrects distribution operational problems using closed loop control of substation transformers, capacitors and reactors by an online computer. This allows the distribution system to be operated close to its capacity without sacrificing the quality of power supply. Such operations help defer the additional cost of installing new substations. The technique integrates the Distribution Feeder Analysis (DFA) and the Distribution Substation Control (DSC) functions to achieve this. The DFA function provides the topology and power flow results for the feeders using the substation real time measurements. It does not require feeder section measurements. The realtime feeder results are used in detecting any currently existing feeder operational problems such as feeder section voltages and currents outside their limits. The detected feeder problems are transformed into substation distribution bus objectives and then corrected by the DSC function using controls available at the substation. The DSC function has been performing successfully for several years at Potomac Electric Power Company (PEPCO) in Washington, D.C. It uses a closed loop control scheme that controls the substation transformer taps and shunt capacitor and reactor breakers and optimizes the substation operation. By combining the DFA and DSC functions into a single function and with proper transformation of feeder problems into substation objectives, a new closed loop control scheme for the substation controls is achieved. This scheme corrects the detected feeder problems and optimizes the substation operation. This technique is implemented and tested using the actual substation and feeder models of PEPCO.

  8. Blueberry Peel Extracts Inhibit Adipogenesis in 3T3-L1 Cells and Reduce High-Fat Diet-Induced Obesity

    PubMed Central

    Jang, Sun-Hee; Lee, Soo-Jung; Ko, Yeoung-Gyu; Kim, Gon-Sup; Cho, Jae-Hyeon

    2013-01-01

    This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBP?, as well as the C/EBP? and PPAR? genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3?, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPAR? and C/EBP? and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBP?, C/EBP?, and PPAR? and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity. PMID:23936120

  9. Effect of diacylglycerol acyltransferase 2 overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation

    PubMed Central

    2014-01-01

    Background Fatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells. Methods To investigate the role of DGAT2 in regulating lipid accumulation, FA composition and the expression of adipogenic genes, we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2. Cells were then cultured in differentiation medium (DM) without FA, with a mixture of FAs (FA-DM), or containing a 13C stable isotope-labeled FA mixture (IFA-DM). The FA composition of adipocytes was analyzed by gas chromatography–mass spectrometry and gas chromatography-isotope ratio mass spectrometry. Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d. Results The triacylglyceride (TAG) content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells. When cultured in DM or FA-DM for 12 d, cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs (C16:1 and C18:1). However, when cells overexpressing DGAT2 were cultured with FA-DM for 30 min, the FA composition was almost identical to that of controls. Further, the proportion of stable isotope-labeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d. These results collectively indicate that the higher proportion of mono-unsaturated FAs, C16:1 and C18:1, may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium. This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA synthesis (ACACA, FASN, SCD1, and A-FABP) were significantly higher in cells overexpressing DGAT2 than in control cells. Conclusions In conclusion, our study revealed that TAG accumulation, the proportion of MUFAs, and the expression of adipogenic genes were higher in 3T3-L1 cells overexpressing DGAT2 than in control cells. PMID:24982757

  10. Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

    SciTech Connect

    Kusunoki, Chisato, E-mail: yosizaki@belle.shiga-med.ac.jp [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan); Yang, Liu; Yoshizaki, Takeshi; Nakagawa, Fumiyuki; Ishikado, Atsushi; Kondo, Motoyuki; Morino, Katsutaro; Sekine, Osamu; Ugi, Satoshi [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan); Nishio, Yoshihiko [Division of Diabetes, Metabolism and Endocrinology, Department of Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)] [Division of Diabetes, Metabolism and Endocrinology, Department of Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kashiwagi, Atsunori; Maegawa, Hiroshi [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Omega-3 PUFA has a direct anti-oxidant effect in adipocytes. Black-Right-Pointing-Pointer EPA and DHA induce HO-1 expression in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer Omega-3 PUFA and its end-product, 4-HHE, activates the Nrf-2/HO-1 pathway. Black-Right-Pointing-Pointer Omega-3 PUFA protects against oxidative stress-induced cytotoxicity. -- Abstract: Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid ({omega}3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of {omega}3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with {omega}3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). {omega}3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with {omega}3-PUFA prevented H{sub 2}O{sub 2}-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.

  11. Stearoyl-CoA desaturase enzyme 1 inhibition reduces glucose utilization for de novo fatty acid synthesis and cell proliferation in 3T3-L1 adipocytes

    PubMed Central

    Wahjudi, Paulin N.; Vega, Juan; Lim, Shu; Martin, Ashley; Patterson, Mary E.; Cohen, Joshua N.; Mao, Catherine S.; Lee, Wai-Nang P.

    2013-01-01

    Stearoyl-CoA desaturase enzyme 1 (SCD1) is a lipogenic enzyme that is upregulated in obesity, insulin resistance, and cancer. Since glucose is a substrate for both de novo fatty acid synthesis and deoxyribose synthesis, we hypothesized that SCD1 affects these multiple synthetic pathways through changes in glucose utilization. This study determined glucose utilization for fatty acid synthesis and cell proliferation in 3T3-L1 preadipocytes during SCD1 inhibition. The effects of SCD1 on cellular metabolism as mediated by its monounstaurated fatty acid products (palmitoleate and oleate) were also observed. 3T3-L1 preadipocytes underwent differentiation induction in conjunction with one of the following treatments for 4 days: (A) no treatment, (B) SCD1 inhibitor CGX0290, (C) CGX0290 + palmitoleate, or (D) CGX0290 + oleate. All cells received medium with 50 % [U13C]-glucose. Cells were harvested on day 7 for studies of fatty acid metabolism, tricarboxylic acid (TCA) cycle activities, and deoxyribose synthesis. CGX0290 decreased fatty acid desaturation, glucose utilization for fatty acid synthesis (acetyl-CoA enrichment), and de novo synthesis. CGX0290 treatment also led to decreased cell density through increased cell death. Further analysis showed that deoxyribose new synthesis and oxidative pentose phosphate pathway activity were unchanged, while non-oxidative transketolase pathway activity was stimulated. Palmitoleate and oleate supplementation each partially ameliorated the effects of CGX0290. In 3T3-L1 cells, SCD1 promotes glucose utilization for fatty acid synthesis. In cell proliferation, SCD1 may promote cell survival, but does not impact the oxidative pathway of deoxyribose production. These effects may be mediated through the production of palmitoleate and oleate. PMID:24039619

  12. Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3)-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv).

    PubMed

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Chow, Vincent T K; Chua, Kaw Bing

    2014-01-01

    Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro. PMID:24671184

  13. Irisin has no effect on lipolysis in 3T3-L1 adipocytes or fatty acid metabolism in HepG2 hepatocytes.

    PubMed

    Wang, Chuan; Wang, Lingshu; Li, Wenjuan; Yan, Fei; Tian, Meng; Wu, Chuanlong; Qi, Lin; Wang, Xuping; Song, Jun; Hou, Xinguo; Chen, Li

    2015-05-01

    Irisin, a newly identified myokine responsible for browning of white or beige adipocytes, has been reported to be present at reduced levels in diabetic patients and associated with obesity, serum triglyceride (TG) levels, and intrahepatic TG levels. We wondered whether irisin could directly affect fatty acid and TG metabolism in adipocytes and hepatocytes. We examined the effects of various concentrations of irisin on lipolysis (according to Oil Red O staining, free fatty acid release, and glycerol release), protein expression of HSL and ATGL, and mRNA expression of other lipid-related genes (UCP-1, PPAR?, FABP-4, HSL, ATGL, PPAR?, and CPT-1) in mature 3T3-L1 adipocytes, as well as mRNA levels of genes involved in the synthesis (SREBP-1C and FAS) and ?-oxidation (PPAR? and CPT-1) of fatty acids in HepG2 hepatocytes under physiological or hyperglycemic conditions. Our results revealed that although irisin significantly increased the mRNA levels of UCP-1 and PPAR?, it failed to show detectable effects on lipolysis, HSL or ATGL protein levels, or the mRNA expression of other lipid-related genes in mature 3T3-L1 adipocytes. In HepG2 hepatocytes, high glucose induced the upregulation of SREBP-1C and FAS and the downregulation of PPAR?; however, no significant effect of irisin on gene expression was observed under either physiological or hyperglycemic conditions. We therefore conclude that irisin has no significant direct effect on lipolysis in 3T3-L1 adipocytes or on fatty acid metabolism in HepG2 hepatocytes. PMID:25326905

  14. Effect of ambrex (a herbal formulation) on oxidative stress in hyperlipidemic rats and differentiation of 3T3-L1 preadipocytes

    PubMed Central

    Devi, A. Jamuna; Ravindran, Rekha; Sankar, M.; Rajkumar, Johanna

    2014-01-01

    Background: Ambrex is a polyherbal formulation which consists of Withania somnifera, Orchis mascula, Cycas circirnalis, Shorea robusta with amber. Objective: The present study was designed to explore the potential effects of ambrex on the antioxidant status in high fat diet fed rats and to investigate the possible mechanisms focusing on the gene expression involved in adipogenesis and inflammation in 3T3-L1 cell line. Materials and Methods: Male Wistar rats were divided into four groups (n = 6); Group A received normal diet, Group B received high fat diet for 30 days, Group C and D received high fat diet for 30 days and treated with ambrex (40 mg/kg b.w) and atorvastatin (10 mg/kg b.w) for successive 15 days respectively. This study also assesses the effect of ambrex on adipogenesis in 3T3-L1 adipocytes. Results: The serum total cholesterol and triglycerides were significantly decreased in ambrex treated hyperlipidemic animals when compared to untreated animals. The activities of catalase, superoxide dismutase and reduced glutathione were significantly augmented in the serum, liver, and heart of hyperlipidemic rats treated with ambrex when compared to control. Ambrex treated rats had significant reductions in malondiadehyde levels in the serum, liver and heart compared to untreated rats. In addition, we observed that treatment with ambrex resulted in a major inhibition of pre-adipocyte differentiation of 3T3-L1 cells in vitro by suppression of peroxisome proliferator activated receptor gamma, sterol regulatory binding proteins, tumor necrosis factor-?, inducible nitricoxide synthase, leptin, and upregulation of thioredoxin 1 (TRX1) and TRX2 mRNA expression. Conclusion: Therefore, ambrex may be a potential drug for treatment of hyperlipidemia and related disorders. PMID:24914283

  15. Phenotypic and Genotypic Characteristics of Novel Mouse Cell Line (NIH/3T3)-Adapted Human Enterovirus 71 Strains (EV71:TLLm and EV71:TLLmv)

    PubMed Central

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Chow, Vincent T. K.; Chua, Kaw Bing

    2014-01-01

    Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus’s inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136–150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro. PMID:24671184

  16. Gangliosides inhibit platelet-derived growth factor-stimulated receptor dimerization in human glioma U-1242MG and Swiss 3T3 cells.

    PubMed

    Van Brocklyn, J; Bremer, E G; Yates, A J

    1993-07-01

    We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) in a dose-responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [35S]Methionine-labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross-linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti-PDGFR antibody. Using both Swiss 3T3 and human glioma U-1242MG cells, western blots with anti-PDGFR and anti-phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350-kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF-mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor-mediated cell functions. PMID:8515285

  17. Anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidea and var. angustifolia on 3T3-L1 adipocytes*

    PubMed Central

    Woon, Shiau Mei; Seng, Yew Wei; Ling, Anna Pick Kiong; Chye, Soi Moi; Koh, Rhun Yian

    2014-01-01

    Objective: This study examined the anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidia and var. angustifolia, a natural slimming aid, on 3T3-L1 adipocytes. Methods: Methanol and water extracts of leaves of the F. deltoidea varieties were analyzed to determine their total flavonoid content (TFC) and total phenolic content (TPC), respectively. The study was initiated by determining the maximum non-toxic dose (MNTD) of the methanol and water extracts for 3T3-L1 preadipocytes. Possible anti-adipogenic effects were then examined by treating 2-d post confluent 3T3-L1 preadipocytes with either methanol extract or water extract at MNTD and half MNTD (˝MNTD), after which the preadipocytces were induced to form mature adipocytes. Visualisation and quantification of lipid content in mature adipocytes were carried out through oil red O staining and measurement of optical density (OD) at 520 nm, respectively. Results: The TFCs of the methanol extracts were 1.36 and 1.97 g quercetin equivalents (QE)/100 g dry weight (DW), while the TPCs of the water extracts were 5.61 and 2.73 g gallic acid equivalents (GAE)/100 g DW for var. deltoidea and var. angustilofia, respectively. The MNTDs determined for methanol and water extracts were (300.0±28.3) and (225.0±21.2) ?g/ml, respectively, for var. deltoidea, while much lower MNTDs [(60.0±2.0) ?g/ml for methanol extracts and (8.0±1.0) ?g/ml for water extracts] were recorded for var. angustifolia. Studies revealed that the methanol extracts of both varieties and the water extracts of var. angustifolia at either MNTD or ˝MNTD significantly inhibited the maturation of preadipocytes. Conclusions: The inhibition of the formation of mature adipocytes indicated that leaf extracts of F. deltoidea could have potential anti-obesity effects. PMID:24599694

  18. Downregulation of JE and KC genes by glucocorticoids does not prevent the G0----G1 transition in BALB/3T3 cells.

    PubMed Central

    Rameh, L E; Armelin, M C

    1992-01-01

    The effects of glucocorticoid hormones on the expression of the growth factor-inducible genes JE, KC, and c-myc were analyzed in parental BALB/3T3 and polyomavirus middle-T antigen-transfected cell lines. Northern (RNA) blot hybridization and run-on transcription analysis showed that (i) glucocorticoid hormones selectively inhibit JE and KC expression at the transcriptional level and (ii) the downregulatory effect of glucocorticoids on JE and KC expression is partial for serum-stimulated and middle T antigen-transformed cells and total for quiescent and exponentially growing cells. Gel mobility assays using AP-1 oligonucleotides showed a positive correlation between glucocorticoid downregulating effect and presence of the AP-1 complex. JE and KC downregulation by means of the AP-1 complex may play a role in the actions of glucocorticoids as anti-inflammatory and antitumor agents. The ability of glucocorticoids to downregulate JE and KC was used to investigate the relevance of these genes to the mitogenic response to serum growth factors. Hydrocortisone did not alter the basal DNA synthesis level displayed by quiescent 3T3 cells, but it potentiated both the mitogenic effect of platelet-derived growth factor and c-myc induction by serum growth factors. Upon serum restimulation, untreated and dexamethasone-treated quiescent 3T3 cultures entered the S phase after an identical time lag (G1). These results suggest that (i) JE and KC are not necessary for the G0----G1----S transition and (ii) c-myc overexpression is likely to be the basis for the potentiating effect of glucocorticoids on serum growth factors. Images PMID:1406651

  19. Involvement of phosphatidylcholine hydrolysis by phospholipase C in prostaglandin F2?-induced 1,2-diacylglycerol formation in osteoblast-like MC3T3-E1 cells

    Microsoft Academic Search

    Takayuki Sakai; Takatoshi Sugiyama; Yoshiko Banno; Yukihiro Kato; Yoshinori Nozawa

    2004-01-01

    We previously demonstrated that a prostaglandin F2? (PGF2?)-induced, sustained increase in 1,2-diacylglycerol (DAG) production was important for proliferation in osteoblast-like MC3T3-E1 cells. The 1,2-DAG formation is mediated by various enzymes, such as phos-phoinositide (PI)-specific phospholipase C (PLC), phospholipase D (PLD), and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). In the present study, to elucidate the mechanism of the 1,2-DAG formation, we have

  20. Docosahexaenoic acid modulates phorbol ester-induced activation of extracellular signal-regulated kinases 1 and 2 in NIH\\/3T3 cells

    Microsoft Academic Search

    Anne Denys; Aziz Hichami; Bernard Maume; Naim Akhtar Khan

    2001-01-01

    Phosphorylation of extracellular signal-regulated kinases (ERK1\\/ERK2) has been implicated in cell proliferation of mammalian\\u000a cells. In the present study, we investigated the role of docosahexaenoic acid (DHA) in the modulation of ERK1\\/ERK2 phosphorylation,\\u000a stimulated either with phorbol 12-myristate 13-acetate (PMA) or transforming growth factor-alpha (TGF?) in NIH\\/3T3 cells.\\u000a We observed that both PMA and TGF? induced ERK1\\/ERK2 phosphorylation within 5

  1. Radical scavenging activity and anti-obesity effects in 3T3-L1 preadipocyte differentiation of Ssuk ( Artemisia princeps Pamp.) extract

    Microsoft Academic Search

    Mi-Ja Kim; MinHee Park; Min Kyu Jeong; JuDong Yeo; Wan-Il Cho; Pahn-Shick Chang; Joo-Ho Chung; JaeHwan Lee

    2010-01-01

    Radical scavenging activity of ethanol extracts from ssuk (Artemisia princeps Pampan.) was determined by 2,2-diphenyl-1-pycryl-hydrazil (DPPH) and 2,2?-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)\\u000a (ABTS) methods. Also, anti-obesity effects of ssuk extract were tested in vitro using 3T3-L1 preadipocyte cells. Total phenolic contents of ssuk extract were 42 ?M tannic acid equivalent\\/mg and EC50 of ssuk extract for scavenging DPPH and ABTS radicals were 2.02

  2. The study of oleanolic acid on the estrodiol production and the fat production of mouse preadipocyte 3T3-L1 in vitro.

    PubMed

    Wan, Qian; Lu, Hua; Liu, Xia; Yie, Shangmian; Xiang, Junbei; Yao, Zouying

    2015-01-01

    The women during the menopause period have an increased tendency for the obesity, which represents the more fat production than during the premenopausal period. Although this is not beneficial overall, it could provide a compensatory source for the estrogen production for the menopausal women. So it would be meaningful to find an agent that could inhibit the fat production while does not disturb the total estrogen production by fat tissues. In the present study, the effect of oleanolic acid (OA) on the fat production and the total estrogen production of the differentiating mouse preadipocyte 3T3-L1 as well as the mechanisms behind those effects were preliminarily investigated. The cell line 3T3-L1 was chosen as the model cell because it is usually used for the research about the obesity. During the induced differentiation of 3T3-L1 cells, cells were intervened continuously with OA. The fat production was determined with the oil red staining assay and the total estrogen production was measured with the ELISA assay. Finally, the expression patterns for important genes of the fat production and the estrogen production were studied, respectively with the real-time fluorescence quantitative PCR (qPCR). The results showed that for the differentiating 3T3-L1 cells, OA could significantly inhibit the fat production and did not disturb the total estrogen production significantly. In the mechanism studies, OA was found to significantly down-regulate ACC, the key gene for fat synthesis, which could explain the inhibitory effect of OA on the fat production; OA was also found to significantly up-regulate CYP11A1, CYP17, CYP19, the key genes for the estrogen synthesis and significantly down-regulate CYP1A1, the key gene for the estrogen decomposition, which preliminarily explained the lack of the effect of OA on the total estrogen production. In conclusion, OA was found able to inhibit the fat production while maintaining the total estrogen level and the mechanisms for the above findings were preliminarily clarified, which suggests that OA may be useful to treat the menopausal obesity. PMID:25027016

  3. Arctiin inhibits adipogenesis in 3T3-L1 cells and decreases adiposity and body weight in mice fed a high-fat diet

    PubMed Central

    Min, Byulchorong; Lee, Heejin; Song, Ji Hye; Han, Myung Joo

    2014-01-01

    BACKGROUND/OBJECTIVES The purpose of this study was to examine the effects and associated mechanisms of arctiin, a lignan compound found in burdock, on adipogenesis in 3T3-L1 cells. Also, the effects of arctiin supplementation in obese mice fed a high-fat diet on adiposity were examined. MATERIALS/METHODS 3T3-L1 cells were treated with arctiin (12.5 to 100 µM) during differentiation for 8 days. The accumulation of lipid droplets was determined by Oil Red O staining and intracellular triglyceride contents. The expressions of genes related to adipogenesis were measured by real-time RT-PCR and Western blot analyses. For in vivo study, C57BL/6J mice were first fed either a control diet (CON) or high-fat diet (HF) to induce obesity, and then fed CON, HF, or HF with 500 mg/kg BW arctiin (HF + AC) for four weeks. RESULTS Arctiin treatment to 3T3-L1 pre-adipocytes markedly decreased adipogenesis in a dose-dependent manner. The arctiin treatment significantly decreased the protein levels of the key adipogenic regulators PPAR? and C/EBP?, and also significantly inhibited the expression of SREBP-1c, fatty acid synthase, fatty acid-binding protein and lipoprotein lipase. Also, arctiin greatly increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target phosphorylated-acetyl CoA carboxylase. Furthermore, administration of arctiin significantly decreased the body weight in obese mice fed with the high-fat diet. The epididymal, perirenal or total visceral adipose tissue weights of mice were all significantly lower in the HF + AC than in the HF. Arctiin administration also decreased the sizes of lipid droplets in the epididymal adipose tissue. CONCLUSIONS Arctiin inhibited adipogenesis in 3T3-L1 adipocytes through the inhibition of PPAR? and C/EBP? and the activation of AMPK signaling pathways. These findings suggest that arctiin has a potential benefit in preventing obesity. PMID:25489405

  4. AICAR inhibits adipocyte differentiation in 3T3L1 and restores metabolic alterations in diet-induced obesity mice model

    PubMed Central

    Giri, Shailendra; Rattan, Ramandeep; Haq, Ehtishamul; Khan, Mushfiquddin; Yasmin, Rifat; Won, Je-song; Key, Lyndon; Singh, Avtar K; Singh, Inderjit

    2006-01-01

    Background Obesity is one of the principal causative factors involved in the development of metabolic syndrome. AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. The role of AMP-activated protein kinase in adipocyte differentiation is not completely understood, therefore, we examined the effect of 5-aminoimidazole-4-carboxamide-1-?-D-ribofuranoside (AICAR), a pharmacological activator of AMP-activated protein kinase (AMPK) on adipocyte differentiation in 3T3L1 cells and in a mouse Diet induced obesity (DIO) model. Methods To examine the effect of AICAR on adipocyte differentiation in 3T3L1 cells and in a mouse Diet induced obesity (DIO) model, 3T3L1 cells were differentiatied in the presence or absence of different concentration of AICAR and neutral lipid content and expression of various adipocyte-specific transcription factors were examined. In vivo study, treated and untreated mice with AICAR (0.1–0.5 mg/g body weight) were fed high-fat diet (60% kcal% fat) to induce DIO and several parameters were studied. Results AICAR blocked adipogenic conversion in 3T3L1 cells along with significant decrease in the neutral lipid content by downregulating several adipocyte-specific transcription factors including peroxisome proliferators-activated receptor ? (PPAR?), C/EBP? and ADD1/SREBP1, which are critical for adipogenesis in vitro. Moreover, intraperitoneal administration of AICAR (0.5 mg g/body weight) to mice fed with high-fat diet (60% kcal% fat) to induce DIO, significantly blocked the body weight gain and total content of epididymal fat in these mice over a period of 6 weeks. AICAR treatment also restored normal adipokine levels and resulted in significant improvement in glucose tolerance and insulin sensitivity. The reduction in adipose tissue content in AICAR treated DIO mice was due to reduction in lipid accumulation in the pre-existing adipocytes. However, no change was observed in the expression of PPAR?, C/EBP? and ADD1/SREBP1 transcription factors in vivo though PGC1? expression was significantly induced. Conclusion This study suggests that AICAR inhibits adipocyte differentiation via downregulation of expression of adipogenic factors in vitro and reduces adipose tissue content in DIO mice by activating expression of PGC1? without inhibiting adipocyte-specific transcription factors in DIO mice. PMID:16901342

  5. Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

    2002-01-01

    Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

  6. 1,2-Diacylglycerol does not mediate the stimulatory effect of phorbol 12-myristate 13-acetate on phosphatidylcholine synthesis in NIH 3T3 fibroblasts.

    PubMed

    Kiss, Z; Chattopadhyay, J; Garamszegi, N

    1992-08-01

    In HeLa cells, increased 1,2-diacylglycerol (1,2-DAG) has been suggested to mediate the stimulatory effect of phorbol 12-myristate 13-acetate (PMA) on phosphatidylcholine (PtdCho) biosynthesis (A. K. Utal, H. Jamil, and D. E. Vance, 1991, J. Biol. Chem. 266, 24,084-24,091). The aim of this study was to examine if 1,2-DAG might have a similar mediatory role in NIH 3T3 fibroblasts. In these cells, PMA-induced hydrolysis of PtdCho and the formation of secondary product 1,2-DAG was inhibited by exposing the cells to either 300 mM ethanol for 15 min (less than 80% inhibition) or 43 degrees C for 60 min (less than 50% inhibition). In contrast, neither ethanol nor heat-treatment caused significant inhibition of PMA-stimulated PtdCho synthesis. These data indicate that in NIH 3T3 fibroblasts, 1,2-DAG is not a mediator of the stimulatory action of PMA on PtdCho synthesis. PMID:1632638

  7. PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways

    PubMed Central

    Wang, Weizhuo; Li, Fang; Wang, Kunzheng; Cheng, Bin; Guo, Xiong

    2012-01-01

    Several studies have indicated that PAPSS2 (3?-phosphoadenosine-5?-phosphosulfate synthetase 2) activity is important to normal skeletal development. Mouse PAPSS2 is predominantly expressed during the formation of the skeleton and cartilaginous elements of the mouse embryo and in newborn mice. However, the role and mechanism of PAPSS2 in bone formation remains largely unidentified. By analyzing the expression pattern of the PAPSS2 gene, we have found that PAPSS2 is expressed in bone tissue and bone formation. PAPSS2 transcripts increase during osteoblast differentiation and are in less level in RANKL-induced osteoclast like cells. By using lentivirus-mediated RNA interference (RNAi) technology, we knocked down PAPSS2 expression in MC3T3-E1 osteoblast. Silencing of PAPSS2 expression significantly decreases ALP activity and cell mineralization, inhibits expression of osteoblast marker osteopontin (OPN) and collagen I. Conversely, overexpression of PAPSS2 promotes the MC3T3-E1 to differentiate into osteoblast and mineralization. Moreover, compared to that in the control cells, the mRNA level and protein expression of phosphorylated Smad 2/3, which is a key transcriptional factor in the Smad osteoblast differentiation pathway, showed significant decreases in PAPSS2-silenced cells and increases in PAPSS2-overexpression cells. These results suggest that PAPSS2 might regulate osteoblast ALP activity and cell mineralization, probably through Smads signal pathways. PMID:22916269

  8. Amelioration of Mitochondrial Dysfunction-Induced Insulin Resistance in Differentiated 3T3-L1 Adipocytes via Inhibition of NF-?B Pathways

    PubMed Central

    Hafizi Abu Bakar, Mohamad; Sarmidi, Mohamad Roji; Kai, Cheng Kian; Huri, Hasniza Zaman; Yaakob, Harisun

    2014-01-01

    A growing body of evidence suggests that activation of nuclear factor kappa B (NF-?B) signaling pathways is among the inflammatory mechanism involved in the development of insulin resistance and chronic low-grade inflammation in adipose tissues derived from obese animal and human subjects. Nevertheless, little is known about the roles of NF-?B pathways in regulating mitochondrial function of the adipose tissues. In the present study, we sought to investigate the direct effects of celastrol (potent NF-?B inhibitor) upon mitochondrial dysfunction-induced insulin resistance in 3T3-L1 adipocytes. Celastrol ameliorates mitochondrial dysfunction by altering mitochondrial fusion and fission in adipocytes. The levels of oxidative DNA damage, protein carbonylation and lipid peroxidation were down-regulated. Further, the morphology and quantification of intracellular lipid droplets revealed the decrease of intracellular lipid accumulation with reduced lipolysis. Moreover, massive production of the pro-inflammatory mediators tumor necrosis factor-? (TNF-?) and interleukin-1? (IL-1?) were markedly depleted. Insulin-stimulated glucose uptake activity was restored with the enhancement of insulin signaling pathways. This study signified that the treatments modulated towards knockdown of NF-?B transcription factor may counteract these metabolic insults exacerbated in our model of synergy between mitochondrial dysfunction and inflammation. These results demonstrate for the first time that NF-?B inhibition modulates mitochondrial dysfunction induced insulin resistance in 3T3-L1 adipocytes. PMID:25474091

  9. Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis.

    PubMed Central

    Varrault, A; Bockaert, J; Waeber, C

    1992-01-01

    NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition. Images PMID:1330092

  10. Identification of the target proteins of rosiglitazone in 3T3-L1 adipocytes through proteomic analysis of cytosolic and secreted proteins.

    PubMed

    Hwang, Hyun-Ho; Moon, Pyong-Gon; Lee, Jeong-Eun; Kim, Jung-Guk; Lee, Wan; Ryu, Sung-Ho; Baek, Moon-Chang

    2011-03-01

    Rosiglitazone, one of the thiazolidinedione (TZD), is an oral antidiabetic drug that activates a gamma isoform of peroxisome proliferator-activated receptor (PPAR?). To identify target proteins induced by rosiglitazone in adipocytes, we first performed simultaneous in-depth proteomic profiling of cytosolic proteins and secreted proteins (secretome) from 3T3-L1 adipocytes using a label-free quantification method with nano-UPLC MS/MS. In total, we identified 646 proteins from 3T3-L1 adipocytes, of which 172 and 162 proteins were upregulated and downregulated >1.5-fold, respectively, in rosiglitazone-treated cells, as compared to controls. Some differentially expressed proteins in particular, including fatty acid translocase (FAT)/CD36, fatty acid binding protein, lipoprotein lipase, acetyl CoA acyltransferase, carnitine O-palmitoyltransferase 2, sterol carrier protein, adiponectin, and phosphoenolpyruvate carboxykinase could explain the current action mechanism of TZDs. Furthermore, this study is the first to report on two potential target proteins of rosiglitazone, such as adenomatosis polyposis coli 2 (APC2), and eukaryotic translation initiation factor 5A-1 (eIF5A) related to apoptosis and cell division. Our data clearly suggest that in-depth proteomic approaches using cytosolic and secreted proteins are important and necessary for identification of drug targets at the protein level. PMID:21347706

  11. Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells.

    PubMed

    Fatokun, Amos A; Tome, Mercedes; Smith, Robert A; Darlington, L Gail; Stone, Trevor W

    2014-11-26

    Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway. PMID:25427161

  12. Stimulation of alpha 1 (I) procollagen gene expression in NIH-3T3 cells by the human T cell leukemia virus type 1 (HTLV-1) Tax gene.

    PubMed Central

    Muńoz, E; Suri, D; Amini, S; Khalili, K; Jiménez, S A

    1995-01-01

    The mechanisms that regulate the expression of genes encoding extracellular matrix proteins in fibroblasts and other mesenchymal cells have remained elusive. Studies from several laboratories have indicated that Tax, a trans-regulatory protein from the human T cell leukemia virus type I not only augments viral gene expression but also triggers the expression of various cellular genes. Here, we examined the hypothesis that the expression of collagen genes may also be modulated by Tax. NIH-3T3 cells were simultaneously transfected with a Tax expressor plasmid and a chimeric construct containing regulatory sequences (-804 to +42 bp) of the alpha 1(I) procollagen gene (COL1A1) promoter. The results indicated that the promoter activity of the -804 to bp COL1A1 fragment increased up to 12-fold in cells expressing Tax. Deletion analysis revealed that the region of COL1A1 encompassing nucleotides -174 to -84 contained the Tax-responsive elements. A gene segment encompassing nucleotides -187 to -67, which contained this region, proved sufficient to confer Tax inducibility (2.5-fold) to a herpes simplex virus thymidine kinase promoter. Stably transfected NIH-3T3 cell clones that constitutively produce Tax displayed elevated levels of alpha 1(I) procollagen and fibronectin transcripts and increased production and accelerated processing of type I procollagen. These findings suggest that retroviral proteins may be involved in the pathogenesis of idiopathic diseases accompanied by collagen overproduction. Images PMID:7593629

  13. Quercitrin and taxifolin stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells.

    PubMed

    Satué, María; Arriero, Maria del Mar; Monjo, Marta; Ramis, Joana Maria

    2013-11-15

    Flavonoids are natural antioxidants that positively influence bone metabolism. The present study screened among different flavonoids to identify biomolecules for potential use in bone regeneration. For this purpose, we used MC3T3-E1 and RAW264.7 cells to evaluate their effect on cell viability and cell differentiation. First, different doses of chrysin, diosmetin, galangin, quercitrin and taxifolin were analyzed to determine the optimum concentration to induce osteoblast differentiation. After 48h of treatment, doses ?100?M of diosmetin and galangin and also 500?M taxifolin revealed a toxic effect on cells. The same effect was observed in cells treated with doses ?100?M of chrysin after 14 days of treatment. However, the safe doses of quercitrin (200 and 500?M) and taxifolin (100 and 200?M) induced bone sialoprotein and osteocalcin mRNA expression. Also higher osteocalcin secreted levels were determined in 100?M taxifolin osteoblast treated samples when compared with the control ones. On the other hand, quercitrin and taxifolin decreased Rankl gene expression in osteoblasts, suggesting an inhibition of osteoclast formation. Indeed, osteoclastogenesis suppression by quercitrin and taxifolin treatment was observed in RAW264.7 cells. Based on these findings, the present study demonstrates that quercitrin and taxifolin promote osteoblast differentiation in MC3T3-E1 cells and also inhibit osteoclastogenesis in RAW264.7 cells, showing a positive effect of these flavonoids on bone metabolism. PMID:24060614

  14. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells.

    PubMed

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

    2015-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC-MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells. PMID:25685661

  15. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells

    PubMed Central

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

    2014-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC–MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells. PMID:25685661

  16. Effect of endoplasmic reticulum (ER) stress inducer thapsigargin on the expression of extracellular-superoxide dismutase in mouse 3T3-L1 adipocytes

    PubMed Central

    Kamiya, Tetsuro; Hara, Hirokazu; Adachi, Tetsuo

    2013-01-01

    Endoplasmic reticulum stress is related to metabolic disorders, including atherosclerosis and type 2 diabetes. It is known that inflammatory adipocytokines and oxidative stress are increased, while anti-inflammatory adipocytokines such as adiponectin are decreased in adipocytes during above conditions. Extracellular-superoxide dismutase is an anti-inflammatory enzyme that protects cells from oxidative stress. Because plasma extracellular-superoxide dismutase levels in type 2 diabetes patients were inversely related to the body mass index and homeostasis model assessment-insulin resistance index, it is speculated that the regulation of extracellular-superoxide dismutase might lead to the suppression of metabolic disorders. Here, we observed the reduction of extracellular-superoxide dismutase and adiponectin in 3T3-L1 adipocytes treated with thapsigargin, an endoplasmic reticulum stress inducer. Interestingly, tunicamycin, another endoplasmic reticulum stress inducer, did not decrease the expression of extracellular-superoxide dismutase in spite of the induction of glucose regulated protein kinase 78 kDa, an endoplasmic reticulum stress marker. Moreover, eukaryotic translation initiation factor 2? signaling cascade plays a pivotal role in the reduction of extracellular-superoxide dismutase in 3T3-L1 adipocytes during endoplasmic reticulum stress conditions. PMID:23525536

  17. Effects of Panicum miliaceum L. extract on adipogenic transcription factors and fatty acid accumulation in 3T3-L1 adipocytes.

    PubMed

    Park, Mi-Young; Seo, Dong-Won; Lee, Jin-Young; Sung, Mi-Kyung; Lee, Young-Min; Jang, Hwan-Hee; Choi, Hae-Yeon; Kim, Jae-Hyn; Park, Dong-Sik

    2011-06-01

    The dietary intake of whole grains is known to reduce the incidence of chronic diseases such as obesity, diabetes, cardiovascular disease, and cancer. To investigate whether there are anti-adipogenic activities in various Korean cereals, we assessed water extracts of nine cereals. The results showed that treatment of 3T3-L1 adipocytes with Sorghum bicolor L. Moench, Setaria italica Beauvois, or Panicum miliaceum L. extract significantly inhibited adipocyte differentiation, as determined by measuring oil red-O staining, triglyceride accumulation, and glycerol 3-phosphate dehydrogenase activity. Among the nine cereals, P. miliaceum L. showed the highest anti-adipogenic activity. The effects of P. miliaceum L. on mRNA expression of peroxisome proliferator-activated receptor-?, sterol regulatory element-binding protein 1, and the CCAAT/enhancer binding protein-? were evaluated, revealing that the extract significantly decreased the expression of these genes in a dose-dependent manner. Moreover, P. miliaceum L. extract changed the ratio of monounsaturated fatty acids to saturated fatty acids in adipocytes, which is related to biological activity and cell characteristics. These results suggest that some cereals efficiently suppress adipogenesis in 3T3-L1 adipocytes. In particular, the effect of P. miliaceum L. on adipocyte differentiation is associated with the downregulation of adipogenic genes and fatty acid accumulation in adipocytes. PMID:21779521

  18. Effects of Panicum miliaceum L. extract on adipogenic transcription factors and fatty acid accumulation in 3T3-L1 adipocytes

    PubMed Central

    Park, Mi-Young; Seo, Dong-Won; Lee, Jin-Young; Sung, Mi-Kyung; Lee, Young-Min; Jang, Hwan-Hee; Choi, Hae-Yeon; Kim, Jae-Hyn

    2011-01-01

    The dietary intake of whole grains is known to reduce the incidence of chronic diseases such as obesity, diabetes, cardiovascular disease, and cancer. To investigate whether there are anti-adipogenic activities in various Korean cereals, we assessed water extracts of nine cereals. The results showed that treatment of 3T3-L1 adipocytes with Sorghum bicolor L. Moench, Setaria italica Beauvois, or Panicum miliaceum L. extract significantly inhibited adipocyte differentiation, as determined by measuring oil red-O staining, triglyceride accumulation, and glycerol 3-phosphate dehydrogenase activity. Among the nine cereals, P. miliaceum L. showed the highest anti-adipogenic activity. The effects of P. miliaceum L. on mRNA expression of peroxisome proliferator-activated receptor-?, sterol regulatory element-binding protein 1, and the CCAAT/enhancer binding protein-? were evaluated, revealing that the extract significantly decreased the expression of these genes in a dose-dependent manner. Moreover, P. miliaceum L. extract changed the ratio of monounsaturated fatty acids to saturated fatty acids in adipocytes, which is related to biological activity and cell characteristics. These results suggest that some cereals efficiently suppress adipogenesis in 3T3-L1 adipocytes. In particular, the effect of P. miliaceum L. on adipocyte differentiation is associated with the downregulation of adipogenic genes and fatty acid accumulation in adipocytes. PMID:21779521

  19. Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

    PubMed Central

    Sullenger, B A; Lee, T C; Smith, C A; Ungers, G E; Gilboa, E

    1990-01-01

    NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies. Images PMID:2247070

  20. Abingdon Feeder Cattle Association 234 West Valley Street, Suite B

    E-print Network

    Liskiewicz, Maciej

    improvements to improve production, management and marketing of feeder cattle in tobacco dependent communities. By making further upgrades in genetics and cattle handling facilities, producers will be able improve (20%) through an association sponsored Virginia Quality Assured (VQA) Feeder Cattle Sale in January

  1. A novel coal feeder for production of low sulfur fuel

    Microsoft Academic Search

    S. J. Khang; T. C. Keener; L. Lin

    1993-01-01

    A dual-screw feeder was designed and constructed for desulfurization of coal. The feeder consists mainly of two concentric screw tubes, the inner tube acting as a coal pyrolyzer and the outer tube acting as a desulfurizer with hot calcined lime pellets or other renewable sorbent pellets. Two basic processes are involved in the development of this reactor: the mild pyrolysis

  2. 103. CANAL AT THE ENTRANCE TO THE POMPTON FEEDER NEAR ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    103. CANAL AT THE ENTRANCE TO THE POMPTON FEEDER NEAR MOUNTAIN VIEW, NEW JERSEY. THE POMPTON FEEDER BOATS TO TRAVEL NORTH 4.26 MILES TO TO POMPTON AS WELL AS PROVIDING THE MAIN CANAL WITH WATER FROM GREENWOOD LAKE. A MODIFIED QUEEN POST TRUSS BRIDGE SPANS THE CANAL IN THE FOREGROUND. - Morris Canal, Phillipsburg, Warren County, NJ

  3. LOW FLOW RATE LABORATORY FEEDERS FOR AGGLOMERATIVE PARTICLES

    EPA Science Inventory

    The paper describes two particle feeders (fluidizing and entrainment): one was developed by modifying a design used for coal dust suspensions, and the other was developed later in response to shortcomings of the initial system. The two feeders were tested and compared for their p...

  4. 19. DETAIL OF STAMP BATTERY AUTOMATIC FEEDER, LOOKING EAST. THIS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    19. DETAIL OF STAMP BATTERY AUTOMATIC FEEDER, LOOKING EAST. THIS IS THE MIDDLE OF THREE FEEDERS, ONE FOR EACH STAMP BATTERY. THE CHUTE (UPPER RIGHT) INTRODUCED THE CRUSHED ORE FROM THE ORE BIN. FLOW WAS CONTROLLED BY A SLIDING DOOR ON THE UPPER LEVEL. - Skidoo Mine, Park Route 38 (Skidoo Road), Death Valley Junction, Inyo County, CA

  5. Effects of uremic toxin p-cresol on proliferation, apoptosis, differentiation, and glucose uptake in 3T3-L1 cells.

    PubMed

    Tanaka, Sayuri; Yano, Shozo; Sheikh, Abdullah M; Nagai, Atsushi; Sugimoto, Toshitsugu

    2014-07-01

    Malnutrition is a common feature seen in chronic dialysis patients, and the survival rate of obese patients receiving such treatment is higher than that of lean patients. Irrespective of obesity or diabetes, dialysis patients commonly have insulin resistance, and the leading cause of death is cardiovascular (CV) disease. It has been reported that the concentration of p-cresol, a uremic toxin, is highly associated with CV events. As uremic toxin levels are high in dialysis patients, they may be involved in the pathogenesis of insulin resistance and CV disease in this population. However, little is known so far. Thus, we focused on this uremic toxin to examine its effects on adipocytes and their precursors. 3T3-L1 cells, a mouse preadipocyte cell line, were cultured until 90% confluency. The cells were then differentiated with 500??M 3-isobutyl-methylxanthine, 250?nM dexamethasone, and 10??g/mL insulin. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (Brd-U) incorporation assay. Glucose uptake was estimated using radiolabeled 2-deoxyglucose. The range of concentrations of p-cresol used in the experiments was from 2 to 200??M. The investigation of cell proliferation by cell counting revealed that, compared with control, 3T3-L1 cells treated with 100 and 200??M p-cresol were significantly decreased in number at day 3 and day 7 of culture. The Brd-U incorporation assay also demonstrated similar inhibitory effects on cell proliferation, suggesting that p-cresol affected the normal cell cycle. Oil Red O staining at day 7 showed that the number of mature adipocytes was decreased by treatment with 200??M p-cresol. Consistent with that finding, the number of apoptotic cells at day 7 was increased by treatment with 100 and 200??M p-cresol. Peroxisome proliferator-activated receptor ? (PPAR?) mRNA expression increased time-dependently during the differentiation process of 3T3-L1 cells. p-Cresol dose-dependently decreased differentiation-induced mRNA expression of PPAR?. Uptake of 3H-labeled 2-deoxyglucose was markedly decreased by 200??M p-cresol in the presence or in the absence of insulin, mainly because of the decreased number of mature adipocytes. High concentrations of p-cresol disturbed the cell cycle, induced apoptosis, inhibited the differentiation of preadipocytes into mature adipocytes, and decreased glucose uptake at baseline and after insulin stimulation. These findings indicate that accumulated p-cresol may induce reduction in adipose tissue, insulin resistance, and malnutrition, eventually leading to poor outcomes in chronic dialysis patients. PMID:24417700

  6. Identification of differentially expressed proteins by treatment with PUGNAc in 3T3-L1 adipocytes through analysis of ATP-binding proteome.

    PubMed

    Lee, Jeong-Eun; Park, Ja-Hye; Moon, Pyong-Gon; Baek, Moon-Chang

    2013-10-01

    O-GlcNAc (2-acetamino-2-deoxy-?-D-glucopyranose), an important modification for cellular processes, is catalyzed by O-GlcNAc transferase and O-GlcNAcase. O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) is a nonselective inhibitor of O-GlcNAcase, which increases the level of protein O-GlcNAcylation and is known to induce insulin-resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3-L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP-bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP-binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3-L1 adipocytes following treatment with PUGNAc. For label-free quantitation, a gel-assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data-independent (671 proteins identified) and data-dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide-binding proteins and we focused on some nucleotide-binding proteins, ubiquitin-activation enzyme 1 (E1), Hsp70, vasolin-containing protein (Vcp), and Hsp90, involved in ubiquitin-proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time-dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3-L1 cells. PMID:23946262

  7. Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

    SciTech Connect

    Xue, Peng [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States) [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); School of Public Health, China Medical University, Shenyang 110001 (China); Hou, Yongyong; Zhang, Qiang; Woods, Courtney G.; Yarborough, Kathy; Liu, Huiyu [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)] [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Sun, Guifan [School of Public Health, China Medical University, Shenyang 110001 (China)] [School of Public Health, China Medical University, Shenyang 110001 (China); Andersen, Melvin E. [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)] [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: jpi@thehamner.org [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)] [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)

    2011-04-08

    Highlights: {yields} In 3T3-L1 adipocytes iAs{sup 3+} decreases insulin-stimulated glucose uptake. {yields} iAs{sup 3+} attenuates insulin-induced phosphorylation of AKT S473. {yields} iAs{sup 3+} activates the cellular adaptive oxidative stress response. {yields} iAs{sup 3+} impairs insulin-stimulated ROS signaling. {yields} iAs{sup 3+} decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 {mu}M) inorganic arsenite (iAs{sup 3+}) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs{sup 3+} exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs{sup 3+} exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in adipocytes. Taken together our studies suggest that prolonged low-level iAs{sup 3+} exposure activates the cellular adaptive oxidative stress response, which impairs insulin-stimulated ROS signaling that is involved in ISGU, and thus causes insulin resistance in adipocytes.

  8. Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.

    PubMed

    Addison, W N; Nelea, V; Chicatun, F; Chien, Y-C; Tran-Khanh, N; Buschmann, M D; Nazhat, S N; Kaartinen, M T; Vali, H; Tecklenburg, M M; Franceschi, R T; McKee, M D

    2015-02-01

    Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone. PMID:25460184

  9. Dexamethasone-induced insulin resistance in 3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction.

    PubMed

    Sakoda, H; Ogihara, T; Anai, M; Funaki, M; Inukai, K; Katagiri, H; Fukushima, Y; Onishi, Y; Ono, H; Fujishiro, M; Kikuchi, M; Oka, Y; Asano, T

    2000-10-01

    Glucocorticoids reportedly induce insulin resistance. In this study, we investigated the mechanism of glucocorticoid-induced insulin resistance using 3T3-L1 adipocytes in which treatment with dexamethasone has been shown to impair the insulin-induced increase in glucose uptake. In 3T3-L1 adipocytes treated with dexamethasone, the GLUT1 protein expression level was decreased by 30%, which possibly caused decreased basal glucose uptake. On the other hand, dexamethasone treatment did not alter the amount of GLUT4 protein in total cell lysates but decreased the insulin-stimulated GLUT4 translocation to the plasma membrane, which possibly caused decreased insulin-stimulated glucose uptake. Dexamethasone did not alter tyrosine phosphorylation of insulin receptors, and it significantly decreased protein expression and tyrosine phosphorylation of insulin receptor substrate (IRS)-1. Interestingly, however, protein expression and tyrosine phosphorylation of IRS-2 were increased. To investigate whether the reduced IRS-1 content is involved in insulin resistance, IRS-1 was overexpressed in dexamethasone-treated 3T3-L1 adipocytes using an adenovirus transfection system. Despite protein expression and phosphorylation levels of IRS-1 being normalized, insulin-induced 2-deoxy-D-[3H]glucose uptake impaired by dexamethasone showed no significant improvement. Subsequently, we examined the effect of dexamethasone on the glucose uptake increase induced by overexpression of GLUT2-tagged p110alpha, constitutively active Akt (myristoylated Akt), oxidative stress (30 mU glucose oxidase for 2 h), 2 mmol/l 5-aminoimidazole-4-carboxamide ribonucleoside for 30 min, and osmotic shock (600 mmol/l sorbitol for 30 min). Dexamethasone treatment clearly inhibited the increases in glucose uptake produced by these agents. Thus, in conclusion, the GLUT1 decrease may be involved in the dexamethasone-induced decrease in basal glucose transport activity, and the mechanism of dexamethasone-induced insulin resistance in glucose transport activity (rather than the inhibition of phosphatidylinositol 3-kinase activation resulting from a decreased IRS-1 content) is likely to underlie impaired glucose transporter regulation. PMID:11016454

  10. Detection of initiating as well as promoting activity of chemicals by a novel cell transformation assay using v-Ha- ras-transfected BALB\\/c 3T3 cells (Bhas 42 cells)

    Microsoft Academic Search

    Shin Asada; Kiyoshi Sasaki; Noriho Tanaka; Ken Takeda; Makoto Hayashi; Makoto Umeda

    2005-01-01

    Cell transformation assay using BALB\\/c 3T3 cells, C3H10T1\\/2 cells and others, can simulate the two-stage carcinogenesis utilized for formation of transformed foci. A sensitive cell transformation assay for tumor initiators as well as promoters has been developed using a v-Ha-ras-transfected BALB\\/c 3T3 cell line, Bhas 42; these cells are regarded as initiated in the two-stage paradigm of carcinogenesis. To distinguish

  11. The fungal chimerolectin MOA inhibits protein and DNA synthesis in NIH/3T3 cells and may induce BAX-mediated apoptosis.

    PubMed

    Cordara, Gabriele; Winter, Harry C; Goldstein, Irwin J; Krengel, Ute; Sandvig, Kirsten

    2014-05-16

    The Marasmius oreades mushroom agglutinin (MOA) is a blood group B-specific lectin carrying an active proteolytic domain. Its enzymatic activity has recently been shown to be critical for toxicity of MOA toward the fungivorous soil nematode Caenorhabditis elegans. Here we present evidence that MOA also induces cytotoxicity in a cellular model system (murine NIH/3T3 cells), by inhibiting protein synthesis, and that cytotoxicity correlates, at least in part, with proteolytic activity. A peptide-array screen identified the apoptosis mediator BAX as a potential proteolytic substrate and further suggests a variety of bacterial and fungal peptides as potential substrates. These findings are in line with the suggestion that MOA and related proteases may play a role for host defense. PMID:24747075

  12. Enamel Matrix Derivative Inhibits Adipocyte Differentiation of 3T3-L1 Cells via Activation of TGF-?RI Kinase Activity

    PubMed Central

    Gruber, Reinhard; Bosshardt, Dieter D.; Miron, Richard J.; Gemperli, Anja C.; Buser, Daniel; Sculean, Anton

    2013-01-01

    Enamel matrix derivative (EMD), an extract of fetal porcine enamel, and TGF-? can both suppress adipogenic differentiation. However, there have been no studies that functionally link the role of EMD and TGF-? in vitro. Herein, we examined whether TGF-? signaling contributes to EMD-induced suppression of adipogenic differentiation. Adipogenesis was studied with 3T3-L1 preadipocytes in the presence of SB431542, an inhibitor of TGF-?RI kinase activity. SB431542 reversed the inhibitory effect of EMD on adipogenic differentiation, based on Oil Red O staining and mRNA expression of lipid regulated genes. SB431542 also reduced EMD-stimulated expression of connective tissue growth factor (CTGF), an autocrine inhibitor of adipogenic differentiation. Moreover, short interfering (si)RNAs for CTGF partially reversed the EMD-induced suppression of lipid regulated genes. We conclude that the TGF-?RI - CTGF axis is involved in the anti-adipogenic effects of EMD in vitro. PMID:23951076

  13. Reduction of adipogenesis and lipid accumulation by Taraxacum officinale (Dandelion) extracts in 3T3L1 adipocytes: an in vitro study.

    PubMed

    González-Castejón, Marta; García-Carrasco, Belén; Fernández-Dacosta, Raquel; Dávalos, Alberto; Rodriguez-Casado, Arantxa

    2014-05-01

    In this in vitro study, we have investigated the ability of Taraxacum officinale (dandelion) to inhibit adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes. HPLC analysis of the three plant extracts used in this study-leaf and root extracts and a commercial root powder-identified caffeic and chlorogenic acids as the main phenolic constituents. Oil Red O staining and triglyceride levels analysis showed decreased lipid and triglyceride accumulation, respectively. Cytotoxicity was assessed with the MTT assay showing non-toxic effect among the concentrations tested. DNA microarray analysis showed that the extracts regulated the expression of a number of genes and long non-coding RNAs that play a major role in the control of adipogenesis. Taken together, our results indicate that the dandelion extracts used in this study may play a significant role during adipogenesis and lipid metabolism, and thus, supporting their therapeutic interest as potential candidates for the treatment of obesity. PMID:23956107

  14. Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes

    SciTech Connect

    Kuroyanagi, Kayo; Kang, Min-Sook; Goto, Tsuyoshi; Hirai, Shizuka; Ohyama, Kana; Kusudo, Tatsuya [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Yu, Rina [Department of Food Science and Nutrition, University of Ulsan, Ulsan 680-749 (Korea, Republic of); Yano, Masamichi [Department of Citriculture, National Institute of Fruit Tree Science, Ministry of Agriculture, Forestry and Fisheries, Shimizu, Shizuoka 424-0292 (Japan); Sasaki, Takao [ARKRAY Inc., Kyoto 601-8045 (Japan); Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)], E-mail: fat@kais.kyoto-u.ac.jp

    2008-02-01

    Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-{alpha} and PPAR{gamma}. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPAR{gamma} antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPAR{gamma} activations. The results indicate that auraptene activates PPAR{gamma} in adipocytes to control adipocytekines such as adiponectin and MCP-1 and suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.

  15. HuR binds to a single site on the C/EBP{beta} mRNA of 3T3-L1 adipocytes

    SciTech Connect

    Jones, Heath [Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27858 (United States); Carver, Melinda [Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27858 (United States); Pekala, Phillip H. [Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27858 (United States)]. E-mail: pekalap@ecu.edu

    2007-03-30

    HuR is a ligand for nuclear mRNAs containing adenylate-uridylate rich elements in the 3'-untranslated region. Once bound to the mRNA, HuR is recognized by adapter proteins which then facilitate nuclear export of the complex. In the cytosol HuR is thought to function to control stability and translation of its ligand message. In the 3T3-L1 cells HuR is constitutively expressed and localized predominantly to the nucleus in the preadipocytes. However within 30 min of exposure to the differentiation stimulus, the HuR content in the cytosol increases consistent with HuR regulating the availability of relevant mRNAs for translation. Using in vitro RNA gel shifts, we have demonstrated that the C/EBP{beta} message is a ligand for HuR and that the single binding site is an adenylate-uridylate rich element in the 3'-untranslated region.

  16. Arachidonic acid has a dominant effect to regulate lipogenic genes in 3T3-L1 adipocytes compared to omega-3 fatty acids

    PubMed Central

    Vaidya, Hitesh; Cheema, Sukhinder Kaur

    2015-01-01

    Background The effects of long-chain n-3 and n-6 polyunsaturated fatty acids (PUFA) on the regulation of adipocytes metabolism are well known. These fatty acids are generally consumed together in our diets; however, the metabolic regulation of adipocytes in the presence of these fatty acids when given together is not known. Objective To investigate the effects of n-3 PUFA and arachidonic acid (AA), an n-6 PUFA, on the regulation of adipogenic and lipogenic genes in mature 3T3-L1 adipocytes. Methods 3T3-L1 adipocytes were incubated in the presence or absence of 100 µM of eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA; docosapentaenoic acid, DPA and AA, either alone or AA+n-3 PUFA; control cells received bovine serum albumin alone. The mRNA expression of adipogenic and lipogenic genes was measured. The fatty acid composition of adipocytes was analyzed using gas chromatography. Results Individual n-3 PUFA or AA had no effect on the mRNA expression of peroxisome-proliferator-activated receptor-?; however, AA+EPA and AA+DPA significantly increased (P<0.05) the expression compared to control cells (38 and 42%, respectively). AA and AA+EPA increased the mRNA expression of acetyl-CoA carboxylase 1 (P<0.05). AA treatment decreased the mRNA expression of stearoyl-CoA desaturase (SCD1) (P<0.01), while n-3 PUFA, except EPA, had no effect compared to control cells. AA+DHA and AA+DPA inhibited SCD1 gene expression (P<0.05) suggesting a dominant effect of AA. Fatty acids analysis of adipocytes revealed a higher accretion of AA compared to n-3 PUFA. Conclusions Our findings reveal that AA has a dominant effect on the regulation of lipogenic genes in adipocytes. PMID:25797050

  17. Prostaglandin F2 alpha-stimulated phospholipase D activation in osteoblast-like MC3T3-E1 cells: involvement in sustained 1,2-diacylglycerol production.

    PubMed Central

    Sugiyama, T; Sakai, T; Nozawa, Y; Oka, N

    1994-01-01

    In [3H]myristic acid-labelled osteoblast-like MC3T3-E1 cells, prostaglandin F2 alpha (PGF2 alpha)-induced PLD activity was assessed by measuring the [3H]phosphatidylethanol (PEt) formation in the presence of ethanol. Inhibition of the increase in intracellular Ca2+ concentration ([Ca2+]i) by U73122, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), or chelation of extracellular Ca2+ with EGTA or of intracellular Ca2+ with BAPTA, suppressed PGF2 alpha-induced phospholipase D (PLD) activation. Neither protein kinase C (PKC) inhibitors nor PKC down-regulation with phorbol 12-myristate 13-acetate affected PGF2 alpha-induced [3H]PEt formation. In permeabilized cells, guanosine 5'-[gamma-thio]triphosphate enhanced PGF2 alpha 's potency in [3H]PEt formation in the presence of Ca2+. The pretreatment of intact cells with pertussis toxin failed to inhibit PGF2 alpha-induced [3H]PEt formation. PGF2 alpha caused a biphasic production of [3H]1,2-diacylglycerol ([3H]1,2-DAG) in [3H]glycerol-labelled cells. The initial transient phase was decreased by U73122, whereas the late sustained phase was decreased by ethanol and the phosphatidic acid phosphohydrolase inhibitor, propranolol. From these results, it was suggested that PGF2 alpha-induced PLD activation was mediated by the dual control of the [Ca2+]i increase due to PI-PLC activation and activation of pertussis-toxin-insensitive G-protein, but not mediated by PKC, and also that PLD activation was involved in the late sustained 1,2-DAG generation in MC3T3-E1 cells. PMID:8135758

  18. Insulin-like growth factor-I stimulates diacylglycerol production via multiple pathways in Balb/c 3T3 cells.

    PubMed

    Kojima, I; Kitaoka, M; Ogata, E

    1990-10-01

    We previously reported that insulin-like growth factor-I (IGF-I) induced sustained calcium cycling across the plasma membrane in primed competent Balb/c 3T3 cells (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1989) J. Biol. Chem. 263, 16561-16567). The present study was conducted to examine whether IGF-I affected cellular metabolism of 1,2-diacylglycerol (1,2-DAG). In primed competent cells prelabeled with [3H]myristate, 1 nM IGF-I caused a 50% increase in [3H]DAG within 10 min. This increase in [3H]DAG was accompanied by 1) a decrease in radioactivity in the glycosylphosphatidylinositol fraction in [3H]glucosamine-labeled cells and a concomitant increase in [3H]inositol-glycan, and 2) a decrease in [3H]phosphatidylcholine and a concomitant elevation of [3H]phosphorylcholine in [3H]choline-labeled cells. When [3H]choline-labeled cells were treated with 10 nM 12-O-tetradecanoylphorbol-4-acetate (TPA), [3H]phosphatidylcholine was reduced by 50%. The TPA-induced reduction of [3H]phosphatidylcholine was completely blocked by 50 microM sphingosine and 50 microM H-7, inhibitors of protein kinase C. Both sphingosine and H-7 attenuated IGF-I-mediated reduction of [3H]phosphatidylcholine. In addition, treatment with IGF-I for 3 h or more resulted in sustained increase in 1,2-DAG mass, which was attenuated by cycloheximide. The increase in DAG mass was accompanied by enhanced incorporation of [14C]glucose into 1,2-DAG. These results indicate that, in primed competent Balb/c 3T3 cells, IGF-I stimulates 1,2-DAG production via multiple pathways and that IGF-I may induce breakdown of phosphatidylcholine by a mechanism involving protein kinase C. PMID:2120207

  19. Prostaglandin F2 alpha-stimulated phospholipase D activation in osteoblast-like MC3T3-E1 cells: involvement in sustained 1,2-diacylglycerol production.

    PubMed

    Sugiyama, T; Sakai, T; Nozawa, Y; Oka, N

    1994-03-01

    In [3H]myristic acid-labelled osteoblast-like MC3T3-E1 cells, prostaglandin F2 alpha (PGF2 alpha)-induced PLD activity was assessed by measuring the [3H]phosphatidylethanol (PEt) formation in the presence of ethanol. Inhibition of the increase in intracellular Ca2+ concentration ([Ca2+]i) by U73122, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), or chelation of extracellular Ca2+ with EGTA or of intracellular Ca2+ with BAPTA, suppressed PGF2 alpha-induced phospholipase D (PLD) activation. Neither protein kinase C (PKC) inhibitors nor PKC down-regulation with phorbol 12-myristate 13-acetate affected PGF2 alpha-induced [3H]PEt formation. In permeabilized cells, guanosine 5'-[gamma-thio]triphosphate enhanced PGF2 alpha 's potency in [3H]PEt formation in the presence of Ca2+. The pretreatment of intact cells with pertussis toxin failed to inhibit PGF2 alpha-induced [3H]PEt formation. PGF2 alpha caused a biphasic production of [3H]1,2-diacylglycerol ([3H]1,2-DAG) in [3H]glycerol-labelled cells. The initial transient phase was decreased by U73122, whereas the late sustained phase was decreased by ethanol and the phosphatidic acid phosphohydrolase inhibitor, propranolol. From these results, it was suggested that PGF2 alpha-induced PLD activation was mediated by the dual control of the [Ca2+]i increase due to PI-PLC activation and activation of pertussis-toxin-insensitive G-protein, but not mediated by PKC, and also that PLD activation was involved in the late sustained 1,2-DAG generation in MC3T3-E1 cells. PMID:8135758

  20. Phorbol ester and bryostatin differentially regulate the hydrolysis of phosphatidylethanolamine in Ha-ras- and raf-oncogene-transformed NIH 3T3 cells.

    PubMed Central

    Kiss, Z; Rapp, U R; Pettit, G R; Anderson, W B

    1991-01-01

    Previously it was reported that transformation of NIH 3T3 fibroblast by the Ha-ras, v-src, v-fms, and A-raf oncogenes decreased the stimulatory effects of phorbol 12-myristate 13-acetate (PMA; 'TPA'), an activator of protein kinase C (PKC), on the phosphorylation of an endogenous 80 kDa substrate and on 86Rb uptake [Wolfman, Wingrove, Blackshear & Macara (1987) J. Biol. Chem. 262, 16546-16552], as well as on sphingomyelin synthesis [Kiss, Rapp & Anderson (1988) FEBS Lett. 240, 221-226]. Here, we investigated how transformation affects the PMA-stimulated hydrolysis of phosphatidylethanolamine (PtdEtn), a recently characterized mechanism which may contribute to the generation of the second messengers phosphatidic acid and 1,2-diacylglycerol. The effects of PMA were compared with those of bryostatin, a non-tumour-promoter activator of PKC. Transformation of NIH 3T3 cells with Ha-ras, v-raf, or A-raf enhanced the stimulatory effect of PMA on the phospholipase D-mediated hydrolysis of PtdEtn. On the other hand, the effects of bryostatin on PtdEtn hydrolysis were only slightly increased, if at all, in cells transformed with these oncogenes. In crude membrane preparations isolated from these transformed cells, PMA, but not bryostatin, enhanced the combined stimulatory effects of ATP and the GTP analogue guanosine 5'-[gamma-thio]triphosphate on phospholipase D-mediated PtdEtn hydrolysis. The PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine inhibited the stimulatory effect of PMA only in intact cells. These results indicate that transformation of cells by certain oncogenes differentially affects phospholipase D-mediated hydrolysis of PtdEtn induced by PMA and bryostatin, suggesting that the action of PMA might involve two different mechanisms. PMID:2049075

  1. Transformer 2? homolog (Drosophila) (TRA2B) regulates protein kinase C ?I (PKC?I) splice variant expression during 3T3L1 preadipocyte cell cycle.

    PubMed

    Patel, Rekha S; Carter, Gay; Cooper, Denise R; Apostolatos, Hercules; Patel, Niketa A

    2014-11-14

    Obesity is characterized by adipocyte hyperplasia and hypertrophy. We previously showed that PKC? expression is dysregulated in obesity (Carter, G., Apostolatos, A., Patel, R., Mathur, A., Cooper, D., Murr, M., and Patel, N. A. (2013) ISRN Obes. 2013, 161345). Using 3T3L1 preadipocytes, we studied adipogenesis in vitro and showed that expression of PKC? splice variants, PKC?I and PKC?II, have different expression patterns during adipogenesis (Patel, R., Apostolatos, A., Carter, G., Ajmo, J., Gali, M., Cooper, D. R., You, M., Bisht, K. S., and Patel, N. A. (2013) J. Biol. Chem. 288, 26834-26846). Here, we evaluated the role of PKC?I splice variant during adipogenesis. Our results indicate that PKC?I expression level is high in preadipocytes and decreasing PKC?I accelerated terminal differentiation. Our results indicate that PKC?I is required for mitotic clonal expansion of preadipocytes. We next evaluated the splice factor regulating the expression of PKC?I during 3T3L1 adipogenesis. Our results show TRA2B increased PKC?I expression. To investigate the molecular mechanism, we cloned a heterologous splicing PKC? minigene and showed that inclusion of PKC? exon 9 is increased by TRA2B. Using mutagenesis and a RNA-immunoprecipitation assay, we evaluated the binding of Tra2? on PKC?I exon 9 and show that its association is required for PKC?I splicing. These results provide a better understanding of the role of PKC?I in adipogenesis. Determination of this molecular mechanism of alternative splicing presents a novel therapeutic target in the management of obesity and its co-morbidities. PMID:25261467

  2. Role of Gas1 down-regulation in mitogenic stimulation of quiescent NIH3T3 cells by v-Src.

    PubMed

    Grossi, M; La Rocca, S A; Pierluigi, G; Vannucchi, S; Ruaro, E M; Schneider, C; Tatň, F

    1998-10-01

    Quiescent mammalian fibroblasts can be induced to reenter the cell cycle by growth factors and oncoproteins. We studied the pathway(s) through which v-Src, the oncogenic tyrosine kinase encoded by the v-src oncogene of Rous sarcoma virus, forces serum-starved NIH3T3 cells to enter S-phase. To this purpose, we isolated and characterized a polyclonal population of NIH3T3 cells transformed by the MR31 retroviral vector, encoding G418 resistance and the v-src temperature-sensitive allele from the mutant ts LA31 PR-A. NIH(MR31) cells displayed a temperature-conditional transformed phenotype and could be made quiescent by serum deprivation at the restrictive temperature. Serum stimulation or thermolabile v-Src reactivation induced entry into S-phase to a comparable extent, although with different kinetics. The data suggest that v-Src mitogenic activity involves early activation of the Erk1/Erk2 MAP kinases with very little tyrosine phosphorylation of the Shc adaptor proteins at least during the early stages of v-Src reactivation and that v-Src-induced S-phase entry was strongly inhibited by drugs affecting MEK or PI 3-kinase. Our results also suggest that down-regulation of gas1 gene expression plays an important role in regulating the efficiency of entry into S-phase triggered by reactivated v-Src and that Gas1 down-regulation does not require PI 3-kinase dependent signals. PMID:9796692

  3. PPAR? agonist fenofibrate attenuates TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway

    SciTech Connect

    Wang, Weirong [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Lin, Qinqin [Physical Education College, Yanshan University, Qinhuangdao, Hebei 066004 (China); Lin, Rong, E-mail: linrong63@yahoo.com.cn [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Zhang, Jiye [Faculty of Pharmacy, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Ren, Feng; Zhang, Jianfeng; Ji, Meixi; Li, Yanxiang [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China)

    2013-06-10

    The ligand-activated transcription factor peroxisome proliferator-activated receptor-? (PPAR?) participates in the regulation of cellular inflammation. More recent studies indicated that sirtuin1 (SIRT1), a NAD{sup +}-dependent deacetylase, regulates the inflammatory response in adipocytes. However, whether the role of PPAR? in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPAR? agonist fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 and underlying mechanisms in 3T3-L1 adipocytes. We found that fenofibrate inhibited CD40 expression and up-regulated SIRT1 expression in tumor necrosis factor-? (TNF-?)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPAR? antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-?-induced CD40 expression in adipocytes. Importantly, NF-?B inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression in adipocytes. Further study found that fenofibrate decreased the expression of acetylated-NF-?B p65 (Ac-NF-?B p65) in TNF-?-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-?-stimulated adipocytes. Taken together, these findings indicate that PPAR? agonist fenofibrate inhibits TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. -- Highlights: • Fenofibrate up-regulates SIRT1 expression in TNF-?-stimulated adipocytes. • Fenofibrate inhibits CD40 expression through SIRT1 in adipocytes. • The effects of fenofibrate on CD40 and SIRT1 expressions are dependent on PPAR?. • Fenofibrate inhibits CD40 expression via SIRT1-dependent deacetylation of NF-?B. • Fenofibrate increases SIRT1 expression through PPAR? and AMPK in adipocytes.

  4. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3

    PubMed Central

    2013-01-01

    Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Results Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Conclusion Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health. PMID:24119256

  5. Correlations between radiation-induced double strand breaks, cell division delay, and cyclin-dependent signaling in x-irradiated NIH3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Cariveau, Mickael J.

    2005-07-01

    Molecular responses to radiation-induced DNA double strand breaks (DSB) are mediated by the phosphorylation of the histone variant H2AX which forms identifiable gamma-H2AX foci at the site of the DSB. This event is thought to be linked with the down-regulation of signaling proteins contributing to the checkpoints regulating cell cycle progression and, vis-a-vis , the induction of cell division delay. However, it is unclear whether this division delay is directly related to the number of DSB (gamma-H2AX foci) sustained by an irradiated cell and, if so, whether this number drives cells into cell cycle delay or apoptosis. For this reason, studies were conducted in the immortalized NIH/3T3 fibroblast cell in order to establish correlations between the temporal appearance of the gamma-H2AX foci (a DSB) and the expression of the cell cycle regulatory proteins, cyclin E, A, B1, and their cyclin kinase inhibitor, p21. Cell cycle kinetics and flow cytometry were used to establish radiation-induced division delay over a dose range of 1--6 Gy where a mitotic delay of 2.65 min/cGy was established. Correlations between the expression of cyclin E, A, B1, p21, and the generation of DSB were established in NIH/3T3 cells exposed to 2 or 4 Gy x-irradiation. The data suggest that the G1/S and S phase delay (cyclin E and cyclin A protein levels) are dependent on the dose of radiation while the G2/M (cyclin B1 protein levels) delay is dependent on the quantity of DSB sustained by the irradiated cell.

  6. miR-135a-5p inhibits 3T3-L1 adipogenesis through activation of canonical Wnt/?-catenin signaling.

    PubMed

    Chen, Chen; Peng, Yongdong; Peng, Yinglin; Peng, Jian; Jiang, Siwen

    2014-06-01

    MicroRNAs are endogenous, conserved, and non-coding small RNAs that function as post-transcriptional regulators of fat development and adipogenesis. Adipogenic marker genes, such as CCAAT/enhancer binding protein ? (Cebpa), peroxisome proliferator-activated receptor ? (Pparg), adipocyte fatty acid binding protein (Ap2), and fatty acid synthase (Fas), are regarded as the essential transcriptional regulators of preadipocyte differentiation and lipid storage in mature adipocytes. Canonical Wnt/?-catenin signaling is recognized as a negative molecular switch during adipogenesis. In the present work we found that miR-135a-5p is markedly downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-135a-5p impairs the expressions of adipogenic marker genes as well as lipid droplet accumulation and triglyceride content, indicating the importance of miR-135a-5p for adipogenic differentiation and adipogenesis. Further studies show that miR-135a-5p directly targets adenomatous polyposis coli (Apc), contributes to the translocation of ?-catenin from cytoplasm to nucleus, and then activates the expressions of cyclin D1 (Ccnd1) and Cmyc, indicating the induction of canonical Wnt/?-catenin signaling. In addition, inhibition of APC with siRNA exhibits the same effects as overexpression of miR-135a-5p. Our findings demonstrate that miR-135a-5p suppresses 3T3-L1 preadipocyte differentiation and adipogenesis through the activation of canonical Wnt/?-catenin signaling by directly targeting Apc. Taken together, these results offer profound insights into the adipogenesis mechanism and the development of adipose tissue. PMID:24850830

  7. Extracellular-signal-related kinase 1/2 is responsible for inhibition of osteogenesis in three-dimensional cultured MC3T3-E1 cells.

    PubMed

    Im, Daniel D; Kruger, Erwin A; Huang, Weibiao R; Sayer, Gregory; Rudkin, George H; Yamaguchi, Dean T; Jarrahy, Reza; Miller, Timothy A

    2010-11-01

    We have previously demonstrated that osteogenic differentiation is inhibited and angiogenic expression is enhanced in murine preosteoblasts (MC3T3-E1) cultured on three-dimensional (3D) poly-L-lactide-co-glycolide (PLGA) scaffolds when compared to two-dimensional (2D) PLGA films. In the present work we investigated the role of the extracellular signal-related kinase 1/2 (ERK1/2) pathway in modulating osteogenic and angiogenic differentiation in 2D and 3D systems made of two distinct biomaterials-type I collagen and PLGA. The addition of a third dimension, regardless of biomaterials, substantially increased ERK1/2 activation as demonstrated by an increase in phosphorylated ERK1/2. Western blot analysis showed significant increases in phosphorylation of ERK1/2 in cells grown in 3D versus 2D cultures at day 4 (5- and 7.7-fold increases 3D vs. 2D in collagens and PLGA, respectively) and day 7 (4.7- and 4.6-fold increases 3D vs. 2D in collagen and PLGA, respectively). Using an ERK-specific inhibitor, PD 98059, we established a correlation between ERK activation and inhibited osteogenic differentiation. Inhibition of ERK activation in 3D cultures significantly enhanced osteogenic differentiation. It in fact restored osteogenic differentiation to a level equal to that of 2D cultured cells. Inhibition of ERK1/2, however, showed little effect on angiogenic gene expression, indicating that two distinct mechanisms are involved in osteogenic and angiogenic differentiation. Taken together, these results allow us to report a mechanistic model in MC3T3-E1 cells in which distinct activation of ERK1/2 in 3D culture has an inhibitory effect on osteogenic differentiation. PMID:20590408

  8. Isolation and characterization of Cox17p from porcine heart by determining its survival-promoting activity in NIH3T3 cells.

    PubMed

    Takenouchi, T; Fujimoto, M; Shimamoto, A; Munekata, E

    1999-11-16

    We have found that the gel filtration fraction of porcine heart extract clearly promoted the survival of NIH3T3 fibroblast cells in the serum-free medium condition. A structural analysis showed that the active fraction contained a novel peptide, porcine Cox17p (p-Cox17p), which was recently reported by Chen et al. as dopuin (Z. W. Chen et al., Eur. J. Biochem. 249 (1997) 518-522). Porcine Cox17p/dopuin possesses high sequence homology to the product of human COX17 gene (h-Cox17p). Although Cox17p has been implied to be involved in copper recruitment to mitochondria and in the functional assembly of cytochrome oxidase in yeast, its role in mammalian cells is unknown. In this study, we chemically synthesized p-Cox17p to investigate its biological effects. Refolding experiments of synthesized linear p-Cox17p revealed the existence of mostly one pattern of three intrachain disulfide bridges similar to that of native p-Cox17p, because the main oxidized p-Cox17p was completely co-eluted with the natural product. The addition of heavy metal ions such as copper, zinc and cadmium significantly inhibited the formation of the oxidized form, suggesting that reduced p-Cox17p may interact directly with these metal ions. The reduced and oxidized forms of p-Cox17p were also confirmed to promote the survival of NIH3T3 cells in serum-free medium as observed with the natural product, indicating that Cox17p may be a bioactive peptide. PMID:10564764

  9. Artemisia capillaris inhibits lipid accumulation in 3T3-L1 adipocytes and obesity in C57BL/6J mice fed a high fat diet.

    PubMed

    Hong, Jung-Hee; Hwang, Eun-Young; Kim, Hyun-Jeong; Jeong, Yun-Jeong; Lee, In Seon

    2009-08-01

    The purpose of the current study was to determine the effect of the Artemisia capillaris ethyl acetate (ACE) fraction on diet-induced obesity and to elucidate the underlying mechanism. The ACE fraction treatment decreased the leptin level and fat accumulation in cultured 3T3-L1 adipocytes through the free fatty acids released in the medium. The ACE fraction significantly suppressed the expression of peroxisome proliferator-activated receptor-gamma in cultured 3T3-L1 adipocytes. To determine the effect of the ACE fraction on C57BL/6J male mice, the mice were separated into six groups: normal control (N), N plus 0.1 g/kg body weight ACE (NB), high fat control group (HF), HF plus 0.05 g/kg of body weight ACE (HFA), HF plus 0.1 g/kg of body weight ACE (HFB), and HF plus 0.03 g/kg of body weight rosiglitazone (RG) groups. We speculate that the HFB group exhibits a lipid-lowering effect via increased mitochondrial beta-oxidation, of which the rate-limiting enzyme is carnitine palmitoyl transferase I, the activity of which was significantly increased. Also, the activity of fatty acid synthase, a key enzyme of fatty acid synthesis, was markedly suppressed (19%) in the HFB group, as compared to the HF group, and glycerol-3-phosphate dehydrogenase activity, which is very useful in studying adipogenic differentiation in vitro, was markedly suppressed (30%) in the HFB group compared with the HF group. Furthermore, the HFB group showed lowered hepatic lipid droplet accumulation and adipose tissue weight and size. We suggest that 0.1 g of the ACE fraction/kg of body weight may exert an anti-obesity effect in C57BL/6J mice by enhancing lipid metabolism. PMID:19735172

  10. Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes.

    PubMed

    Yamaguchi, Shinya; Katahira, Hiroshi; Ozawa, Sachihiko; Nakamichi, Yoko; Tanaka, Toshiaki; Shimoyama, Tatsuhiro; Takahashi, Kazuto; Yoshimoto, Katsuhiko; Imaizumi, Mica Ohara; Nagamatsu, Shinya; Ishida, Hitoshi

    2005-10-01

    To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism. PMID:15928020

  11. Peptide derived from desalinated boiled tuna extract inhibits adipogenesis through the downregulation of C/EBP-? and PPAR-? in 3T3-L1 adipocytes.

    PubMed

    Kim, Young-Min; Kim, Eun-Young; Kim, In-Hye; Nam, Taek-Jeong

    2015-05-01

    Recently, obesity has increased due to a variety of reasons, including the availability of 'fast food' and high-fat diets. Developing anti-obesity functional drugs and foods from natural sources may offer solutions to this global concern. Generally, tuna is a high-protein, low-fat and low-calorie food with various bioactive effects. It may improve memory, reduce cholesterol levels and positively affect the development of brain cells. In this study, we screened the anti-obesity potential of peptides derived from tuna protein. We then observed protein bands by the Coomassie blue staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The protein mixture was concentrated and desalted using in-gel trypsin digestion and a C18 nano column and Poros R2 reversed-phase preparation, prior to quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS). We screened the peptides for their ability to affect adipogenesis in 3T3-L1 adipocytes. We also measured glucose uptake, triglyceride levels and lipid droplets using Oil Red O staining. As a result, we confirmed that one peptide inhibited adipocyte differentiation. We also observed the expression of obesity-related genes by western blot analysis and reverse transcription-polymerase chain reaction. The peptide from the tuna extract significantly reduced the expression levels of CCAAT/enhancer-binding protein ? (C/EBP-?) and peroxisome proliferator-activated receptor-? (PPAR-?) adipocyte marker genes. Thus, our data suggest that this peptide from boiled tuna extract reduces lipid components and adipogenesis in 3T3-L1 cells, and these characteristics may be of value in the development of anti-obesity foods. PMID:25761066

  12. Effects of 17?-estradiol and progesterone on the production of adipokines in differentiating 3T3-L1 adipocytes: Role of Rho-kinase.

    PubMed

    Pekta?, Mehtap; Kurt, Akif Hakan; Ün, ?smail; Tiftik, Rukiye Nalan; Büyükaf?ar, Kansu

    2015-04-01

    Effect of female sex hormones on the production/release of adipocyte-derived cytokines has been debatable. Furthermore, whether the cellular signaling triggered by these hormones involve Rho-kinase has not been investigated yet. Therefore, in this study, effects of 17?-estradiol and progesterone as well as the Rho-kinase inhibitor, Y-27632 on the level of adipokines such as resistin, adiponectin, leptin, TNF-? and IL-6 were investigated in 3T3-L1-derived adipocytes. Differentiation was induced in the post-confluent preadipocytes by the standard differentiation medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum together with the mixture of isobutylmethylxanthine, dexamethasone and insulin) in the presence of 17?-estradiol (10(-8)-10(-7)M), progesterone (10(-6)-10(-5)M), the Rho-kinase inhibitor, Y-27632 (10(-5)M) and their combination for 8days. Measurements of the adipokines were performed in the culturing medium by ELISA kits using specific monoclonal antibodies. 17?-estradiol elevated resistin but decreased adiponectin and IL-6 levels; however, it did not alter the concentration of leptin and TNF-?. Y-27632 pretreatment inhibited the rise of resistin and the fall of adiponectin by 17?-estradiol without any effects by its own. Progesterone did not change resistin, leptin and TNF-? level; however, it elevated adiponectin and decreased IL-6 production. Neither 17?-estradiol nor Y-27632 was able to antagonize the increase of adiponectin and the reduction of IL-6 levels by progesterone. While Y-27632 alone lowered IL-6 level, it increased leptin and TNF-? concentration without altering resistin and adiponectin. In conclusion, 17?-estradiol could modify adipokine production in 3T3-L1 adipocytes with the actions some of which involve Rho-kinase mediation. PMID:25647267

  13. Nicotine-induced activation of AMP-activated protein kinase inhibits fatty acid synthase in 3T3L1 adipocytes: a role for oxidant stress.

    PubMed

    An, Zhibo; Wang, Hong; Song, Ping; Zhang, Miao; Geng, Xuemei; Zou, Ming-Hui

    2007-09-14

    Recent studies suggest that the AMP-activated protein kinase (AMPK) acts as a major energy sensor and regulator in adipose tissues. The objective of this study was to investigate the role of AMPK in nicotine-induced lipogenesis and lipolysis in 3T3L1 adipocytes. Exposure of 3T3L1 adipocytes to smoking-related concentrations of nicotine increased lipolysis and inhibited fatty acid synthase (FAS) activity in a time- and dose-dependent manner. The effects of nicotine on FAS activity were accompanied by phosphorylation of both AMPK (Thr(172)) and acetyl-CoA carboxylase (ACC; Ser(79)). Nicotine-induced AMPK phosphorylation appeared to be mediated by reactive oxygen species based on the finding that nicotine significantly increased superoxide anions and 3-nitrotyrosine-positive proteins, exogenous peroxynitrite (ONOO(-)) mimicked the effects of nicotine on AMPK, and N-acetylcysteine (NAC) abolished nicotine-enhanced AMPK phosphorylation. Inhibition of AMPK using either pharmacologic (insulin, compound C) or genetic means (overexpression of dominant negative AMPK; AMPK-DN) abolished FAS inhibition induced by nicotine or ONOO(-). Conversely, activation of AMPK by pharmacologic (nicotine, ONOO(-), metformin, and AICAR) or genetic (overexpression of constitutively active AMPK) means inhibited FAS activity. Notably, AMPK activation increased threonine phosphorylation of FAS, and this effect was blocked by adenovirus encoding dominant negative AMPK. Finally, AMPK-dependent FAS phosphorylation was confirmed by (32)P incorporation into FAS in adipocytes. Taken together, our results strongly suggest that nicotine, via ONOO(-) activates AMPK, resulting in enhanced threonine phosphorylation and consequent inhibition of FAS. PMID:17635921

  14. A novel coal feeder for production of low sulfur fuel

    SciTech Connect

    Khang, Soon-Jai; Keener, T.C.

    1989-01-01

    The first task of the project is to evaluate and model the pyrolysis section of the proposed coal feeder. Literature review on coal pyrolysis have been conducted and a mathematical/computer modeling of this work has begun. Both isothermal and non-isothermal conditions are considered for this model. A pseudo-component approach was adopted for coal pyrolysis kinetics. Initially, the literature values of pyrolysis kinetic are planned to be utilized. Later, actual kinetics will be measured from a feeder-pyrolyzer. A separate feeder-pyrolyzer is under construction for this purpose.

  15. An Online Learning System for the Prediction of Electricity Distribution Feeder Failures

    E-print Network

    Arias, Marta

    Consolidated Edison Company of New York Motivation Electrical feeder cables are an essential part Drop the (-N) models with lowest q value The feature set for each feeder include Static data ­ age, composition of feeder sections Dynamic data ­ electrical load on a feeder and its transformers Dynamic data

  16. Syrian Hamster Embryo (SHE) cell transformation assay with and without X-ray irradiation of feeder cells using Di(2-ethylhexyl)phthalate (DEHP) and N-nitroso-N-methylnitroguanidine (MNNG).

    PubMed

    Pant, K; Sly, J E; Bruce, S W; Scott, A D; Carmichael, P L; San, R H C

    2010-04-30

    The SHE cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The need for an X-ray irradiated feeder cell layer necessitates the maintenance of an X-ray machine and the additional step to seed feeder cells prior to plating target cells. This laboratory has previously reported a method allowing target cells to be seeded in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer (Pant et al. [1,2,4]). In order to expand the data base for chemicals tested using this method, we describe in this paper the results obtained testing Di(2-ethylhexyl)phthalate (DEHP) and N-nitroso-N-methylnitroguanidine (MNNG) which are known to give positive responses in the standard SHE cell transformation assay. With freshly prepared conditioned medium (used within 2 weeks of preparation), there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In more than ten experiments the positive control benzo(a)pyrene [B(a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without X-ray irradiated feeder cells. Compounds, DEHP and MNNG, were tested in the SHE cell transformation assay with and without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable between experiments performed using either method. These results demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and assisting the scoring of morphologically transformed colonies. PMID:20226874

  17. A new angle on microscopic suspension feeders near boundaries.

    PubMed

    Pepper, Rachel E; Roper, Marcus; Ryu, Sangjin; Matsumoto, Nobuyoshi; Nagai, Moeto; Stone, Howard A

    2013-10-15

    Microscopic sessile suspension feeders are a critical component in aquatic ecosystems, acting as an intermediate trophic stage between bacteria and higher eukaryotic taxa. Because they live attached to boundaries, it has long been thought that recirculation of the feeding currents produced by sessile suspension feeders inhibits their ability to access fresh fluid. However, previous models for the feeding flows of these organisms assume that they feed by pushing fluid perpendicular to surfaces they live upon, whereas we observe that sessile suspension feeders often feed at an angle to these boundaries. Using experiments and calculations, we show that living suspension feeders (Vorticella) likely actively regulate the angle that they feed relative to a substratum. We then use theory and simulations to show that angled feeding increases nutrient and particle uptake by reducing the reprocessing of depleted water. This work resolves an open question of how a key class of suspension-feeding organisms escapes physical limitations associated with their sessile lifestyle. PMID:24138855

  18. A New Angle on Microscopic Suspension Feeders near Boundaries

    PubMed Central

    Pepper, Rachel E.; Roper, Marcus; Ryu, Sangjin; Matsumoto, Nobuyoshi; Nagai, Moeto; Stone, Howard A.

    2013-01-01

    Microscopic sessile suspension feeders are a critical component in aquatic ecosystems, acting as an intermediate trophic stage between bacteria and higher eukaryotic taxa. Because they live attached to boundaries, it has long been thought that recirculation of the feeding currents produced by sessile suspension feeders inhibits their ability to access fresh fluid. However, previous models for the feeding flows of these organisms assume that they feed by pushing fluid perpendicular to surfaces they live upon, whereas we observe that sessile suspension feeders often feed at an angle to these boundaries. Using experiments and calculations, we show that living suspension feeders (Vorticella) likely actively regulate the angle that they feed relative to a substratum. We then use theory and simulations to show that angled feeding increases nutrient and particle uptake by reducing the reprocessing of depleted water. This work resolves an open question of how a key class of suspension-feeding organisms escapes physical limitations associated with their sessile lifestyle. PMID:24138855

  19. Analysis of Feeder Bus Network Design and Scheduling Problems

    PubMed Central

    Almasi, Mohammad Hadi; Karim, Mohamed Rehan

    2014-01-01

    A growing concern for public transit is its inability to shift passenger's mode from private to public transport. In order to overcome this problem, a more developed feeder bus network and matched schedules will play important roles. The present paper aims to review some of the studies performed on Feeder Bus Network Design and Scheduling Problem (FNDSP) based on three distinctive parts of the FNDSP setup, namely, problem description, problem characteristics, and solution approaches. The problems consist of different subproblems including data preparation, feeder bus network design, route generation, and feeder bus scheduling. Subsequently, descriptive analysis and classification of previous works are presented to highlight the main characteristics and solution methods. Finally, some of the issues and trends for future research are identified. This paper is targeted at dealing with the FNDSP to exhibit strategic and tactical goals and also contributes to the unification of the field which might be a useful complement to the few existing reviews. PMID:24526890

  20. Analysis of feeder bus network design and scheduling problems.

    PubMed

    Almasi, Mohammad Hadi; Mirzapour Mounes, Sina; Koting, Suhana; Karim, Mohamed Rehan

    2014-01-01

    A growing concern for public transit is its inability to shift passenger's mode from private to public transport. In order to overcome this problem, a more developed feeder bus network and matched schedules will play important roles. The present paper aims to review some of the studies performed on Feeder Bus Network Design and Scheduling Problem (FNDSP) based on three distinctive parts of the FNDSP setup, namely, problem description, problem characteristics, and solution approaches. The problems consist of different subproblems including data preparation, feeder bus network design, route generation, and feeder bus scheduling. Subsequently, descriptive analysis and classification of previous works are presented to highlight the main characteristics and solution methods. Finally, some of the issues and trends for future research are identified. This paper is targeted at dealing with the FNDSP to exhibit strategic and tactical goals and also contributes to the unification of the field which might be a useful complement to the few existing reviews. PMID:24526890

  1. VIEW OF FEEDER TABLE WITH THE BOOM OF THE UNLOADER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF FEEDER TABLE WITH THE BOOM OF THE UNLOADER IN BACKGROUND. THE CASE HYDRAULIC BOOM HOIST IS TO THE RIGHT. VIEW FROM THE SOUTHWEST - Kekaha Sugar Company, Sugar Mill Building, 8315 Kekaha Road, Kekaha, Kauai County, HI

  2. 46 CFR 111.75-1 - Lighting feeders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...a fire in any main vertical and horizontal fire zone does not interfere with the lighting in any other fire zone. This requirement is met if main and emergency feeders passing through any zone are separated both vertically and horizontally...

  3. 11. MOVABLE BED SEDIMENTATION MODELS. AUTOMATIC SEDIMENT FEEDER DESIGNED AND ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. MOVABLE BED SEDIMENTATION MODELS. AUTOMATIC SEDIMENT FEEDER DESIGNED AND BUILT BY WES. - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

  4. 39. CLOSE UP DETAIL OF THE FEEDER AND STAMP CONNECTION. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    39. CLOSE UP DETAIL OF THE FEEDER AND STAMP CONNECTION. THE STAMP AN MORTAR BOX ARE ON THE LEFT AND THE FEEDER WITH ITS FEEDER DISK IS ON THE RIGHT. NOTE THE COLLAR ON THE CENTER STAMP STEM (UPPER LEFT CORNER OF THE IMAGE) THAT ACTIVATES THE LEVER IN THE CENTER OF THE PHOTO. THE COLLAR IS POSITIONED SUCH THAT WHEN THE LEVEL OF THE MATERIAL REACHES A LOW POINT IN THE MORTAR BOX IT PUSHES DOWN ON THE LEVER WHICH IN TURN ACTIVATES THE AUTOMATIC FEEDER DRIVE MECHANISM WHICH THEM DELIVERS ORE INTO THE BACKSIDE OF THE MORTAR BOX. - Standard Gold Mill, East of Bodie Creek, Northeast of Bodie, Bodie, Mono County, CA

  5. Real-time monitoring of inflammation status in 3T3-L1 adipocytes possessing a secretory Gaussia luciferase gene under the control of nuclear factor-kappa B response element

    SciTech Connect

    Nagasaki, Haruka; Yoshimura, Takeshi [Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507 (Japan)] [Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507 (Japan); Aoki, Naohito, E-mail: n-aoki@bio.mie-u.ac.jp [Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507 (Japan)] [Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507 (Japan)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Inflammation status in adipocytes can be monitored by the new assay system. Black-Right-Pointing-Pointer Only an aliquot of conditioned medium is required without cell lysis. Black-Right-Pointing-Pointer Inflammation-attenuating compounds can be screened more conveniently. -- Abstract: We have established 3T3-L1 cells possessing a secretory Gaussia luciferase (GLuc) gene under the control of nuclear factor-kappa B (NF-{kappa}B) response element. The 3T3-L1 cells named 3T3-L1-NF-{kappa}B-RE-GLuc could differentiate into adipocyte as comparably as parental 3T3-L1 cells. Inflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1{beta} induced GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes in a concentration- and time-dependent manner. GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes was also induced when cultured with RAW264.7 macrophages and was dramatically enhanced by lipopolysaccharide (LPS)-activated macrophages. An NF-{kappa}B activation inhibitor BAY-11-7085 and an antioxidant N-acetyl cysteine significantly suppressed GLuc secretion induced by macrophages. Finally, we found that rosemary-derived carnosic acid strongly suppressed GLuc secretion induced by macrophages and on the contrary up-regulated adiponectin secretion. Collectively, by using 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes, inflammation status can be monitored in real time and inflammation-attenuating compounds can be screened more conveniently.

  6. A novel coal feeder for production of low sulfur fuel

    SciTech Connect

    Lin, L.; Khang, S.J.; Keener, T.C.

    1991-01-01

    In this project, a dual-screw feeder was designed for desulfurization of coal. The key parts of this reactor are two screw tubes which are used to feed coal and calcined lime particles separately, the inner tube acting as a coal pyrolyzer and the outer tube acting as a desulfurizer with hot calcined lime pellets or other renewable sorbent pellets. The objective of this project is to study the feasibility of an advanced concept of desulfurization in the coal feeder. (VC)

  7. A versatile technique to minimize electrical losses in distribution feeders

    SciTech Connect

    Kyaruzi, A.L.

    1994-12-31

    This dissertation presents a method of minimizing electrical losses in radial distribution feeders by the use of shunt capacitors. The engineering benefits of reducing peak electrical power and energy losses are compared to the costs associated with the current engineering practice of buying, installing and servicing capacitor banks in the distribution feeders. The present analysis defines this cost-benefit problem and the formulation of the problem of nonuniform feeders with different wire gauges at various feeder sections. Standard utility capacitor bank sizes are used to give a more realistic model. An original computer solution methodology based on techniques developed for this study determines: (i) Whether it is economical to install compensating capacitor banks on a particular radial distribution feeder or not. (ii) The locations at which capacitor banks should be installed. (iii) The types and sizes of capacitor banks to be installed. (iv) The time setting of switched capacitor banks. The techniques have been applied to a typical radial distribution feeder in Dar-es-Salaam, Tanzania. The results and the engineering implications of this work are discussed and recommendations for the engineering community made.

  8. Regulation of epidermal growth factor receptor by activated H-ras and V-myc oncogenes in mouse Balb/3T3 cells: possible roles of AP-1.

    PubMed

    Okimoto, T; Kohno, K; Kuwano, M; Gopas, J; Kung, H F; Ono, M

    1996-04-18

    We previously reported that introduction of H-ras oncogene decreases the epidermal growth factor (EGF) binding activity to cell surface EGF receptor in mouse Balb/3T3. In this study, we have further isolated four H-ras transfectants, four v-myc transfectants and three both H-ras and v-myc (H-ras/v-myc) transfectants of mouse Balb/3T3 cells. In comparison with introduction of v-myc alone or both H-ras and v-myc oncogene, introduction of H-ras alone resulted in a loss of [125I]EGF binding activity to the cell surface EGF receptor. RT-PCR analysis also showed much lower levels of EGF receptor gene expression in H-ras transfectants compared to that of parental untransformed cells (Balb-Neo1), v-myc and H-ras/v-myc transfectants. Our results demonstrated the activated binding of a transcription factor, Stat1 p84/p91, which directly interacts with EGF receptor, to c-sis-inducible element (SIE) in both v-myc and H-rs/v-myc transfectants, but not in H-ras transfectants. Among transcription factors which we have analysed, activator protein 1 (AP-1) but not SP-1 was modulated by H-ras. Gel shift assays demonstrated the mobility pattern of TPA-responsive element (TRE) binding complex with AP-1 derived from H-ras transfectants migrated faster than those from Balb-Neo1, v-myc and H-ras/v-myc. Expression of c-Jun and Fra-1 was increased more than threefold in H-ras transfectants compared with Balb-Neo1, v-myc and H-ras/v-myc transfectants, but that of c-Fos, Jun B and SP-1 was unchanged. Both transient and permanent expression of H-ras enhanced AP-1 activity in mouse cells, but further co-introduction of dominant negative c-jun mutant encoding a transcriptionally inactive product inhibited the H-ras dependent AP-1 induction. Transfection of the dominant negative c-jun mutant also restored down-regulation of EGF binding by activated H-ras oncogene. Down-regulation of EGf receptor by activated H-ras and the possible involvement of a transcription factor, AP-1 will be discussed. PMID:8622882

  9. Autocrine/Paracrine Function of Globular Adiponectin: Inhibition of Lipid Metabolism and Inflammatory Response in 3T3-L1 Adipocytes.

    PubMed

    Lazra, Yulia; Falach, Alona; Frenkel, Lital; Rozenberg, Konstantin; Sampson, Sanford; Rosenzweig, Tovit

    2015-05-01

    Adiponectin is an adipose-derived hormone, with beneficial effects on insulin sensitivity and inflammation. The aim of this study was to clarify the autocrine/paracrine effects of globular adiponectin (gAd) administrated during differentiation on the function of the mature adipocytes. Experiments were performed on 3T3-L1 preadipocytes treated with gAd (10?nM), starting at an early stage of differentiation. gAd treatment during differentiation was without effect on mRNA expression of adiponectin and AdipoR2, but increased AdipoR1 expression. PPARgamma, perillipin and FABP4 mRNA expressions were lower in gAd-treated adipocytes, accompanied by a reduction in lipid accumulation. While mRNA expression of HSL was not affected by gAd compared to untreated adipocytes, both ATGL and FAS were reduced, indicating that gAd regulates both lipolysis and lipogenesis. PPAR?, ACOX2 and UCPs mRNA expressions were not affected by gAd, indicating that the reduction in lipid content is not attributed to an increase in fatty-acid oxidation. In accord with the lower PPAR? expression, there was reduced Glut4 mRNA expression; however, insulin-induced PKB phosphorylation was enhanced by gAd, and glucose uptake was not altered. To investigate the effect of gAd on LPS-induced inflammatory response, cells were treated with gAd either during differentiation or 24?h before induction of inflammation in differentiated adipocytes. LPS-induced inflammatory response, as indicated by increase in IL6 and MCP-1 mRNA expression. gAd given through differentiation was much more effective in abrogating LPS-dependent cytokines production than gAd given to the mature adipocyte. In conclusion, elevated gAd at differentiation of 3T3-L1 cells leads to reduced lipid content, reduced lipid metabolism and high resistance to inflammation. J. Cell. Biochem. 116: 754-766, 2015. © 2014 Wiley Periodicals, Inc. PMID:25491932

  10. Ghrelin augments the expressions and secretions of proinflammatory adipokines, VEGF120 and MCP-1, in differentiated 3T3-L1 adipocytes.

    PubMed

    Kitahara, Atsuko; Takahashi, Kazuto; Moriya, Rie; Onuma, Hirohisa; Handa, Keiko; Sumitani, Yoshikazu; Tanaka, Toshiaki; Katsuta, Hidenori; Nishida, Susumu; Sakurai, Takuya; Inukai, Kouichi; Ohno, Hideki; Ishida, Hitoshi

    2015-01-01

    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 (VEGF(120)) releases (p?3T3-L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor-? (TNF-?), interleukin-1? (IL-1?), 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?

  11. Sheets of Vertically Aligned BaTiO3 Nanotubes Reduce Cell Proliferation but Not Viability of NIH-3T3 Cells

    PubMed Central

    Giannini, Marianna; Giannaccini, Martina; Sibillano, Teresa; Giannini, Cinzia; Liu, Dun; Wang, Zhigang; Baů, Andrea; Dente, Luciana; Cuschieri, Alfred; Raffa, Vittoria

    2014-01-01

    All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Nanotechnology has the potential to address this problem by virtue of the ability of some nanomaterials to modulate interactions with cells, thereby inducing specific biological responses to implanted foreign materials. To this effect in the present study, we have characterised the growth of fibroblasts on nano-structured sheets constituted by BaTiO3, a material extensively used in biomedical applications. We found that sheets of vertically aligned BaTiO3 nanotubes inhibit cell cycle progression - without impairing cell viability - of NIH-3T3 fibroblast cells. We postulate that the 3D organization of the material surface acts by increasing the availability of adhesion sites, promoting cell attachment and inhibition of cell proliferation. This finding could be of relevance for biomedical applications designed to prevent or minimize fibrous encasement by uncontrolled proliferation of fibroblastic cells with loss of material-tissue interface underpinning long-term function of implants. PMID:25506693

  12. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    SciTech Connect

    Chung, Le Thi Kim, E-mail: ngocanh@nutr.med.tokushima-u.ac.jp [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Hosaka, Toshio [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan)] [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Teshigawara, Kiyoshi [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan); Sakai, Tohru [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan)] [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Nakaya, Yutaka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Funaki, Makoto, E-mail: m-funaki@clin.med.tokushima-u.ac.jp [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  13. Influence of heating and cyclic tension on the induction of heat shock proteins and bone-related proteins by MC3T3-E1 cells.

    PubMed

    Chung, Eunna; Sampson, Alana Cherrell; Rylander, Marissa Nichole

    2014-01-01

    Stress conditioning (e.g., thermal, shear, and tensile stress) of bone cells has been shown to enhance healing. However, prior studies have not investigated whether combined stress could synergistically promote bone regeneration. This study explored the impact of combined thermal and tensile stress on the induction of heat shock proteins (HSPs) and bone-related proteins by a murine preosteoblast cell line (MC3T3-E1). Cells were exposed to thermal stress using a water bath (44°C for 4 or 8 minutes) with postheating incubation (37°C for 4 hours) followed by exposure to cyclic strain (equibiaxial 3%, 0.2?Hz, cycle of 10-second tensile stress followed by 10-second rest). Combined thermal stress and tensile stress induced mRNA expression of HSP27 (1.41 relative fold induction (RFI) compared to sham-treated control), HSP70 (5.55?RFI), and osteopontin (1.44?RFI) but suppressed matrix metalloproteinase-9 (0.6?RFI) compared to the control. Combined thermal and tensile stress increased vascular endothelial growth factor (VEGF) secretion into the culture supernatant (1.54-fold increase compared to the control). Therefore, combined thermal and mechanical stress preconditioning can enhance HSP induction and influence protein expression important for bone tissue healing. PMID:25013774

  14. Purification and Characterization of Aporphine Alkaloids from Leaves of Nelumbo nucifera Gaertn and Their Effects on Glucose Consumption in 3T3-L1 Adipocytes

    PubMed Central

    Ma, Chengjun; Wang, Jinjun; Chu, Hongmei; Zhang, Xiaoxiao; Wang, Zhenhua; Wang, Honglun; Li, Gang

    2014-01-01

    Aporphine alkaloids from the leaves of Nelumbo nucifera Gaertn are substances of great interest because of their important pharmacological activities, particularly anti-diabetic, anti-obesity, anti-hyperlipidemic, anti-oxidant, and anti-HIV’s activities. In order to produce large amounts of pure alkaloid for research purposes, a novel method using high-speed counter-current chromatography (HSCCC) was developed. Without any initial cleanup steps, four main aporphine alkaloids, including 2-hydroxy-1-methoxyaporphine, pronuciferine, nuciferine and roemerine were successfully purified from the crude extract by HSCCC in one step. The separation was performed with a simple two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (5:3:3:2.5:5, v/v/v/v/v). In each operation, 100 mg crude extracts was separated and yielded 6.3 mg of 2-hydroxy-1-methoxyaporphine (95.1% purity), 1.1 mg of pronuciferine (96.8% purity), 8.5 mg of nuciferine (98.9% purity), and 2.7 mg of roemerine (97.4%) respectively. The chemical structure of four aporphine alkaloids are identified by means of electrospray ionization MS (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Moreover, the effects of four separated aporphine alkaloids on insulin-stimulated glucose consumption were examined in 3T3-L1 adipocytes. The results showed that 2-hydroxy-1-methoxyaporphine and pronuciferine increased the glucose consumption significantly as rosiglitazone did. PMID:24577311

  15. Purification and characterization of aporphine alkaloids from leaves of Nelumbo nucifera Gaertn and their effects on glucose consumption in 3T3-L1 adipocytes.

    PubMed

    Ma, Chengjun; Wang, Jinjun; Chu, Hongmei; Zhang, Xiaoxiao; Wang, Zhenhua; Wang, Honglun; Li, Gang

    2014-01-01

    Aporphine alkaloids from the leaves of Nelumbo nucifera Gaertn are substances of great interest because of their important pharmacological activities, particularly anti-diabetic, anti-obesity, anti-hyperlipidemic, anti-oxidant, and anti-HIV's activities. In order to produce large amounts of pure alkaloid for research purposes, a novel method using high-speed counter-current chromatography (HSCCC) was developed. Without any initial cleanup steps, four main aporphine alkaloids, including 2-hydroxy-1-methoxyaporphine, pronuciferine, nuciferine and roemerine were successfully purified from the crude extract by HSCCC in one step. The separation was performed with a simple two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (5:3:3:2.5:5, v/v/v/v/v). In each operation, 100 mg crude extracts was separated and yielded 6.3 mg of 2-hydroxy-1-methoxyaporphine (95.1% purity), 1.1 mg of pronuciferine (96.8% purity), 8.5 mg of nuciferine (98.9% purity), and 2.7 mg of roemerine (97.4%) respectively. The chemical structure of four aporphine alkaloids are identified by means of electrospray ionization MS (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Moreover, the effects of four separated aporphine alkaloids on insulin-stimulated glucose consumption were examined in 3T3-L1 adipocytes. The results showed that 2-hydroxy-1-methoxyaporphine and pronuciferine increased the glucose consumption significantly as rosiglitazone did. PMID:24577311

  16. Platelet-derived growth factor stimulates synthesis of 1,2-diacylglycerol from monoacylglycerol in Balb/c 3T3 cells.

    PubMed Central

    Hata, Y; Ogata, E; Kojima, I

    1989-01-01

    1,2-Diacylglycerol (1,2,-DAG) plays an important role in the protein kinase C-mediated signal-transduction system. Several reports have shown that 1,2-DAG is generated through various pathways other than classical phospholipid hydrolysis. We observed a rapid incorporation of [3H]myristate into 1,2-DAG in platelet-derived-growth-factor (PDGF)-treated Balb/c 3T3 cells. [14C]Palmitate was similarly incorporated into 1,2-DAG. The effect of PDGF, which was inhibited by cycloheximide, became maximal after 60 min treatment with PDGF, and disappeared 300 min after removal of PDGF. Treatment with triacylglycerol lipase revealed that labelled saturated fatty acid was incorporated into the sn-1 position. PDGF barely stimulated incorporation of [3H]glycerol or [14C]glucose into 1,2-DAG. Incorporation of [3H]myristate into 1,2-DAG preceded that into triacyglycerol and phospholipids. These results suggest that synthesis of 1,2-DAG from monoacylglycerol is enhanced in PDGF-treated cells. However, there is no significant difference in the activity of monoacylglycerol acyltransferase measured in vitro in quiescent and PDGF-treated cells. The reason for this discrepancy is discussed. PMID:2556114

  17. Platelet-derived growth factor stimulates synthesis of 1,2-diacylglycerol from monoacylglycerol in Balb/c 3T3 cells.

    PubMed

    Hata, Y; Ogata, E; Kojima, I

    1989-09-15

    1,2-Diacylglycerol (1,2,-DAG) plays an important role in the protein kinase C-mediated signal-transduction system. Several reports have shown that 1,2-DAG is generated through various pathways other than classical phospholipid hydrolysis. We observed a rapid incorporation of [3H]myristate into 1,2-DAG in platelet-derived-growth-factor (PDGF)-treated Balb/c 3T3 cells. [14C]Palmitate was similarly incorporated into 1,2-DAG. The effect of PDGF, which was inhibited by cycloheximide, became maximal after 60 min treatment with PDGF, and disappeared 300 min after removal of PDGF. Treatment with triacylglycerol lipase revealed that labelled saturated fatty acid was incorporated into the sn-1 position. PDGF barely stimulated incorporation of [3H]glycerol or [14C]glucose into 1,2-DAG. Incorporation of [3H]myristate into 1,2-DAG preceded that into triacyglycerol and phospholipids. These results suggest that synthesis of 1,2-DAG from monoacylglycerol is enhanced in PDGF-treated cells. However, there is no significant difference in the activity of monoacylglycerol acyltransferase measured in vitro in quiescent and PDGF-treated cells. The reason for this discrepancy is discussed. PMID:2556114

  18. RELAXIN enhances differentiation and matrix mineralization through Relaxin/insulin-like family peptide receptor 2 (Rxfp2) in MC3T3-E1 cells in vitro.

    PubMed

    Duarte, Carolina; Kobayashi, Yukiho; Kawamoto, Tatsuo; Moriyama, Keiji

    2014-08-01

    RELAXIN (RLN) is a polypeptide hormone of the insulin-like hormone family; it facilitates birth by softening and widening the pubic symphysis and cervix in many mammals, including humans. The role of RLN in bone metabolism was recently suggested by its ability to induce osteoclastogenesis and activate osteoclast function. RLN binds to RELAXIN/INSULIN-LIKE FAMILY PEPTIDE 1 (RXFP1) and 2 (RXFP2), with varying species-specific affinities. Young men with mutated RXFP2 are at high risk for osteoporosis, as RXFP2 influences osteoblast metabolism by binding to INSULIN-LIKE PEPTIDE 3 (INSL3). However, there have been no reports on RLN function in osteoblast differentiation and mineralization or on the functionally dominant receptors for RLN in osteoblasts. We previously described Rxfp1 and 2 expression patterns in developing mouse oral components, including the maxillary and mandibular bones, Meckel's cartilage, tongue, and tooth primordia. We hypothesized that Rln/Rxfp signaling is a key mediator of skeletal development and metabolism. Here, we present the gene expression patterns of Rxfp1 and 2 in developing mouse calvarial frontal bones as determined by in situ hybridization. In addition, RLN enhanced osteoblastic differentiation and caused abnormal mineralization and extracellular matrix metabolism through Rxfp2, which was predominant over Rxfp1 in MC3T3-E1 mouse calvarial osteoblasts. Our data suggest a novel role for Rln in craniofacial skeletal development and metabolism through Rxfp2. PMID:24857857

  19. Rearrangement and expression of myeov and hst in NIH/3T3 transfectants: a caveat for the interpretation of DNA transfection analyses.

    PubMed

    Brecht, Marcus; Steenvoorden, Agatha C M; Luf, Susanne; Bartram, Claus R; Janssen, Johannes W G

    2007-05-01

    By means of the tumorigenicity assay applying DNA from a patient with a gastric carcinoma (MA) we have already reported the identification of the putative oncogene myeov. In addition we have shown its involvement in t(11;14)-positive multiple myelomas and amplifications of breast tumours and esophageal carcinomas. The failure of myeov cDNA to induce tumour formation in NIH/3T3 cells prompted us to analyze the human sequences present in our MA-T1a1 tertiary transfectants. Sequence analysis revealed the presence of the human oncogene hst (fgf4) at a distance of approximately 9kb from the myeov gene in our MA-T1a1 tertiary transfectants. Both myeov and hst (fgf4) are normally situated approximately 475-kb apart at band 11q13, a region that is frequently amplified and overexpressed in various tumours. Southern and Northern blot analyses confirmed our sequence data and showed rearrangement of hst sequences during the transfection process and its expression in our MA-T1a1 tertiary transfectants. PMID:17390055

  20. New vinegar produced by tomato suppresses adipocyte differentiation and fat accumulation in 3T3-L1 cells and obese rat model.

    PubMed

    Lee, Ju-Hye; Cho, Hyun-Dong; Jeong, Ji-Hye; Lee, Mi-Kyung; Jeong, Yong-Ki; Shim, Ki-Hwan; Seo, Kwon-Il

    2013-12-01

    There is an increasing surplus of tomatoes that are abandoned due to their failure to meet customer standards. Therefore, to allow both value additions and the effective reuse of surplus tomatoes, we developed tomato vinegar (TV) containing phytochemicals and evaluated its anti-obesity effects in vitro and in vivo. TV inhibited adipocyte differentiation of 3T3-L1 preadipocyte and lipid accumulation during differentiation. TV supplementation in rats fed a high-fat diet (HFD) markedly decreased visceral fat weights without changing the food and calories intakes. TV significantly decreased hepatic triglyceride and cholesterol levels compared to the HFD group. Furthermore, TV lowered plasma LDL-cholesterol level and antherogenic index compared to the HFD group, whereas elevated HDL-cholesterol to total cholesterol ratio. These results show that TV prevented obesity by suppressing visceral fat and lipid accumulation in adipocyte and obese rats, and suggest that TV can be used as an anti-obesity therapeutic agent or functional food. PMID:23871083

  1. Radix Dipsaci total saponins stimulate MC3T3-E1 cell differentiation via the bone morphogenetic protein-2/MAPK/Smad-dependent Runx2 pathway.

    PubMed

    Niu, Yin-Bo; Kong, Xiang-He; Li, Yu-Hua; Fan, Li; Pan, Ya-Lei; Li, Chen-Rui; Wu, Xiang-Long; Lu, Ting-Li; Mei, Qi-Bing

    2015-06-01

    Radix Dipsaci total saponins (RTS) are primary active components of Radix Dipsaci, which is administered orally for the treatment of osteoporosis according to Chinese Medicine. RTS have also been shown to reduce the risk of bone fractures in rats. However, the detailed molecular mechanisms underlying their action remain elusive. In the present study, the ability of RTS to increase alkaline phosphatase activity, osteocalcin levels and the degree of mineralization was investigated in MC3T3?E1 mouse osteoblast precursor cells. In addition, the associated molecular mechanism was detected. The results revealed that RTS exerted an effect on osteoblastic maturation and differentiation. Induction of differentiation by RTS was associated with an increase in the expression levels of bone morphogenetic protein?2 (BMP?2), phosphorylated (P)?Smad1/5/8, P?ERK1/2, P?p38 and Runt?related transcription factor 2 (Runx2). Blocking BMP?2 expression with noggin significantly reduced the levels of osteoblastic differentiation and subsequently attenuated the expression levels of P?Smad1/5/8, P?ERK1/2, P?p38 and Runx2. This indicated that RTS induced osteoblastic differentiation through BMP?2/mitogen?activated protein kinase/Smad1/5/8?dependent Runx2 signaling pathways and that it may be a promising agent for enhancing bone formation. PMID:25625570

  2. In Vitro and In Vivo Enhancement of Adipogenesis by Italian Ryegrass (Lolium multiflorum) in 3T3-L1 Cells and Mice

    PubMed Central

    Kim, Da Hye; Gun Roh, Sang; Lee, Jeong-Chae; Choi, Ki Choon

    2014-01-01

    Adipogenesis is very much important in improving the quality of meat in animals. The aim of the present study was to investigate the in vitro and in vivo adipogenesis regulation properties of Lolium multiflorum on 3T3-L1 pre-adipocytes and mice. Chemical composition of petroleum ether extract of L. multiflorum (PET-LM) confirmed the presence of fatty acids, such as ?-linolenic acid, docosahexaenoic acid, oleic acid, docosatetraenoic acid, and caprylic acid, as the major compounds. PET-LM treatment increased viability, lipid accumulation, lipolysis, cell cycle progression, and DNA synthesis in the cells. PET-LM treatment also augmented peroxysome proliferator activated receptor (PPAR)-?2, CCAAT/enhancer binding protein-?, adiponectin, adipocyte binding protein, glucose transporter-4, fatty acid synthase, and sterol regulatory element binding protein-1 expression at mRNA and protein levels in differentiated adipocytes. In addition, mice administered with 200 mg/kg body weight PET-LM for 8 weeks showed greater body weight than control mice. These findings suggest that PET-LM facilitates adipogenesis by stimulating PPAR?-mediated signaling cascades in adipocytes which could be useful for quality meat development in animals. PMID:24454838

  3. Eicosapentaenoic acid upregulates VEGF-A through both GPR120 and PPAR? mediated pathways in 3T3-L1 adipocytes.

    PubMed

    Hasan, Arif U; Ohmori, Koji; Konishi, Kumi; Igarashi, Junsuke; Hashimoto, Takeshi; Kamitori, Kazuyo; Yamaguchi, Fuminori; Tsukamoto, Ikuko; Uyama, Toru; Ishihara, Yasuhiro; Noma, Takahisa; Tokuda, Masaaki; Kohno, Masakazu

    2015-05-01

    Vascular endothelial growth factor-A (VEGF-A) released from adipocytes promotes angiogenesis; and thereby ameliorates the local hypoxia-induced adipose inflammation and insulin resistance. Here, we newly found that eicosapentaenoic acid (EPA) upregulated both mRNA expression and release of VEGF-A in mature 3T3-L1 adipocytes. Silencing mRNA of G-protein coupled receptor 120 (GPR120) and specific inhibition of peroxisome proliferator-activated receptor ? (PPAR?) by GW9662 respectively attenuated the EPA-induced augmentation of VEGF-A release by adipocytes. Furthermore, transfection of GPR120 gene alone and PPAR? gene alone to HEK293 cells respectively increased the promoter activity of VEGF-A as assessed by luciferase reporter assay, which was further augmented when both genes were co-transfected. Promoter deletion analysis and chromatin immunoprecipitation assay revealed that co-transfection of GPR120 enhanced EPA-induced PPAR? binding to PPAR-response element in VEGF-A promoter region. Thus, by the synchronized activation of a membrane receptor GRP120 and a nuclear receptor PPAR?, EPA enhances VEGF-A production in adipocytes. PMID:25697344

  4. Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

    PubMed

    Shen, Yan; Honma, Natsumi; Kobayashi, Katsuya; Jia, Liu Nan; Hosono, Takashi; Shindo, Kazutoshi; Ariga, Toyohiko; Seki, Taiichiro

    2014-01-01

    We previously demonstrated that cinnamon extract (CE) ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4) translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s) with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK) signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK. PMID:24551069

  5. Expression of cell adhesion and differentiation related genes in MC3T3 osteoblasts plated on titanium alloys: role of surface properties.

    PubMed

    Sista, Subhash; Wen, Cuie; Hodgson, Peter D; Pande, Gopal

    2013-04-01

    It is important to understand the cellular and molecular events that take place at the cell-material interface of implants used for bone repair. An understanding of the mechanisms involved in the initial stages of osteoblast interactions with the surface of the implant material is fundamental in deciding the fate of the cells that come in contact with it. In this study, we compared the relative gene expression of markers that are known to be associated with cell adhesion and differentiation in MC3T3 osteoblast cells, at various time points after plating the cells on surfaces of titanium (Ti) and its two alloys, titanium-zirconium (TiZr) and titanium-niobium (TiNb) by using Quantitative Real Time Polymerase Chain Reaction (RT-PCR). Our analysis indicated that expression of adhesion supporting genes was higher on TiZr surface as compared to Ti and TiNb. The behavior of these genes is possibly driven by a higher surface energy of TiZr. However no significant difference in the expression of differentiation related genes could be seen between the two alloys, although on both substrates it was higher as compared to unalloyed Ti. We propose that substrate composition of the alloys can influence the adhesion and differentiation related gene expression and that Ti alloys are better substrates for inducing osteogenesis as compared to unalloyed Ti. PMID:23827610

  6. Screening of medicinal plants for PPAR? and PPAR? activation and evaluation of their effects on glucose uptake and 3T3-L1 adipogenesis.

    PubMed

    Yang, Min Hye; Avula, Bharathi; Smillie, Troy; Khan, Ikhlas A; Khan, Shabana I

    2013-08-01

    Medicinal plants are a rich source of ligands for nuclear receptors. The present study was aimed to screen a collection of plant extracts for PPAR?/?-activating properties and identify the active extract that can stimulate cellular glucose uptake without enhancing the adipogenesis. A reporter gene assay was performed to screen ethanolic extracts of 263 plant species, belonging to 94 families, for activation of PPAR? and PPAR?. Eight extracts showed activation of PPAR?, while 22 extracts showed activation of PPAR?. The extracts of five plants (Daphne gnidium, Illicium anisatum, Juniperus virginiana, Terminalia chebula, and Thymelaea hirsuta) showed activation of both PPAR? and PPAR? and out of them, D. gnidium and T. hirsuta markedly increased PPAR?/? protein expression. All five extracts showed an increase in cellular glucose uptake. Of the five dual agonists, T. chebula and T. hirsuta did not show any increase in differentiation of 3T3-L1 preadipocytes, but I. anisatum caused an increase in adipogenesis, while D. gnidium and J. virginiana were toxic to adipocytes. The adipogenic effect of rosiglitazone was antagonized by T. chebula and T. hirsuta. It was concluded that T. hirsuta and T. chebula retain the property of elevating glucose uptake as PPAR?/? dual agonists without the undesired side effect of adipogenesis. This is the first report to reveal the PPAR?/? dual agonistic action and glucose uptake enhancing property of T. hirsuta and T. chebula. PMID:23877921

  7. Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization

    SciTech Connect

    Tanner, L.; Lienhard, G.

    1987-05-01

    Incubation of 3T3-L1 adipocytes with insulin at 37C resulted in a 2-fold increase in transferrin binding to surface receptors, as measured by a subsequent incubation of cells at 4C with SVI-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 sec. Scatchard analysis of SVI-transferrin binding to cells at 4C showed that the insulin-induced increase in transferrin binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 with SVI-transferrin to achieve steady-state binding and then exposed to insulin, there was a 2-fold increase in surface-bound transferrin and a corresponding decrease in intracellularly bound transferrin. Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin was a 2-fold increase in the rate of VZFe uptake from VZFe-transferrin. The rate of externalization of intracellular SVI-transferrin-receptor complex at 37 was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 2-fold. The effect of insulin on the rate constant for externalization is sufficient to account entirely for the increase in surface transferrin receptors.

  8. Labisia pumila Upregulates Peroxisome Proliferator-Activated Receptor Gamma Expression in Rat Adipose Tissues and 3T3-L1 Adipocytes

    PubMed Central

    Gu, Harvest F.; Östenson, Claes-Göran; Mannerĺs-Holm, Louise; Stener-Victorin, Elisabet; Wan Mohamud, Wan Nazaimoon

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that regulates lipid and glucose metabolism. We investigated the effects of Labisia pumila (LP) standardized water extract on PPARgamma transcriptional activity in adipocytes in vitro and in vivo. We used a rat model of dihydrotestosterone- (DHT-) induced polycystic ovary syndrome (PCOS), a condition characterized by insulin resistance. At 9 weeks of age, the PCOS rats were randomly subdivided into two groups: PCOS-LP (50?mg/kg/day of LP) and PCOS-control (1?mL of deionised water) for 4-5 weeks on the same schedule. Real-time RT-PCR was performed to determine the PPARgamma mRNA levels. LP upregulated PPARgamma mRNA level by 40% in the PCOS rats. Western blot analysis further demonstrated the increased PPARgamma protein levels in parallel with upregulation in mRNA. These observations were further proven by adipocytes culture. Differentiated 3T3-L1 adipocytes were treated with final concentration of 100??g/mL LP and compared to untreated control and 10??M of rosiglitazone (in type of thiazolidinediones). LP increased PPARgamma expressions at both mRNA and protein levels and enhanced the effect of glucose uptake in the insulin-resistant cells. The data suggest that LP may ameliorate insulin resistance in adipocytes via the upregulation of PPARgamma pathway. PMID:23935612

  9. SPARC is over-expressed in adipose tissues of diet-induced obese rats and causes insulin resistance in 3T3-L1 adipocytes.

    PubMed

    Shen, Yang; Zhao, Yuyan; Yuan, Lizhi; Yi, Wei; Zhao, Rui; Yi, Qianru; Yong, Tongwu

    2014-01-01

    Secreted protein acidic and rich in cysteine (SPARC) is a secretory multifunctional matricellular glycoprotein. High circulating levels of SPARC have been reported to be associated with obesity and insulin resistance. The aim of the present study was to investigate whether SPARC induces insulin resistance and mitochondrial dysfunction in adipocytes. Our results showed that feeding high fat diet to rats for 12 weeks significantly increased SPARC expression in adipose tissues at both mRNA and protein levels. Moreover, SPARC overexpression in stably transfected 3T3-L1 cells induced insulin resistance and mitochondrial dysfunction, as evidenced by inhibition of insulin-stimulated glucose transport, lower ATP synthesis and mitochondrial membrane potential, reduced expression of glucose transporter 4 (GLUT4), and increased levels of reactive oxygen species (ROS) in mature adipocytes. Finally, overexpression of SPARC also modulated the expression levels of several inflammatory cytokines, which play important roles in insulin resistance, glucose and lipid metabolism during adipogenesis. In conclusion, our data suggest that SPARC is involved in obesity-induced adipose insulin resistance and may serve as a potential target in the treatment of obesity and obesity-related insulin resistance. PMID:23910024

  10. An active extract of Ulmus pumila inhibits adipogenesis through regulation of cell cycle progression in 3T3-L1 cells.

    PubMed

    Ghosh, Chiranjit; Chung, Ha-Yull; Nandre, Rahul M; Lee, John Hwa; Jeon, Tae-Il; Kim, In-Sook; Yang, Seung Hak; Hwang, Seong-Gu

    2012-06-01

    Obesity and its associated metabolic disorders has become a major obstacle in improving the average life span. In this regard therapeutic approach using natural compounds are currently receiving much attention. Herbal compounds rich in triterpenes are well known to regulate glucose and lipid metabolism. Here, we have found that Ulmus pumila (UP) contained at least four different triterpenoids and inhibited adipogenesis of 3T3-L1 cells. The cell viability was dose dependently decreased by UP showing the increase of cell accumulation in G1 phase while reducing in S and G2/M phase of cell cycle. UP treatment also significantly decreased the GPDH activity and intracellular lipid accumulation. In addition, UP inhibited the mRNA levels of adipogenic transcription factors and lipogenic genes such as PPAR?, C/EBP?, SREBP1c and FAS while showing no effects on C/EBP-? and C/EBP-?. Importantly enough, treatment of cells with UP suppressed the TNF-? induced activation of NF-?B signaling. Collectively, our results indicate that UP extract effectively attenuated adipogenesis by controlling cell cycle progression and down regulating adipogenic gene expression. PMID:22445738

  11. The effect of glucose concentration and sodium phenylbutyrate treatment on mitochondrial bioenergetics and ER stress in 3T3-L1 adipocytes.

    PubMed

    Tanis, Ross M; Piroli, Gerardo G; Day, Stani D; Frizzell, Norma

    2015-01-01

    While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions. Basal respiration was ~1.7-fold greater in adipocytes that had matured in 30 mM glucose; however, their ability to increase oxygen consumption in response to stress was impaired. Adipogenesis proceeded in both normal and high glucose with concomitant activation of the UPR, but only high glucose was associated with increased levels of ER stress and mitochondrial stress as observed by parallel increases in CHOP and protein succination. Treatment of adipocytes with sodium phenylbutyrate relieved mitochondrial stress through a reduction in mitochondrial respiration. Our data suggests that mitochondrial stress, protein succination and ER stress are uniquely linked in adipocytes matured in high glucose. PMID:25448036

  12. ?-Mangostin Improves Glucose Uptake and Inhibits Adipocytes Differentiation in 3T3-L1 Cells via PPAR?, GLUT4, and Leptin Expressions

    PubMed Central

    Taher, Muhammad; Mohamed Amiroudine, Mohamed Zaffar Ali; Tengku Zakaria, Tengku Muhamad Faris Syafiq; Ichwan, Solachuddin J. A.; Kaderi, Mohd Arifin; Ahmed, Qamar Uddin; Zakaria, Zainul Amiruddin

    2015-01-01

    Obesity has been often associated with the occurrence of cardiovascular diseases, type 2 diabetes, and cancer. The development of obesity is also accompanied by significant differentiation of preadipocytes into adipocytes. In this study, we investigated the activity of ?-mangostin, a major xanthone component isolated from the stem bark of G. malaccensis, on glucose uptake and adipocyte differentiation of 3T3-L1 cells focusing on PPAR?, GLUT4, and leptin expressions. ?-Mangostin was found to inhibit cytoplasmic lipid accumulation and adipogenic differentiation. Cells treated with 50??M of ?-mangostin reduced intracellular fat accumulation dose-dependently up to 44.4% relative to MDI-treated cells. Analyses of 2-deoxy-D-[3H] glucose uptake activity showed that ?-mangostin significantly improved the glucose uptake (P < 0.05) with highest activity found at 25??M. In addition, ?-mangostin increased the amount of free fatty acids (FFA) released. The highest glycerol release level was observed at 50??M of ?-mangostin. qRT-PCR analysis showed reduced lipid accumulation via inhibition of PPAR? gene expression. Induction of glucose uptake and free fatty acid release by ?-mangostin were accompanied by increasing mRNA expression of GLUT4 and leptin. These evidences propose that ?-mangostin might be possible candidate for the effective management of obesity in future.

  13. Regulation of Insulin Receptor Substrate 1 (IRS-1)/AKT Kinase-mediated Insulin Signaling by O-Linked ?-N-Acetylglucosamine in 3T3-L1 Adipocytes*

    PubMed Central

    Whelan, Stephen A.; Dias, Wagner B.; Thiruneelakantapillai, Lakshmanan; Lane, M. Daniel; Hart, Gerald W.

    2010-01-01

    Increased O-linked ?-N-acetylglucosamine (O-GlcNAc) is associated with insulin resistance in muscle and adipocytes. Upon insulin treatment of insulin-responsive adipocytes, O-GlcNAcylation of several proteins is increased. Key insulin signaling proteins, including IRS-1, IRS-2, and PDK1, are substrates for OGT, suggesting potential O-GlcNAc control points within the pathway. To elucidate the roles of O-GlcNAc in dampening insulin signaling (Vosseller, K., Wells, L., Lane, M. D., and Hart, G. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5313–5318), we focused on the pathway upstream of AKT. Increasing O-GlcNAc in 3T3-L1 adipocytes decreases phosphoinositide 3-kinase (PI3K) interactions with both IRS-1 and IRS-2. Elevated O-GlcNAc also reduces phosphorylation of the PI3K p85 binding motifs (YXXM) of IRS-1 and results in a concomitant reduction in tyrosine phosphorylation of Y608XXM in IRS-1, one of the two main PI3K p85 binding motifs. Additionally, insulin signaling stimulates the interaction of OGT with PDK1. We conclude that one of the steps at which O-GlcNAc contributes to insulin resistance is by inhibiting phosphorylation at the Y608XXM PI3K p85 binding motif in IRS-1 and possibly at PDK1 as well. PMID:20018868

  14. Ezrin, radixin, and moesin phosphorylation in NIH3T3 cells revealed angiotensin II type 1 receptor cell-type-dependent biased signaling.

    PubMed

    Ibrahim, Islam A A E-H; Nakaya, Michio; Kurose, Hitoshi

    2013-01-01

    ?-Arrestin-biased agonists are a new class of drugs with promising therapeutic effects. The molecular mechanisms of ?-arrestin-biased agonists are still not completely identified. Here, we investigated the effect of angiotensin II (AngII) and [Sar1,Ile4,Ile8] AngII (SII), a ?-arrestin-biased agonist, on ezrin-radixin-moesin (ERM) phosphorylation in NIH3T3 cells (a fibroblast cell line) stably expressing AngII type 1A receptor. ERM proteins are cross-linkers between the plasma membrane and the actin cytoskeleton and control a number of signaling pathways. We also investigated the role of G?q protein and ?-arrestins in mediating ERM phosphorylation. We found that AngII stimulates ERM phosphorylation by acting as a ?-arrestin-biased agonist and AngII-stimulated ERM phosphorylation is mediated by ?-arrestin2 not ?-arrestin1. We also found that SII inhibits ERM phosphorylation by acting as a G?q protein-biased agonist. We concluded that ERM phosphorylation is a unique ?-arrestin-biased agonism signal. Both AngII and SII can activate either G?q protein or ?-arrestin-mediated signaling as functional biased agonists according to the type of the cell on which they act. PMID:23575451

  15. Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells

    PubMed Central

    Valentino, Rossella; D’Esposito, Vittoria; Passaretti, Federica; Liotti, Antonietta; Cabaro, Serena; Longo, Michele; Perruolo, Giuseppe; Oriente, Francesco; Beguinot, Francesco; Formisano, Pietro

    2013-01-01

    Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPAR? and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-?, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function. PMID:24349194

  16. Neoplastic transformation and tumorigenesis associated with overexpression of imup-1 and imup-2 genes in cultured NIH/3T3 mouse fibroblasts

    SciTech Connect

    Ryoo, Zae Young [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of)]. E-mail: jaewoong64@hanmail.net; Jung, Boo Kyoung [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 110-799 (Korea, Republic of); Lee, Sang Ryeul [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Kim, Myoung Ok [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Kim, Sung Hyun [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Kim, Hyo Jin [CHA Research Institute, Pochon CHA University, Sungnam 463-836 (Korea, Republic of); Ahn, Jung Yong [CHA Research Institute, Pochon CHA University, Sungnam 463-836 (Korea, Republic of); Lee, Tae-Hoon [Department of Oral Biochemistry, College of Dentistry, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Cho, Youl Hee [Department of Medical Genetics, College of Medicine, Hanyang University, Seoul 133-791 (Korea, Republic of); Park, Jae Hak [Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 110-799 (Korea, Republic of); Kim, Jin Kyeoung [CHA Research Institute, Pochon CHA University, Sungnam 463-836 (Korea, Republic of); Graduate School of Life Science and Biotechnology, College of Medicine, Pochon CHA University, Sungnam 463-836 (Korea, Republic of)

    2006-10-27

    Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation.

  17. Anti-obesity effects of Boussingaulti gracilis Miers var. pseudobaselloides Bailey via activation of AMP-activated protein kinase in 3T3-L1 cells.

    PubMed

    Kim, Hana; Choung, Se-Young

    2012-09-01

    In a previous study, we demonstrated the anti-obesity and hypolipidemic effects of Boussingaulti gracilis Miers var. pseudobaselloides Bailey in high-fat diet-induced obese rats. The present study investigated the molecular mechanisms by which B. gracilis Miers var. pseudobaselloides Bailey ethanol extract (BGE) conferred antidifferentiation and anti-adipogenic effects in the 3T3-L1 preadipocyte differentiation model. BGE treatment significantly and dose-dependently suppressed lipid accumulation and down-regulated the expression of major transcription factors involved in adipogenesis, such as peroxisome proliferator-activated receptor-?, CCAAT/enhancer binding protein ?, sterol regulatory element-binding proteins, and their target genes. It is important that treatment with BGE increased phosphorylation of AMP-activated protein kinase (AMPK), which is one of the rate-limiting enzymes in the fatty acid synthesis pathway, and its direct downstream protein, acetyl-coenzyme A carboxylase. These results suggest that BGE may exert anti-adipogenic effects through regulation of AMPK activity and expression of genes involved in lipogenesis. PMID:22871035

  18. Nonivamide Enhances miRNA let-7d Expression and Decreases Adipogenesis PPAR? Expression in 3T3-L1 Cells.

    PubMed

    Rohm, Barbara; Holik, Ann-Katrin; Kretschy, Nicole; Somoza, Mark M; Ley, Jakob P; Widder, Sabine; Krammer, Gerhard E; Marko, Doris; Somoza, Veronika

    2015-06-01

    Red pepper and its major pungent principle, capsaicin (CAP), have been shown to be effective anti-obesity agents by reducing energy intake, enhancing energy metabolism, decreasing serum triacylglycerol content, and inhibiting adipogenesis via activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the binding of CAP to the TRPV1 receptor is also responsible for its pungent sensation, strongly limiting its dietary intake. Here, the effects of a less pungent structural CAP-analog, nonivamide, on adipogenesis and underlying mechanisms in 3T3-L1 cells were studied. Nonivamide was found to reduce mean lipid accumulation, a marker of adipogenesis, to a similar extent as CAP, up to 10.4% (P?

  19. Interactions between sub-10-nm iron and cerium oxide nanoparticles and 3T3 fibroblasts: the role of the coating and aggregation state

    NASA Astrophysics Data System (ADS)

    Safi, M.; Sarrouj, H.; Sandre, O.; Mignet, N.; Berret, J.-F.

    2010-04-01

    Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of cerium and iron oxide sub-10-nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 g mol - 1). Electrostatically adsorbed on the surfaces, the organic moieties provide a negatively charged coating in physiological conditions. We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. Only the bare and the citrate-coated nanoceria exhibit a slight decrease in mitochondrial activity at very high cerium concentrations (>1 g l - 1). We also observe that the citrate-coated particles are internalized/adsorbed by the cells in large amounts, typically 250 pg/cell after 24 h incubation for iron oxide. In contrast, the polymer-coated particles are taken up at much lower rates (<30 pg/cell). The strong uptake shown by the citrated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes. In conclusion, we show that the uptake of nanomaterials by living cells depends on the coating of the particles and on its ability to preserve the colloidal nature of the dispersions.

  20. Cinnamon Extract Enhances Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myocytes by Inducing LKB1-AMP-Activated Protein Kinase Signaling

    PubMed Central

    Shen, Yan; Honma, Natsumi; Kobayashi, Katsuya; Jia, Liu Nan; Hosono, Takashi; Shindo, Kazutoshi; Ariga, Toyohiko; Seki, Taiichiro

    2014-01-01

    We previously demonstrated that cinnamon extract (CE) ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4) translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s) with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK) signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK. PMID:24551069

  1. (-)-Hydroxycitric acid attenuates endoplasmic reticulum stress-mediated alterations in 3T3-L1 adipocytes by protecting mitochondria and downregulating inflammatory markers.

    PubMed

    Nisha, V M; Priyanka, A; Anusree, S S; Raghu, K G

    2014-11-01

    Endoplasmic reticulum (ER) stress is an emerging potential therapeutic target for metabolic syndrome due to its role in synthesis, secretion, and folding of proteins. It leads to an increased production of reactive oxygen species (ROS) which, along with mitochondrial dysfunction and reduced antioxidant defense, causes chronic cell injury. The present investigation aims to observe the alterations in adipocytes due to ER stress and the protective effect of hydroxycitric acid (HCA), a bioactive from Garcinia species, to develop the same as a nutraceutical. ER stress was induced in mature 3T3-L1 adipocytes by treating them with tunicamycin (2?g/ml) for 18 h. Alterations in cell viability, innate antioxidant system (superoxide dismutase, glutathione peroxidase, and glutathione reductase), mitochondria (membrane potential, biogenesis, and transition pore opening), and inflammatory cytokines (tumor necrosis factor, monocyte chemoattractant protein, interferon-?, interleukin (IL)-10, IL-6, and IL-1?) during ER stress, and co-treatment with HCA were analyzed. Endocrine function of adipocytes was also assessed by measuring adiponectin and leptin secretion levels. HCA protected the cells from ER stress by improving the antioxidant status and mitochondrial functions. The results validate nutraceutical properties of the edible bioactive, commonly used for culinary purpose. A more detailed study on the mechanism of action of HCA is required for developing it as a therapeutic agent for metabolic syndrome. PMID:25175938

  2. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A/sub 2/

    SciTech Connect

    Burch, R.M.; Axelrod, J.

    1987-09-01

    In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E/sub 2/ (PGE/sub 2/) synthesis. The EC/sub 50/ values for stimulation of PGE/sub 2/ synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-(..gamma..-thio)triphosphate stimulated PGE/sub 2/ synthesis and InsP formation, and guanosine-5'-(..beta..-thio)diphosphate inhibited both PGE/sub 2/ synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE/sub 2/ synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE/sub 2/ synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE/sub 2/ synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE/sub 2/ synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with (/sup 3/H) choline, the phospholipase A/sub 2/ products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A/sub 2/ and that phospholipase A/sub 2/ is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.

  3. Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway.

    PubMed Central

    Pettitt, T R; Zaqqa, M; Wakelam, M J

    1994-01-01

    Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF. PMID:8141780

  4. MicroRNA-1 Participates in Nitric Oxide-Induced Apoptotic Insults to MC3T3-E1 Cells by Targeting Heat-Shock Protein-70

    PubMed Central

    Lee, Yong-Eng; Hong, Chung-Ye; Lin, Yi-Ling; Chen, Ruei-Ming

    2015-01-01

    Our previous studies showed that nitric oxide (NO) could induce osteoblast apoptosis. MicroRNA-1 (miR-1), a skeletal- and cardiac muscle-specific small non-coding RNA, contributes to the regulation of multiple cell activities. In this study, we evaluated the roles of miR-1 in NO-induced insults to osteoblasts and the possible mechanisms. Exposure of mouse MC3T3-E1 cells to sodium nitroprusside (SNP) increased amounts of cellular NO and intracellular reactive oxygen species. Sequentially, SNP decreased cell survival but induced caspase-3 activation, DNA fragmentation, and cell apoptosis. In parallel, treatment with SNP induced miR-1 expression in a time-dependent manner. Application of miR-1 antisense inhibitors to osteoblasts caused significant inhibition of SNP-induced miR-1 expression. Knocking down miR-1 concurrently attenuated SNP-induced alterations in cell morphology and survival. Consecutively, SNP time-dependently inhibited heat-shock protein (HSP)-70 messenger (m)RNA and protein expressions. A bioinformatic search predicted the existence of miR-1-specific binding elements in the 3'-untranslational region of HSP-70 mRNA. Downregulation of miR-1 expression simultaneously lessened SNP-induced inhibition of HSP-70 mRNA and protein expressions. Consequently, SNP-induced modifications in the mitochondrial membrane potential, caspase-3 activation, DNA fragmentation, and apoptotic insults were significantly alleviated by miR-1 antisense inhibitors. Therefore, this study showed that miR-1 participates in NO-induced apoptotic insults through targeting HSP-70 gene expression. PMID:25678843

  5. Bilobalide attenuates hypoxia induced oxidative stress, inflammation, and mitochondrial dysfunctions in 3T3-L1 adipocytes via its antioxidant potential.

    PubMed

    Priyanka, A; Nisha, V M; Anusree, S S; Raghu, K G

    2014-10-01

    Excessive expansion of white adipose tissue leads to hypoxia which is considered as a key factor responsible for adipose tissue dysfunction in obesity. Hypoxia induces inflammation, insulin resistance, and other obesity related complications. So the hypoxia-signalling pathway is expected to provide a new target for the treatment of obesity-associated complications. Inhibition or downregulation of the HIF-1 pathway could be an effective target for the treatment of obesity related hypoxia. In the present study, we evaluated the effect of hypoxia on functions of 3T3-L1 adipocytes emphasising on oxidative stress, antioxidant status, inflammation and mitochondrial functions. We have also evaluated the protective role of bilobalide, a bioactive from Gingko biloba, on hypoxia induced alterations. The results revealed that hypoxia significantly altered all the vital parameters of adipocyte biology like HIF-1? expression (103.47% ?), lactate and glycerol release (184.34% and 69.1% ?, respectively), reactive oxygen species (ROS) production (432.53% ?), lipid and protein oxidation (376.6% and 566.6% ?, respectively), reduction in antioxidant enzymes (superoxide dismutase and catalase) status, secretion of inflammatory markers (TNF-?, IL-6, IL-1? and IFN-?) and mitochondrial functions (mitochondrial mass, membrane potential, permeability transition pore integrity, superoxide generation). Bilobalide significantly protected adipocytes from adverse effects of hypoxia in a dose-dependent manner by attenuating oxidative stress, inflammation and protecting mitochondria. Acriflavine (HIF-1 inhibitor) was used as positive control. On the basis of this study, a detailed investigation is needed to delineate the mechanism of action of bilobalide to develop it as therapeutic target for obesity. PMID:25039303

  6. Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples

    PubMed Central

    Dessaux, Sophie; Besse, Anthony; Brahimi-Adouane, Sabrina; Emile, Jean-François; Blay, Jean-Yves; Alberti, Laurent

    2013-01-01

    Objective Most gain of function mutations of tyrosine kinase receptors in human tumours are hemizygous. Gastrointestinal stromal tumours (GIST) with homozygous mutations have a worse prognosis. We aimed to identify genes differentially regulated by hemizygous and heterozygous KIT mutations. Materials and Methods Expression of 94 genes and 384 miRNA was analysed with low density arrays in five NIH3T3 cell lines expressing the full-length human KIT cDNA wild-type (WT), hemizygous KIT mutation with del557-558 (D6) or del564-581 (D54) and heterozygous WT/D6 or WT/D54. Expression of 5 of these genes and 384 miRNA was then analysed in GISTs samples. Results Unsupervised and supervised hierarchical clustering of the mRNA and miRNA profiles showed that heterozygous mutants clustered with KIT WT expressing cells while hemizygous mutants were distinct. Among hemizygous cells, D6 and D54 expressing cells clustered separately. Most deregulated genes have been reported as potentially implicated in cancer and severals, as ANXA8 and FBN1, are highlighted by both, mRNA and miRNA analyses. MiRNA and mRNA analyses in GISTs samples confirmed that their expressions varied according to the mutation of the alleles. Interestingly, RGS16, a membrane protein of the regulator of G protein family, correlate with the subcellular localization of KIT mutants and might be responsible for regulation of the PI3K/AKT signalling pathway. Conclusion Patterns of mRNA and miRNA expression in cells and tumours depend on heterozygous/hemizygous status of KIT mutations, and deletion/presence of TYR568 & TYR570 residues. Thus each mutation of KIT may drive specific oncogenic pathways. PMID:23593401

  7. Transient expression of interferon-inducible p204 in the early stage is required for adipogenesis in 3T3-L1 cells.

    PubMed

    Xiao, Jing; Sun, Bing; Cai, Guo-ping

    2010-07-01

    A member of the interferon-inducible p200 family of proteins, p204, has recently been reported to function in the development of many mesoderm-derived tissues, such as bone, muscle, and cartilage. However, no published study has yet investigated the role of p204 in adipogenesis. Our preliminary experiments showed that p204 can be found in 3T3-L1 preadipocytes, and its expression was up-regulated in a differentiation-dependent manner. As such, we hypothesized that p204 is associated with adipogenesis and focused on the influence of p204 on adipogenesis. In the present study, we investigated the transient elevated expression and cytoplasm-to-nucleus translocation of p204 in the early stage of adipogenesis. To determine the effect of p204 on adipogenesis, p204-siRNA and expression vector were produced for p204 suppression and overexpression, respectively. The knockdown of p204 resulted in a significantly depressed adipocyte differentiation, whereas p204 overexpression promoted adipocyte differentiation. The mRNA expression of adipogenic markers, such as peroxisome-proliferator-activated receptor (PPAR)gamma, CCAAT/enhancer-binding-protein (C/EBP)alpha, lipoprotein lipase, and adipsin, was decreased by p204 suppression and increased by p204 overexpression. A coimmunoprecipitation assay coupled with an indirect immunofluorescence assay also indicated that p204 interacted and colocalized with C/EBPdelta in the nucleus. Furthermore, the knockdown of p204 disrupted the interaction between p204 and C/EBPdelta and partially suppressed the PPARgamma transcriptional activity by dissociating C/EBPdelta with the PPARgamma promoter element. Collectively, our data indicate that the transient expression of p204 in the early stage is indispensable for adipocyte differentiation. Disruption of p204 expression patterns at this stage leads to irreversible damage in fat formation. PMID:20444940

  8. Individual trans 18:1 isomers are metabolised differently and have distinct effects on lipogenesis in 3T3-L1 adipocytes.

    PubMed

    Vahmani, P; Meadus, W J; Turner, T D; Duff, P; Rolland, D C; Mapiye, C; Dugan, M E R

    2015-02-01

    The objective of this research was to study the metabolism of individual trans fatty acids (FAs) that can be found in ruminant fat or partially hydrogenated vegetable oils (PHVO) and determine their effects on FA composition and lipogenic gene expression in adipocytes. Differentiated 3T3-L1 adipocytes were treated with 200 µM of either trans-9-18:1, trans-11-18:1, trans-13-18:1, cis-9-18:1 or BSA vehicle control for 120 h. Trans-9-18:1 increased total cell FA content (µmole/well) compared to other FA treatments, which was mainly related to the accumulation of trans-9-18:1 in the cells. Adipocytes were able to desaturate a significant proportion of absorbed trans-11-18:1 and trans-13-18:1 (~20 and 30% respectively) to cis-9,trans-11-18:2 and cis-9,trans-13-18:2, whereas trans-9-18:1 was mostly incorporated intact resulting in a greater lipophilic index (i.e. decreased mean FA fluidity) of adipocytes. Trans-9-18:1 up-regulated (P < 0.05) the expression of lipogenic genes including acetyl-CoA carboxylase (1.65 fold), FA synthase (1.45 fold), FA elongase-5 (1.52 fold) and stearoyl-CoA desaturase-1 (1.49 fold), compared to the control, whereas trans-11-18:1 and trans-13-18:1 did not affect the expression of these genes compared to control. Our results suggest that the metabolism and lipogenic properties of trans-11-18:1 and trans-13-18:1, typically the most abundant trans FA in beef from cattle fed forage-based diets, are similar and are different from those of trans-9-18:1, the predominant trans FA in PHVO. PMID:25544125

  9. Polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) alter leptin signaling and lipid metabolism in differentiated 3T3-L1 adipocytes.

    PubMed

    Ferrante, Maria C; Amero, Paola; Santoro, Anna; Monnolo, Anna; Simeoli, Raffaele; Di Guida, Francesca; Mattace Raso, Giuseppina; Meli, Rosaria

    2014-09-15

    Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) are highly lipophilic environmental contaminants that accumulate in lipid-rich tissues, such as adipose tissue. Here, we reported the effects induced by PCBs 101, 153 and 180, three of the six NDL-PCBs defined as indicators, on mature 3T3-L1 adipocytes. We observed an increase in lipid content, in leptin gene expression and a reduction of leptin receptor expression and signaling, when cells were exposed to PCBs, alone or in combination. These modifications were consistent with the occurrence of "leptin-resistance" in adipose tissue, a typical metabolic alteration related to obesity. Therefore, we investigated how PCBs affect the expression of pivotal proteins involved in the signaling of leptin receptor. We evaluated the PCB effect on the intracellular pathway JAK/STAT, determining the phosphorylation of STAT3, a downstream activator of the transcription of leptin gene targets, and the expression of SOCS3 and PTP1B, two important regulators of leptin resistance. In particular, PCBs 153 and 180 or all PCB combinations induced a significant reduction in pSTAT3/STAT3 ratio and an increase in PTP1B and SOCS3, evidencing an additive effect. The impairment of leptin signaling was associated with the reduction of AMPK/ACC pathway activation, leading to the increase in lipid content. These pollutants were also able to increase the transcription of inflammatory cytokines (IL-6 and TNF?). It is worthy to note that the PCB concentrations used are comparable to levels detectable in human adipose tissue. Our data strongly support the hypothesis that NDL-PCBs may interfere with the lipid metabolism contributing to the development of obesity and related diseases. PMID:24978599

  10. 1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine pre-osteoblastic MC3T3-E1 cells by direct regulation of cystathionine ?-synthase

    PubMed Central

    Kriebitzsch, Carsten; Verlinden, Lieve; Eelen, Guy; van Schoor, Natasja M.; Swart, Karin; Lips, Paul; Meyer, Mark B.; Pike, J Wesley; Boonen, Steven; Carlberg, Carsten; Vitvitsky, Victor; Bouillon, Roger; Banerjee, Ruma; Verstuyf, Annemieke

    2011-01-01

    High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY due to its negative impact on collagen maturation. HCY is cleared by cystathionine ?-synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3-E1 murine pre-osteoblasts treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH)2D3. Quantitative real-time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH)2D3 treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH)2D3-treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knock-out osteoblasts, in which up-regulation of cbs in response to 1,25(OH)2D3 was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs. To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D3 levels] and HCY levels. In conclusion, this study demonstrated that cbs is a primary 1,25(OH)2D3 target gene which renders HCY metabolism responsive to 1,25(OH)2D3. PMID:21898591

  11. Prostaglandin E2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 cells in a PKA-dependent manner

    PubMed Central

    Gong, Xiaoyuan; Yang, Weidong; Wang, Liyun; Duncan, Randall L.; Pan, Jun

    2014-01-01

    The effect of prostaglandin E2 (PGE2) on bone mass has been well-established in vivo. Previous studies have showed that PGE2 increases differentiation, proliferation, and regulates cell morphology through F-actin stress fiber in statically cultured osteoblasts. However, the effect of PGE2 on osteoblasts in the presence of fluid shear stress (FSS), which could better uncover the anabolic effect of PGE2 in vivo, has yet to be examined. Here, we hypothesized that PGE2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 osteoblastic cells through protein kinase A (PKA) pathway. Furthermore, this PGE2-induced F-actin remodeling was associated with the recovery of cellular mechanosensitivity. Our data showed that treatment with 10 nM dmPGE2 for 15 min significantly suppressed the F-actin stress fiber intensity in FSS-stimulated cells in a PKA-dependent manner. In addition, dmPGE2 treatment enhanced the cells' calcium peak magnitude and the percentage of responding cells in the second FSS stimulation, though these effects were abolished and attenuated by co-treatment with phalloidin. Our results demonstrated that 10 nM dmPGE2 was able to accelerate the ‘reset’ process of F-actin stress fiber to its pre-stimulated level partially through PKA pathway, and thus promoted the recovery of cellular mechanosensitivity. Our finding provided a novel cellular mechanism by which PGE2 increased bone formation as shown in vivo, suggesting that PGE2 could be a potential target for treatments of bone formation-related diseases. PMID:24296051

  12. Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts.

    PubMed

    You, J; Reilly, G C; Zhen, X; Yellowley, C E; Chen, Q; Donahue, H J; Jacobs, C R

    2001-04-20

    Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca(2+)i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3-E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to -2 Newtons/m(2) at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca(2+)i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca(2+)i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway. PMID:11278573

  13. Effects of Varying Degrees of Intermittent Hypoxia on Proinflammatory Cytokines and Adipokines in Rats and 3T3-L1 Adipocytes

    PubMed Central

    Zhou, Qin; Zhu, Hui; Niu, Wen-yan; Feng, Jing; Wang, Yan; Cao, Jie; Chen, Bao-yuan

    2014-01-01

    Objectives Intermittent hypoxia (IH), resulted from recurring episodes of upper airway obstruction, is the hallmark feature and the most important pathophysiologic pathway of obstructive sleep apnea (OSA). IH is believed to be the most important factor causing systemic inflammation. Studies suggest that insulin resistance (IR) is positively associated with OSA. In this study, we hypothesized that the recurrence of IH might result in cellular and systemic inflammation, which was manifested through the levels of proinflammatory cytokines and adipokines after IH exposure, and because IR is linked with inflammation tightly, this inflammatory situation may implicate an IR status. Methods We developed an IH 3T3-L1 adipocyte and rat model respectively, recapitulating the nocturnal oxygen profile in OSA. In IH cells, nuclear factor kappa B (NF-?B) DNA binding reactions, hypoxia-inducible factor-1? (HIF-1?), glucose transporter-1 (Glut-1), necrosis factor alpha (TNF-?), interleukin (IL) -6, leptin, adiponectin mRNA transcriptional activities and protein expressions were measured. In IH rats, blood glucose, insulin, TNF-?, IL-6, leptin and adiponectin levels were analyzed. Results The insulin and blood glucose levels in rats and NF-?B DNA binding activities in cells had significantly statistical results described as severe IH>moderate IH>mild IH>sustained hypoxia>control. The mRNA and protein levels of HIF-1? and Glut-1 in severe IH group were the highest. In cellular and animal models, both the mRNA and protein levels of TNF-?, IL-6 and leptin were the highest in severe IH group, when the lowest in severe IH group for adiponectin. Conclusions Oxidative stress and the release of pro-inflammatory cytokines/adipokines, which are the systemic inflammatory markers, are associated with IH closely and are proportional to the severity of IH. Because IR and glucose intolerance are linked with inflammation tightly, our results may implicate the clinical relationships between OSA and IR. PMID:24466027

  14. Effects of Long-Term 50Hz Power-Line Frequency Electromagnetic Field on Cell Behavior in Balb/c 3T3 Cells

    PubMed Central

    An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

    2015-01-01

    Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn’t change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein. PMID:25695503

  15. Saikosaponin a, an active compound of Radix Bupleuri, attenuates inflammation in hypertrophied 3T3-L1 adipocytes via ERK/NF-?B signaling pathways.

    PubMed

    Kim, Sung Ok; Park, Ji Yeoung; Jeon, Seo Young; Yang, Chea Ha; Kim, Mi Ryeo

    2015-04-01

    Bupleurum falcatum L. is employed in oriental medicine in Korea. This root has been used for anti?inflammatory, anti?pyretic, and anti?hepatotoxic effects in the treatments of common cold, fever, and hepatitis. One of major bioactive compounds of Radix Bupleuri is the saikosaponin a (SSNa). However, little is known concerning the effects of SSNa on obesity associated with a state of low?grade inflammation. Consequently, this study was conducted to determine the inhibition of the inflammation pathway of SSNa in obesity. MTT assay was conducted for cytotoxicity and viability; nuclear and cytoplasmic fractions were extracted from adipocytes for translocation of nuclear factor??B cells (NF??B); nitric oxide (NO) production and secretion using Griess reagent; reverse transcription?polymerase chain reaction (RT?PCR) and immunoblotting for mRNA and protein levels associated with inflammation in the hypertrophied adipocytes. The results revealed that SSNa significantly decreased the expression of tumor necrosis factor?? (TNF?), interleukin (IL)?1? and IL?6 as proinflammatory cytokines, compared to that of non?treated control cells. Inducible nitric oxide synthase (iNOS) and cyclooxygenase?2 (COX?2) as inflammatory factors were reduced by treatment of these cells with SSNa and also suppressed NO production. Phosphorylation of I?B? was inhibited and translocation of NF??B was suppressed via the ERK pathway in response to SSNa treatment. In conclusion, the results demonstrated that SSNa can inhibit the expression of inflammatory?associatied genes in hypertrophied 3T3?L1 adipocytes and is a potent inhibitor of NF??B activation. Thus these results suggest that SSNa is a novel therapeutic agent against that can be used against obesity?associated inflammation. PMID:25672367

  16. Assessing the impact of As-Cd-Pb metal mixture on cell transformation by two-stage Balb/c 3T3 cell assay.

    PubMed

    Rodríguez-Sastre, M A; Rojas, E; Valverde, M

    2014-07-01

    Human beings are exposed to metals as a consequence of various industrial activities, including glass production, agrochemical production, metallurgy and battery manufacture. New data about the possible mechanisms involved in the carcinogenic activity of these metals are constantly being reported. Exposure to complex mixtures of metals is more likely to occur than exposure to a single metal alone. Among these elements, arsenic, cadmium and lead are ubiquitous air and water pollutants that continue to threaten the quality of public health around the world. The aim of the present study was to evaluate the capability of a mixture of 2 µM NaAsO2, 2 µM CdCl2 and 5 µM Pb(C2H3O2)2·3H2O at relevant epidemiological concentrations to induce cell transformation processes. Transforming potential was determined by a murine two-stage Balb/c 3T3 cell assay. Cell viability, reactive oxygen species (ROS), DNA damage, cell cycle analysis, senescence, generation time and metallothionein expression were also evaluated. The results showed that the metal mixture induced morphological cell transformation only when acting as initiator stimuli of the process. A decrease in cell viability was observed at the promotion stage, a time during which ROS increase, especially when a metal mixture was applied as a promoter stimulant. Changes in DNA damage were not observed throughout the assay; however, we observed G1 cell cycle arrest. The metal mixture, acting as a promoter, is capable of inducing senescence, but metals employed as initiators with 12-O-tetradecanoylphorbol-13-acetate as a promoter are capable of causing avoidance of senescence and triggering the transformation potential of the cells. PMID:24782466

  17. A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK 1/2 pathway

    NASA Technical Reports Server (NTRS)

    Hatton, Jason P.; Pooran, Milad; Li, Chai-Fei; Luzzio, Chris; Hughes-Fulford, Millie

    2003-01-01

    Physiological mechanical loading is crucial for maintenance of bone integrity and architecture. We have calculated the strain caused by gravity stress on osteoblasts and found that 4-30g corresponds to physiological levels of 40-300 microstrain. Short-term gravity loading (15 minutes) induced a 15-fold increase in expression of growth-related immediate early gene c-fos, a 5-fold increase in egr-1, and a 3-fold increase in autocrine bFGF. The non-growth-related genes EP-1, TGF-beta, and 18s were unaffected by gravity loading. Short-term physiological loading induced extracellular signal-regulated kinase (ERK 1/2) phosphorylation in a dose-dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12g (p < 0.001) with no effect on total ERK. The phosphorylation of focal adhesion kinase (FAK) was unaffected by mechanical force. g-Loading did not activate P38 MAPK or c-jun N-terminal kinase (JNK). Additionally, a gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus; this did not occur in unloaded cells. The induction of c-fos was inhibited 74% by the MEK1/2 inhibitor U0126 (p < 0.001) but was not affected by MEK1 or p38 MAPK-specific inhibitors. The long-term consequence of a single 15-minute gravity pulse was a 64% increase in cell growth (p < 0.001). U0126 significantly inhibited gravity-induced growth by 50% (p < 0.001). These studies suggest that short periods of physiological mechanical stress induce immediate early gene expression and growth in MC3T3-E1 osteoblasts primarily through an ERK 1/2-mediated pathway.

  18. A magnetic flux leakage NDE system for CANDU feeder pipes

    NASA Astrophysics Data System (ADS)

    Mak, Thomas Don

    This work examines the application of different magnetic flux leakage (MFL) inspection concepts to the non destructive evaluation (NDE) of residual (elastic) stresses in CANDURTM reactor feeder pipes. The stress sensitivity of three MFL inspection techniques was examined with flat plate samples, with stress-induced magnetic anisotropy (SMA) demonstrating the greatest stress sensitivity. A prototype SMA testing system was developed to apply magnetic NDE to feeders. The system consists of a flux controller that incorporates feedback from a wire coil and a Hall sensor (FCV2), and a magnetic anisotropy prototype (MAP) probe. The combination of FCV2 and the MAP probe was shown to provide SMA measurements on feeder pipe samples and predict stresses from SMA measurements with a mean accuracy of +/-38MPa.

  19. An automatic 14-day paste diet feeder for animals

    NASA Technical Reports Server (NTRS)

    Vasques, Marilyn; Mulenburg, Jerry; Gundo, Dan; Griffith, Jon

    1994-01-01

    During a centrifuge experiment, any interruption that requires stopping the centrifuge may influence the results. Centrifuges often must be stopped for animal maintenance (food, water and waste removal), especially in cases of timed feedings. To eliminate the need for stopping the centrifuge while still providing timed feeding, an automatic paste diet feeder was developed. The feeder is based on a constant volume concept and can deliver a predetermined amount of paste diet at specified time intervals. This unit was supported by water delivery and waste collection systems. The entire system performed reliably and maintained the animals well for a continuous centrifugation experiment of 14 days.

  20. Selection of a biopolymer based on attachment, morphology and proliferation of fibroblast NIH\\/3T3 cells for the development of a biodegradable tissue regeneration template: Alginate, bacterial cellulose and gelatin

    Microsoft Academic Search

    Nitar Nwe; Tetsuya Furuike; Hiroshi Tamura

    2010-01-01

    Material selection is one of the most important aspects to construct a novel template for tissue regeneration. In this research alginate, bacterial cellulose (BC) and gelatin were selected based on available material and published data to prepare membranes and then the morphology of Swiss mouse embryo fibroblast NIH\\/3T3 cells on the surface of these membranes was examined to select the

  1. Cell-free Embryonic Stem Cell Extract-Mediated Derivation of Multi-potent Stem Cells from NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair

    PubMed Central

    Rajasingh, Johnson; Lambers, Erin; Hamada, Hiromichi; Bord, Evelyn; Thorne, Tina; Goukassian, Ilona; Krishnamurthy, Prasanna; Rosen, Kenneth M.; Ahluwalia, Deepali; Zhu, Yan; Qin, Gangjian; Losordo, Douglas W.; Kishore, Raj

    2008-01-01

    The oocyte-independent source for the generation of pluripotent stem cells is one of the ultimate goals in regenerative medicine. We report that upon exposure to mouse ES cell (ESC) extracts, reversibly permeabilized NIH3T3 cells undergo de-differentiation followed by stimulus-induced re-differentiation into multiple lineage cell types. Genome-wide expression profiling revealed significant differences between NIH3T3 control and ESC extract treated NIH3T3 cells including the re-activation of ESC specific transcripts. Epigenetically, ESC extracts induced CpG de-methylation of Oct4 promoter, hyper-acetylation of histones 3 and 4 and decreased lysine 9 (K-9) dimethylation of histone 3. In mouse models of surgically-induced hind limb ischemia (HLI) or acute myocardial infarction (AMI) transplantation of reprogrammed NIH3T3 cells significantly improv