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Comparative analysis of human-derived feeder layers with 3T3 fibroblasts for the ex vivo expansion of human limbal and oral epithelium.  


Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and I?1 and p63 (putative stem cell markers) in all culture conditions. The level of I?1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents. PMID:21964568

Sharma, Sandhya M; Fuchsluger, Thomas; Ahmad, Sajjad; Katikireddy, Kishore R; Armant, Myriam; Dana, Reza; Jurkunas, Ula V



Modulation of Keratin and Connexin Expression in Limbal Epithelium Expanded on Denuded Amniotic Membrane with and without a 3T3 Fibroblast Feeder Layer  

Microsoft Academic Search

PURPOSE. Based on the knowledge that limbal epithelial stem cells (SCs) do not express keratin-3 (K3), connexin (Cx)43, and Cx50, a study was conducted to investigate amniotic mem- brane (AM) culturing conditions that promote limbal SC ex- pansion. METHODS. Human limbal epithelium was expanded on intact and epithelially denuded AM, with or without a 3T3 feeder layer, and subsequently transplanted

Martin Grueterich; Edgar M. Espana; Scheffer C. G. Tseng



Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration  

PubMed Central

There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5–14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1×104 in a 35-mm dish (9.6 cm2) grew to confluence (about 1.87–2.41×106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin ?1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.

Lu, Rong; Bian, Fang; Lin, Jing; Su, Zhitao; Qu, Yangluowa; Pflugfelder, Stephen C.; Li, De-Quan



Profiling of extracellular matrix and cadherin family gene expression in mouse feeder layer cells: type VI collagen is a candidate molecule inducing the colony formation of epithelial cells.  


Mouse 3T3 feeder layer has been utilized for epidermal and corneal epithelial cell culture to promote tissue-like cell stratification. However, the molecular mechanism underlying epithelial-feeder layer interactions remains poorly understood. Here, the feeder layer activity of six different mouse cell lines was examined in terms of the colony-forming efficiency (CFE) of primary limbal epithelial cells, including corneal epithelial stem/progenitor cells. When epithelial cells and feeder layers were separated by culture inserts, the CFE was significantly lower than that of epithelial cells, which were cultured with feeder cells on the same dish surfaces, implying that direct contacts between these cells and/or pericellular extracellular matrix (ECM) deposition by feeder layers have an important role in feeder layer activity. With TaqMan polymerase chain reaction assay, the gene expression of 29 ECM molecules and 32 cadherin family genes was profiled in two highest and two lowest cell lines in the CFE for limbal and oral mucosal epithelial cells. A significant difference in the expression correlated with the CFE was observed in six ECM molecules and four kinds of cadherin family genes. In these results, type VI collagen was confirmed to be able to promote the colony formation of epithelial cells in vitro effectively. PMID:22784000

Takagi, Ryo; Yamato, Masayuki; Kushida, Ai; Nishida, Kohji; Okano, Teruo



Evaluation of unrestricted somatic stem cells as a feeder layer to support undifferentiated embryonic stem cells.  


The use of unrestricted somatic stem cells (USSCs) holds great promise for future clinical applications. Conventionally, mouse embryonic fibroblasts (MEFs) or other animal-based feeder layers are used to support embryonic stem cell (ESC) growth; the use of such feeder cells increases the risk of retroviral and other pathogenic infection in clinical trials. Implementation of a human-based feeder layer, such as hUSSCs that are isolated from human sources, lowers such risks. Isolated cord blood USSCs derived from various donors were used as a novel, supportive feeder layer for growth of C4mES cells (Royan C4 ESCs). Complete cellular characterization using immunocytochemical and flow cytometric methods were performed on murine ESCs (mESCs) and hUSSCs. mESCs cultured on hUSSCs showed similar cellular morphology and presented the same cell markers of undifferentiated mESC as would have been observed in mESCs grown on MEFs. Our data revealed these cells had negative expression of Stat3, Sox2, and Fgf4 genes while showing positive expression for Pou5f1, Nanog, Rex1, Brachyury, Lif, Lifr, Tert, B2m, and Bmp4 genes. Moreover, mESCs cultured on hUSSCs exhibited proven differentiation potential to germ cell layers showing normal karyotype. The major advantage of hUSSCs is their ability to be continuously cultured for at least 50 passages. We have also found that hUSSCs have the potential to provide ESC support from the early moments of isolation. Further study of hUSSC as a novel human feeder layer may lead to their incorporation into clinical methods, making them a vital part of the application of human ESCs in clinical cell therapy. PMID:22888050

Keshel, Saeed Heidari; Soleimani, Masoud; Tavirani, Mostafa Rezaei; Ebrahimi, Maryam; Raeisossadati, Reza; Yasaei, Hemad; Afsharzadeh, Danial; Behroz, Mahmoud Jabbarvand; Atashi, Amir; Amanpour, Saeid; Khoshzaban, Ahad; Roozafzoon, Reza; Behrouzi, Gholam Reza



Transgenic human fetal fibroblasts as feeder layer for human embryonic stem cell lineage selection.  


Successful gene targeting in human embryonic stem (hES) cells requires the use of primary fibroblast feeder layers, which assist in the maintenance of the pluripotent state of hES cells. Such feeder layers must also survive any further selection strategy for hES cells. Here we report the production of a novel transgenic human fetal fibroblast (tHFF) as a feeder layer that is resistant to puromycin and can be used for gene targeting and selection of positive clones in hES cells. tHFFs survive under a wide range of puromycin concentrations (0.5-2 microg/ml) and also supports the undifferentiated growth of hES cells. We have demonstrated here that tHFFs are suitable for selecting Envy-hES cells that were transfected with a green fluorescent protein-small interfering RNA (GFP-siRNA) plasmid construct to induce GFP gene down-regulation. The later studies were designed to isolate and propagate stably knockdown cells. tHFFs thus can be used for targeting other genes that would serve as a model to select and understand the differentiation process in hES cells. PMID:17105409

Sidhu, Kuldip S; Lie, Khun Hong D; Tuch, Bernard E



The 3T3-L1 adipocyte glycogen proteome  

PubMed Central

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.



Bird Feeders  

NSDL National Science Digital Library

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.

Science, Lawrence H.



Resistin promotes 3T3-L1 preadipocyte differentiation  

Microsoft Academic Search

Objective: To investigate the relationship between resistin (a potential link between obesity and type 2 diabetes) and preadipocyte differentiation. Design: A rat resistin expression vector was transfected into 3T3-L1 preadipocytes and differentiation was compared between normal 3T3-L1 cells, rat resistin-transfected cells and non-transfected cells grown in conditioned medium taken from resistin-expressing cultures. Methods: The rat resistin gene was inserted into

Haixia Gong; Yuhui Ni; Xirong Guo; L i Fei; Xiaoqin Pan; Mei Guo; Ronghua Chen



Amniocytes can serve a dual function as a source of iPS cells and feeder layers  

PubMed Central

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5–7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4+ or c-Kit+ amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.

Anchan, Raymond M.; Quaas, Philipp; Gerami-Naini, Behzad; Bartake, Hrishikesh; Griffin, Adam; Zhou, Yilan; Day, Daniel; Eaton, Jennifer L.; George, Liji L.; Naber, Catherine; Turbe-Doan, Annick; Park, Peter J.; Hornstein, Mark D.; Maas, Richard L.



Phosphatidylcholine induces apoptosis of 3T3-L1 adipocytes  

PubMed Central

Background Phosphatidylcholine (PPC) formulation is used for lipolytic injection, even though its mechanism of action is not well understood. Methods The viability of 3T3-L1 pre-adipocytes and differentiated 3T3-L1 cells was measured after treatment of PPC alone, its vehicle sodium deoxycholate (SD), and a PPC formulation. Western blot analysis was performed to examine PPC-induced signaling pathways. Results PPC, SD, and PPC formulation significantly decreased 3T3-L1 cell viability in a concentration-dependent manner. PPC alone was not cytotoxic to CCD-25Sk human fibroblasts at concentrations <1 mg/ml, whereas SD and PPC formulation were cytotoxic. Western blot analysis demonstrated that PPC alone led to the phosphorylation of the stress signaling proteins, such as p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, and activated caspase-9, -8, -3 as well as cleavage of poly(ADP-ribose) polymerase. However, SD did not activate the apoptotic pathways. Instead, SD and PPC formulation induced cell membrane lysis, which may lead to necrosis of cells. Conclusions PPC results in apoptosis of 3T3-L1 cells.



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


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

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



Midkine induces the transformation of NIH3T3 cells.  

PubMed Central

Midkine (MK) is a heparin-binding growth factor and is frequently expressed at high levels in many human carcinomas. To investigate further the roles of MK in the regulation of cell growth, we introduced MK expression in NIH3T3 cells. A mixture of transfectants of an MK expression vector, but not a control vector, formed colonies in soft agar, showed an elevated cell number at confluence, and formed tumours in nude mice. An interesting characteristic of the transformed cells was that they became spontaneously detached from the culture dish substratum. In the transformed cells, MK was not only secreted, but also localized, in the perinuclear region as spots. The present data indicate that MK has the potential to transform NIH3T3 cells and suggest that overexpression of the MK gene may promote unregulated cell growth in vivo. Images Figure 2 Figure 3 Figure 4

Kadomatsu, K.; Hagihara, M.; Akhter, S.; Fan, Q. W.; Muramatsu, H.; Muramatsu, T.



Bird Feeder  

NSDL National Science Digital Library

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.

Science, Lawrence H.



Methotrexate enhances 3T3-L1 adipocytes hypertrophy.  


Methotrexate (MTX) is broadly used in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). The prevalence of metabolic syndrome (MeS) in patients with this condition is relatively high. Given the importance of adipose tissue in the development of obesity metabolic complications, this study aimed to investigate the effect of methotrexate on preadipocyte proliferation, adipogenesis, and glucose uptake by adipocytes. 3T3-L1 preadipocytes proliferation was evaluated by sulforhodamine B staining and (3)H-thymidine incorporation, after 24 or 48 h of treatment with MTX (0.1 and 10 ?M). Preadipocytes were induced to differentiate with an appropriate adipogenic cocktail in the presence or absence of MTX. Adipogenesis was determined by measuring lipid accumulation after staining with oil red O. (3)H-Deoxyglucose ((3)H-DG) uptake was determined by liquid scintillation counting. MTX treatment reduced culture protein content in a concentration-dependent manner and (3)H-thymidine incorporation (P?

Marques, Cláudia; Teixeira, Diana; Cunha, Ana; Meireles, Manuela; Pestana, Diogo; Keating, Elisa; Calhau, Conceiçăo; Monteiro, Rosário; Faria, Ana



Fluoroaluminate Induces Pertussis Toxin-Sensitive Protein Phosphorylation: Differences in MC3T3-E1 Osteoblastic and NIH3T3 Fibroblastic Cells  

Microsoft Academic Search

Fluoride is an acknowledged bone-forming agent that may act through stimulation of osteoblast proliferation. Fluoride's action on osteoblasts and bone is potentiated by aluminum, which can form a complex with fluoride (fluoroaluminate) and activate heterotrimeric G proteins. Here we examined signaling pathways activated by fluoroaluminate in MC3T3-E1 osteoblastic and in NIH3T3 fibroblastic cells. In MC3T3-E1 cells, fluoroaluminate induced a decrease

Mira Šuša; Gesche J. R. Standke; Margit Jeschke; Daisy Rohner



Feeder layer-free in vitro assay for screening antitrypanosomal compounds against Trypanosoma brucei brucei and T. b. evansi.  

PubMed Central

A drug-susceptible Trypanosoma brucei brucei stock, a multidrug-resistant T. b. brucei stock, and a T. b. evansi stock resistant to two commercial trypanocides were adapted to a feeder layer-free culture system. Bloodstream forms were grown continuously in a liquid medium at 37 degrees C in 4% CO2 in air. Samples of trypanosome populations in the logarithmic growth phase were incubated with various concentrations of commercial and experimental compounds. Growth inhibition was monitored after a 24-h incubation and quantified by comparing the number of generations between control and drug-treated cultures. Some of the experimental compounds [taxol, formicin B, thioridazine, Ro 15-0216, and DL-alpha-(difluoromethyl)ornithine hydrochloride monohydrate] showed activity against both drug-susceptible and drug-resistant trypanosomes. Other compounds [sinefungin, 1,3,5-triacetylbenzene tris(guanylhydrazone)trimethanesulfonate hydrate, and 9-deazainosine] which inhibited the growth of drug-susceptible trypanosomes showed little or no effect upon drug-resistant parasites. Gossypol, however, had no antitrypanosomal effect on either trypanosome stock. The results obtained in this study correlate with observations obtained from drug screening in mice. The main advantages of the described in vitro screening assay are as follows: (i) lower amounts of drugs are required, (ii) results are obtained more rapidly, (iii) animals are not necessary, and (iv) the method is less labor intensive. These advantages result in an economical and rapid assay for primary drug screening.

Kaminsky, R; Zweygarth, E



Mesenchymal stem cells feeder layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells  

Microsoft Academic Search

Ex vivo expansion of hematopoietic stem cells was suggested as the best way of overcoming problems caused by limited hematopoietic\\u000a cell number for cord blood transplantation. In this study, we quantified and characterized an ex vivo expansion capacity of\\u000a umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) as a cell feeder layer for support of UCB-derived committed\\u000a hematopoietic progenitor cells

Yun Kyung Jang; Dai Hyun Jung; Mee Hyun Jung; Dong Hyun Kim; Keon Hee Yoo; Ki Woong Sung; Hong Hoe Koo; Wonil Oh; Yoon Sun Yang; Sung-Eun Yang



Triiodothyronine inhibits multilayer formation of the osteoblastic cell line, MC3T3-E1, by promoting apoptosis  

Microsoft Academic Search

Cell death through apoptosis is a well-known mechanism for maintaining homoeostasis in many developmental and pathological processes. We have recently presented evi- dence for the occurrence of apoptosis during the formation of bone-like tissue in vitro. MC3T3-E1 osteoblast-like cells in culture develop features of the osteoblastic phenotype and form many cell layers embedded in extracellular matrix which can mineralise. Tri-iodothyronine

F Varga; E Luegmayr; N Fratzl-Zelman; H Glantschnig; A Ellinger; D Prinz; M Rumpler; K Klaushofer



Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth.  

PubMed Central

Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an insufficient number of stromal precursor cells in HUCB to form a hematopoietic-supporting feeder layer in primary cultures. The present study shows that HUCB does contain a significant accessory cell population that routinely develops into a confluent, adherent cell layer under defined primary culture conditions. HUCB-derived adherent layers were shown to support long-term hematopoietic activity for an average of 4 months. This was achieved by using a customized coverslip with a modified surface structure as the cell attachment substratum and using a specialized culture feeding regime. We have characterized the various cell types (including fibroblasts, macrophages, and endothelial cells) and extracellular matrix proteins (including fibronectin, collagen III, and laminin) that were present in abundance in the HUCB-derived adherent cell layer. In contrast, oil red O-staining fat cells were rarely detected. ELISA and bioassays showed that stem cell factor and interleukin 6 were produced by the HUCB stromal cell cultures, but interleukin 3 or granulocyte/macrophage colony-stimulating factor was not detected. Application of this hematopoietic culture system to transgenic and gene therapy studies of stem cells is discussed. Images

Ye, Z Q; Burkholder, J K; Qiu, P; Schultz, J C; Shahidi, N T; Yang, N S



Regulation of Na+-H+ exchange in normal NIH-3T3 cells and in NIH-3T3 cells expressing the ras oncogene  

SciTech Connect

Our laboratory and others have demonstrated that Na+-H+ exchange can be regulated by two different pathways; one that is mediated by an inositol trisphosphate-stimulated increase in intracellular calcium activity, and one that is mediated by an increase in protein kinase C activity. To determine whether one of these pathways is more important than the other, or whether one pathway is physiologically relevant, we employed normal NIH-3T3 cells (3T3 cells) and NIH-3T3 cells expressing the EJ human bladder ras oncogene (EJ cells). The EJ cells were chosen because they provide a genetic model that does not exhibit serum- or platelet-derived growth factor (PDGF)-stimulated inositol trisphosphate release or Ca2+ mobilization. It was found that serum- or PDGF-stimulated Na+-H+ exchange was more pronounced in EJ cells than in control 3T3 cells. As expected, serum- or PDGF-stimulated Na+-H+ exchange in 3T3 cells was inhibited by chelating intracellular Ca2+ with the intracellular Ca2+ chelator quin2, by the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and by the calmodulin antagonist trifluoperazine. In contrast, these agents did not inhibit serum- or PDGF-stimulated Na+-H+ exchange in EJ cells. Activators of protein kinase C (e.g., 1-oleoyl-2-acetylglycerol or biologically active phorbol esters) were found to stimulate Na+-H+ exchange in EJ cells to the same extent as serum. However, these agents were considerably less effective than serum in control 3T3 cells. Despite these findings, PDGF did not stimulate diacylglycerol levels in EJ cells.

Owen, N.E.; Knapik, J.; Strebel, F.; Tarpley, W.G.; Gorman, R.R.



Effect of alpha-lipoic acid on 3T3 and 3T3SV40 fibroblasts: Comparison with N-acetylcysteine  

Microsoft Academic Search

In this study, we have investigated the intracellular level of reduced glutathione, cell-cycle phase distribution, and microfilament\\u000a and microtubule structures in normal (3T3) and transformed (3T3-SV40) fibroblasts exposed to alpha-lipoic acid (ALA) in concentrations\\u000a of 0.7–5 mM. It was found that ALA treatment increased the glutathione content in transformed cells, but did not affect its\\u000a level in normal cells; moreover,

E. A. Vakhromova; Yu. S. Polozov; K. M. Kirpichnikova; N. D. Aksenov; I. A. Gamaley



Precision powder feeder  


A new class of precision powder feeders is disclosed. These feeders provide a precision flow of a wide range of powdered materials, while remaining robust against jamming or damage. These feeders can be precisely controlled by feedback mechanisms.

Schlienger, M. Eric (Albuquerque, NM); Schmale, David T. (Albuquerque, NM); Oliver, Michael S. (Sandia Park, NM)



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

Microsoft Academic Search

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,

Kenichi Mizutani; Katsumi Ikeda; Yasuhiro Kawai; Yukio Yamori



Bombesin Stimulation of DNA Synthesis and Cell Division in Cultures of Swiss 3T3 Cells  

Microsoft Academic Search

Bombesin is shown to be a potent mitogen for Swiss 3T3 cells. At nanomolar concentrations the peptide markedly enhances the ability of fresh serum to stimulate DNA synthesis in confluent and quiescent cultures of these cells. In the presence of a low concentration (3.5%) of serum, bombesin stimulates 3T3 cell proliferation. In serum-free medium, bombesin induces DNA synthesis in the

Enrique Rozengurt; James Sinnett-Smith



ADAM15 Overexpression in NIH3T3 Cells Enhances Cell–Cell Interactions  

Microsoft Academic Search

ADAM15 is a member of the family of metalloprotease-disintegrins that have been shown to interact with integrins in an RGD- and non-RGD-dependent manner. In the present study, we examined the effects of ADAM15 overexpression on cell–matrix and cell–cell interactions in NIH3T3 cells. Tetracycline-regulated ADAM15 overexpression in NIH3T3 cells leads to an inhibition of migration on a fibronectin-coated filter in a

Barbara Herren; Kyle J. Garton; Scott Coats; Daniel F. Bowen-Pope; Russell Ross; Elaine W. Raines



The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells  

Microsoft Academic Search

AIM The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in

Meng Sen LI; Ping Feng LI; Fei Yi YANG; Shi Peng HE; Guo Guang DU; Gang LI



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

Microsoft Academic Search

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,

Ming?Chung Jiang



Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.  


Differentiating mouse 3T3-L1 preadipocytes have been used as a model system to study the ability of type beta transforming growth factor (TGF-beta) to modulate cell development. We find that TGF-beta inhibits potently (ID50 approximately equal to 25 pM) the adipogenic conversion of 3T3-L1 cells. Inhibition is observed only when cells are exposed to TGF-beta before they become committed to differentiation. Even a transient (4 hr) exposure to TGF-beta immediately before the commitment point is sufficient to prevent differentiation. This point coincides with the time point immediately preceding the onset of coordinate expression of differentiation-specific proteins in 3T3-L1 cells. TGF-beta interacts with cell surface receptors in 3T3-L1 cells that have structural and binding properties similar to TGF-beta receptors in other cell types in which TGF-beta acts as a growth activator or a growth inhibitor. However, TGF-beta does not markedly alter differentiation-related mitosis in 3T3-L1 cells. The action of TGF-beta on 3T3-L1 cells does not involve changes in cAMP or prostaglandin E levels. These results suggest that TGF-beta is a unique modulator of adipogenic differentiation of fibroblasts. PMID:3001708

Ignotz, R A; Massagué, J



Project Feeder Watch  

NSDL National Science Digital Library

Project FeederWatch is a winter-long survey of birds that visit feeders at backyards, nature centers, community areas, and other locales in North America. FeederWatchers periodically count the birds they see at their feeders from November through early April and send their counts to Project FeederWatch. FeederWatch data help scientists track broadscale movements of winter bird populations and long-term trends in bird distribution and abundance. Anyone with an interest in birds can participate. FeederWatch is conducted by people of all skill levels and backgrounds, including children, families, individuals, classrooms, retired persons, youth groups, nature centers, and bird clubs



Detecting effects of low levels of cytochalasin B in 3T3 fibroblast cultures by analysis of electrical noise obtained from cellular micromotion  

Microsoft Academic Search

We performed micromotion experiments using electric cell–substrate impedance sensing (ECIS) on a confluent layer of 3T3 fibroblasts exposed to different low levels of the toxin cytochalasin B. This toxin is know to affect actin polymerization and to disrupt cytoskeletal structure and function in cells, changing the morphology of confluent cell cultures and altering the nature of the cellular micromotion, which

Douglas C. Lovelady; Jennifer Friedman; Sonali Patel; David A. Rabson; Chun-Min Lo



TRPM7 Channels Regulate Proliferation and Adipogenesis in 3T3-L1 Preadipocytes.  


Transient receptor potential melastatin-7 (TRPM7) channels are involved in many cellular physiological and pathological processes. The present study was designed to investigate the expression of TRPM7 channels and the potential role in regulating cell proliferation and adipogenesis in 3T3-L1 preadipocytes with approaches of whole-cell patch voltage-clamp, molecular biology, cell proliferation, adipogenesis, etc. We found that a TRPM7-like current was recorded with Mg(2+) -free pipette solution in 3T3-L1 preadipocytes, and the current was inhibited by intercellular free Mg(2+) . The TRPM7-like current was potentiated by acidic pH and inhibited by 2-aminoethoxydiphenyl borate (2-APB). RT-PCR, Western blot and immunocytochemistry revealed that gene and protein of TRPM7 channels were abundant in 3T3-L1 preadipocytes. Blockade of TRPM7 channels with 2-APB inhibited cell proliferation in 3T3-L1 cells. In addition, knockdown of TRPM7 with specific siRNA inhibited both proliferation and adipogenesis. The present study demonstrates for the first time that TRPM7 channels regulate cell cycle and adipogenesis of 3T3-L1 preadipocytes. J. Cell. Physiol. 229: 60-67, 2014. © 2013 Wiley Periodicals, Inc. PMID:23765921

Chen, Kui-Hao; Xu, Xiao-Hui; Liu, Yi; Hu, Yan; Jin, Man-Wen; Li, Gui-Rong



An Altered Rate of Uridine Transport in Membrane Vesicles Isolated from Growing and Quiescent Mouse 3T3 Fibroblast Cells  

PubMed Central

Balb/c 3T3 and simian virus-40-transformed Balb/c 3T3 (SV-3T3) cells were examined for their ability to transport nucleosides. Confluent (quiescent) 3T3 cells transported uridine at a rate 3-4 fold lower than did subconfluent cells. Adenosine uptake was independent of cell population density. Both adenosine and uridine were transported at the same rate by confluent and sub-confluent SV-3T3 cells. A membrane vesicle population (plasma membrane and endoplasmic reticulum) was isolated from 3T3 and SV-3T3 cells by nitrogen cavitation. With membrane vesicles derived from SV-3T3 cells it was determined that uptake occurred by a mediated process. Uptake of adenosine and uridine by membrane vesicles isolated from 3T3 and SV-3T3 cells demonstrated the same pattern as found in whole cells; that is, membrane vesicles from confluent 3T3 cells transported uridine at a rate 3-fold lower than did membrane vesicles from subconfluent 3T3 cells. Much of the adenosine taken up was converted to inosine, hypoxanthine, and ribose 1-phosphate, whereas uridine transport resulted only in the accumulation of uridine. Results obtained with membrane vesicles indicate that the lowered rate of uridine transport by confluent 3T3 cells seems due to an alteration in the membrane itself or a component(s) thereof, rather than to changes in subsequent cellular metabolic processes.

Quinlan, Dennis C.; Hochstadt, Joy



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


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

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



Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells  

SciTech Connect

Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.

Sutherland, B.M.; Bennett, P.B.; Freeman, A.G.; Moore, S.P.; Strickland, P.T.



11 beta-hydroxysteroid dehydrogenase type 1 promotes differentiation of 3T3-L1 preadipocyte  

Microsoft Academic Search

Aim:To investigate the relationship between 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1), a potential link between obesity and type 2 diabetes, and preadipocyte differentiation.Methods:Mouse 11 beta-HSD1 siRNA plasmids were transfected into 3T3-L1 preadipocytes (a cell line derived from mouse Swiss3T3 cells that were isolated from mouse embryo), for examination of the effect of targeted 11 beta-HSD1 inhibition on differentiation of

Yun Liu; Yan Sun; Ting Zhu; Yu Xie; Jing Yu; Wen-lan Sun; Guo-xian Ding; Gang Hu



Ligand Activation of Overexpressed Epidermal Growth Factor Receptors Transforms NIH 3T3 Mouse Fibroblasts  

NASA Astrophysics Data System (ADS)

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.

Riedel, Heimo; Massoglia, Sharon; Schlessinger, Joseph; Ullrich, Axel



?-cryptoxanthin suppresses the adipogenesis of 3T3-L1 cells via RAR activation.  


We recently reported that the oral intake of ?-cryptoxanthin exerted anti-obesity effects by lowering visceral fat levels. In the present study, we characterized the molecular mechanisms underlying the lipid-lowering effects of ?-cryptoxanthin on 3T3-L1 cells. Consistent with our previous findings, ?-cryptoxanthin rapidly reduced the level of intracellular lipids in 3T3-L1 cells as assessed by Oil red O staining. Using an in vitro nuclear receptor binding assay, we demonstrated the ability of ?-cryptoxanthin to bind to and activate members of the retinoic acid receptor (RAR) family. Accordingly, treatment of cells with LE540, an RAR antagonist, abolished the ?-cryptoxanthin-dependent suppression of 3T3-L1 adipogenesis, suggesting that ?-cryptoxanthin mediates its effects on 3T3-L1 cells via RAR activation. In addition, real-time RT-PCR analysis revealed that ?-cryptoxanthin down-regulates mRNA expression of PPAR?, a key regulator of adipocyte differentiation, and that this inhibition was blocked by LE540 treatment. Taken together, these data indicate that RAR activation contributes to the molecular mechanism by which ?-cryptoxanthin prevents obesity. PMID:22472285

Shirakura, Yoshiyuki; Takayanagi, Katsuhiko; Mukai, Katsuyuki; Tanabe, Hiroki; Inoue, Makoto



Expression of an Exogenous Eukaryotic DNA Methyltransferase Gene Induces Transformation of NIH 3T3 Cells  

Microsoft Academic Search

Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous

Jianjun Wu; Jean-Pierre Issa; James Herman; Douglas E. Bassett Jr.; Barry D. Nelkin; Stephen B. Baylin



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

PubMed Central

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.

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



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


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

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



Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells  

SciTech Connect

The mutant c-fgr protein (p58{sup c-fgr/F523}) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58{sup c-fgr} (p58{sup c-fgr/wt}) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive {alpha}-naphthyl butyrate esterase ({alpha}-NBE), marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive {alpha}-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1{alpha}, 25-dihydroxyvitamin D{sub 3}-treated WEHI-3B cells. Immunoblotting studies with antophosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive {alpha}-NBE and cell transformation by p58{sup c-fgr}.

Inoue, Kazushi; Akiyama, Tetsu; Toyoshima, Kumao (Osaka Univ. (Japan)); Wongsasant, Budsaba (Univ. of Tokyo (Japan))



Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen  

SciTech Connect

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10/sup 6/ cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.

Noda, T.; Satake, M.; Robins, T.; Ito, Y.



Proteomic analysis of rosiglitazone and guggulsterone treated 3T3-L1 preadipocytes.  


Adipogenesis is the differentiation of preadipocytes to adipocytes which is marked by the accumulation of lipid droplets. Adipogenic differentiation of 3T3-L1 cells is achieved by exposing the cells to Insulin, Dexamethasone and IBMX for 5-7 days. Thiazolidinedione drugs, like rosiglitazone are potent insulin sensitizing agents and have been shown to enhance lipid droplet formation in 3T3-L1 cells, a model cell line for preadipocyte differentiation. Guggulsterone is a natural drug extracted from the gum resin of tree Commiphora mukul. Guggulsterone has been shown to inhibit adipogenesis and induce apoptosis in 3T3-L1 cells. In this study we treated the 3T3-L1 preadipocytes with rosiglitazone and guggulsterone and assessed the protein expression profile using 2D gel electrophoresis-based proteomics to find out differential target proteins of these drugs. The proteins that were identified upon rosiglitazone treatment generally regulate cell proliferation and/or exhibit anti-inflammatory effect which strengthens its differentiation-inducing property. Guggulsterone treatment resulted in the identification of the apoptosis-inducing proteins to be up regulated which rightly is in agreement with the apoptosis-inducing property of guggulsterone in 3T3-L1 cells. Some of the proteins identified in our proteomic screen such as Galectin1, AnnexinA2 & TCTP were further confirmed by Real Time qPCR. Thus, the present study provides a better outlook of proteins being differentially regulated/expressed upon treatment with rosiglitazone and guggulsterone. The detailed study of the differentially expressed proteins identified in this proteomic screen may further provide the better molecular insight into the mode of action of these anti-diabetic drugs rosiglitazone and guggulsterone. PMID:23275126

Pal, Pooja; Kanaujiya, Jitendra K; Lochab, Savita; Tripathi, Shashi B; Sanyal, Sabyasachi; Behre, Gerhard; Trivedi, Arun K



A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells  

PubMed Central

Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions.



Influence of multilayer rhBMP-2 DNA coating on the proliferation and differentiation of MC3T3-E1 cells seeded on roughed titanium surface.  


For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation. PMID:22623077

Jiang, Qiao-Hong; Liu, Li; Shen, Jian-Wei; Peel, Sean; Yang, Guo-Li; Zhao, Shi-Fang; He, Fu-Ming



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

SciTech Connect

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.

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



Hypoxia Increases Serum Amyloid A3 (SAA3) in Differentiated 3T3-L1 Adipocytes.  


Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue. PMID:23605472

de Oliveira, Edson Mendes; Sandri, Silvana; Knebel, Franciele Hinterholz; Contesini, Caroline Garcia Iglesias; Campa, Ana; Filippin-Monteiro, Fabíola Branco



Ligand Activation of Overexpressed Epidermal Growth Factor Receptors Transforms NIH 3T3 Mouse Fibroblasts  

Microsoft Academic Search

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human

Heimo Riedel; Sharon Massoglia; Joseph Schlessinger; Axel Ullrich



Heat Shock Induces the Release of Fibroblast Growth Factor 1 from NIH 3T3 Cells  

Microsoft Academic Search

Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene

Anthony Jackson; Stanley Friedman; Xi Zhan; Kurt A. Engleka; Reza Forough; Thomas Maciag



Binding Kinetics of Ecotropic (Moloney) Murine Leukemia Retrovirus with NIH 3T3 Cells  

Microsoft Academic Search

A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescenceflow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (66.4) 310 212 Mat4 &C and was three- to sixfold lower at temperatures above 15&C.




Transformation of NIH\\/3T3 Cells by Ornithine Decarboxylase Overexpression1  

Microsoft Academic Search

Ornithine decarboxylase (ODO plays a rate-limiting role in polyamine biosynthesis and is intimately associated with cell proliferation and func tion. Although elevated levels of ODC mRNA, protein, and enzyme activity are consistently detected in transformed cells and tumors, the question remains as to whether ODC gene overexpression has a causative role in tumorigenesis. We have stably transfected MH\\/3T3 fibroblasts with

Jeffrey A. Moshier; Julie Dosescu; Magdalena Skunca; Gordon D. Luk


Transformation of NIH 3T3 cells by microinjection of Haras p21 protein  

Microsoft Academic Search

Alteration in gene structure has been shown to occur in some human tumours1-5. These altered genes, termed oncogenes, were originally identified by their ability to induce foci of transformed cells on transfected mouse 3T3 cultures. The oncogene identified in the EJ\\/T24 human bladder carcinoma is similar to the transforming gene of BALB and Harvey murine sarcoma virus (MSV)6,7 and differs

Dennis W. Stacey; Hsiang-Fu Kung



Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells  

Microsoft Academic Search

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

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



Production and Secretion of Adiponectin from 3T3–L1 Adipocytes: Comparison of Antihypertensive Drugs  

Microsoft Academic Search

BackgroundAdiponectin is an important vascular protective adipocytokine that possesses antidiabetic, antiatherogenic, and anti-inflammatory properties. The aim of this study was to evaluate the effect of various antihypertensive drugs on the production and secretion of adiponectin from adipocytes.Methods3T3–L1 adipocytes were incubated for 6 h with increased doses of the following drugs: hydrochlorothiazide, atenolol, losartan, telmisartan, captopril, and nifedipine. Adiponectin levels, as

Raphaelle Brody; Edna Peleg; Ehud Grossman; Yehonatan Sharabi



Modification of transplasma membrane oxidoreduction by SV40 transformation of 3T3 cells  

Microsoft Academic Search

Transformation of 3T3 cells by SV40 virus changes the properties of the transplasma membrane electron transport activity which can be assayed by reduction of external ferric salts. After 42 h of culture and before the growth rate is maximum, the transformed cells have a much slower rate of ferric reduction. The change in activity is expressed both by change inKm

Hans Löw; Frederick L. Crane; Carin Grebing; Monica Isaksson; Annika Lindgren; Iris L. Sun



Genistein directly inhibits GLUT4-mediated glucose uptake in 3T3-L1 adipocytes  

Microsoft Academic Search

The isoflavone-derivative genistein is commonly applied as an inhibitor of tyrosine kinases. In this report we analyze the effect of genistein on insulin-stimulated glucose uptake in 3T3-L1 adipocytes. In these cells insulin-induced glucose uptake is primarily mediated by the GLUT4 glucose transporter. We observed that pre-treatment with genistein did not affect insulin-induced tyrosine kinase activity of the insulin receptor or

Merlijn Bazuine; Peter J. A. van den Broek; J. Antonie Maassen



Intracellular calcium mobilization induces period genes via MAP kinase pathways in NIH3T3 cells  

Microsoft Academic Search

Mammalian period genes have a pivotal role in generating circadian rhythms and are rapidly induced by several stimuli in mammalian cells. In the present study, we revealed that treatment with thapsigargin significantly induced transcripts of mouse period 1 and 2 (mPer1 and mPer2) but not mPer3 among circadian related genes in NIH3T3 cells. Thapsigargin-induced mPer1 and mPer2 mRNA expressions took

Kentaro Oh-hashi; Yoshihisa Naruse; Masaki Tanaka



Reactive Oxygen Species Regulate Swelling-induced Taurine Efflux in NIH3T3 Mouse Fibroblasts  

Microsoft Academic Search

NIH3T3 mouse fibroblasts generate reactive oxygen species (ROS) and release taurine following exposure to hypotonic medium and to isotonic medium containing the lipase activator melittin. The swelling-induced taurine release is potentiated by H2O2, the calmodulin antagonist W7, and ATP, but inhibited by the antioxidant butulated hydroxytoluene (BHT), the NAD(P)H oxidase inhibitor diphenylene iodonium (DI), and the iPLA2 inhibitor bromoenol lactone

I. H. Lambert



Elevation of apoptotic potential by anoxia-hyperoxia shift in NIH3T3 cells  

Microsoft Academic Search

Apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathway. In this study, we demonstrated the induction of cellular apoptosis by anoxia-hyperoxia shift, but not by anoxia or hyperoxia alone in NIH3T3 cells. The decrement of ROS by anoxia thus appears to be an essential early event leading to apoptosis. G1 arrest was detected

Yen-Chou Chen; Shu-Huei Tsai; Shoei-Yn Lin-Shiau



Tumorigenicity and Gene Amplification Potentials of Cyclin D1-Overexpressing NIH3T3 Cells  

Microsoft Academic Search

Cyclin D1 is a key regulator of the G1-S transition in cell cycle, and its gene is amplified and overexpressed in many cancers. To address the gene amplification potential of the cells in which the cyclin D1 gene expression is deregulated, we have established NIH3T3 clones with various levels of cyclin D1 transgene message. Those transfectants showed anchorage independent growth

K. Asano; H. Sakamoto; H. Sasaki; T. Ochiya; T. Yoshida; Y. Ohishi; T. Machida; T. Kakizoe; T. Sugimura; M. Terada



Nanog transforms NIH3T3 cells and targets cell-type restricted genes  

Microsoft Academic Search

The transcription factor Nanog is uniquely expressed in embryonic stem (ES) cells 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 and a transformed phenotype as demonstrated by foci formation and colony growth in soft agar.

Dan Piestun; Bose S. Kochupurakkal; Jasmine Jacob-Hirsch; Sharon Zeligson; Mark Koudritsky; Eytan Domany; Ninette Amariglio; Gideon Rechavi; David Givol



in BALB\\/c 3T3 and CHO cell lines  

Microsoft Academic Search

The cytotoxicity of extracts (water, Ham's F 12 medium and water\\/ethanol) of the animal feed additive Pharmastim® 8% premix, containing flavophospholipol (Moenomycin, Bambermycin) as an active sub- stance has been studied in in vitro. The continuous cell lines BALB\\/c 3T3 clone 31 and CHO, of mouse and hamster origin, respectively, were used. The cytotoxic effect was measured by the cell



Effect of insulin detemir, compared to human insulin, on 3T3-L1 adipogenesis  

Microsoft Academic Search

Insulin detemir (DET) represents a myristic acid (MA)-coupled insulin derivative with protracted action due to reversible albumin binding. As compared to human insulin (HI), DET provokes no or only minor body weight gain in vivo. Therefore, we compared DET's and HI's adipogenic effects. 3T3-L1 preadipocytes were differentiated with 5 nmol\\/l HI, 5 nmol\\/l DET (=DETequimolar), or 20 nmol\\/l DET (=DETequipotent; equipotent in terms

Anja Böhm; Harald Staiger; Anita M. Hennige; Carina Haas; Fausto Machicao; Hans-Ulrich Häring



Sunflower seed extract enhances the differentiation of osteoblastic MC3T3-E1 cells  

Microsoft Academic Search

Osteoporosis is recognised as one of the major hormonal deficiency diseases, especially in menopausal women and the elderly. The present study investigated whether treatment with sunflower (Helianthus annuus L.) seed extract (SSE) may affect the function of MC3T3-E1 osteogenic cells. In order to determine the growth and differentiation of osteoblast, MTT (3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay, alkaline phosphatase (ALP) activity, collagen

Eun Mi Choi



The effect of undernutrition on circadian genes and rhythmic induction in NIH3T3 cells  

Microsoft Academic Search

Nutrition is essential for health, and it has been widely studied and demonstrated to be related with circadian rhythm. To verify the effect of nutrient deficiency on the in vitro cultured cells, we had NIH3T3 cells cultured in diluted medium for 5 days, and detected the expression levels of Bmal1, Clock and Cry1 on each day. Phorbol 12-myristate 13-acetate (1PMA) was

Shuting Cheng; Wang Hou; Shiping Li; Shuhong Yang; Yanyou Liu; Zhou Jiang; Yuhui Wang; Jing Xiao; Huiling Guo; Zhengrong Wang



Stimulatory effect of daidzein in osteoblastic MC3T3-E1 cells  

Microsoft Academic Search

Daidzein is a natural isoflavone found in Leguminosae. The effect of daidzein on osteoblastic MC3T3-E1 cells was investigated. Cells were cultured in a serum-free medium for 48 hr in the presence of daidzein (10?7–10?5 M). Daidzein (10?6 and 10?5 M) caused a significant elevation of protein content, alkaline phosphatase activity, and DNA content in cells; those increases were about 1.4-,

Emi Sugimoto; Masayoshi Yamaguchi



Corticosteroids as long-term regulators of the insulin effectiveness in mouse 3T3 adipocytes  

Microsoft Academic Search

Summary  Since corticosteroid treatment is often accompanied by insulin resistance, we explored the role of corticosteroids in the regulation of the insulin effectiveness in cultured 3T3 (mouse) adipocytes. Exposure of the fat cells to dexamethasone or corticosterone (0–5 days) induced a time-, concentration-, and protein synthesis-dependent and reversible decrease in insulin binding and in basal and insulin-stimulated 2-deoxyglucose uptake. The decrease

J. P. M. van Putten; Tj. Wieringa; H. M. J. Krans



Hindered Diffusion of Inert Tracer Particles in the Cytoplasm of Mouse 3T3 Cells  

Microsoft Academic Search

Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size-fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent

Katherine Luby-Phelps; Philip E. Castle; D. Lansing Taylor; Frederick Lanni



Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3-L1 preadipocytes  

Microsoft Academic Search

Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation\\u000a and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either\\u000a albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt.\\u000a 1), cells were cultured in media containing a crude

M. Evans; C. Geigerman; J. Cook; L. Curtis; B. Kuebler; M. McIntosh



Nuclear localization and signalling activity of phosphoinositidase Cbeta in Swiss 3T3 cells  

Microsoft Academic Search

THE hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2and phosphatidylinositol 4-phosphate (PtdlnsP) in vitro1and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are

Alberto M. Martelli; R. Stewart Gilmour; Valeria Bertagnolo; Luca M. Neri; Lucia Manzoli; Lucio Cocco



Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells  

Microsoft Academic Search

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

Phimon Atsawasuwan; Yoshiyuki Mochida; Duenpim Parisuthiman; Mitsuo Yamauchi



Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy  

Microsoft Academic Search

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water- soluble cationic dye, diI-C3-(3), fluoresced only as the




Inhibition of Hms Oncogene Transformation of NIH3T3 Cells by Protease Inhibitors1  

Microsoft Academic Search

The protease inhibitors antipain, leupeptin, «,-antitrvpsin,and .-ami- nocaproic acid were found to inhibit transformation of NIH3T3 cells after transfection with an activated H-nu oncogene. Inhibition of focus formation by protease inhibitors was concentration dependent and maximal at 50% of control values. Transfection of a gene for neomycin resistance was not affected by protease inhibitors. Antipain was inactive if present only

Diane D. Currie; Walter Troll


Functional ion channels and cell proliferation in 3T3-L1 preadipocytes.  


Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K(+) current (I(Kir), recorded in 15% of cells) inhibited by Ba(2+), a Ca(2+)-activated intermediate K(+) current (IK(Ca), recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (I(Cl)) inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8 T) insult, implicating a volume-sensitive I(Cl) (I(Cl.vol)). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IK(Ca)), Kir2.1 (for I(Kir)), and Clcn3 (for I(Cl.vol)). Blockade of IK(Ca) with TRAM-34 or I(Cl.vol) with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, I(KCa), I(Cl.vol), and I(Kir), are heterogeneously present in 3T3-L1 preadipocytes. I(KCa) and I(Cl.vol) participate in the regulation of cell proliferation. PMID:21732368

Zhang, Xiao-Hua; Zhang, Ying-Ying; Sun, Hai-Ying; Jin, Man-Wen; Li, Gui-Rong



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


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

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



Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells.  

PubMed Central

Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development.

Mercier, N; Moldes, M; El Hadri, K; Feve, B



Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.  

PubMed Central

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex. Images Fig. 1 Fig. 2 Fig. 3

Yeh, W C; Bierer, B E; McKnight, S L



Exogenous MC3T3 preosteoblasts migrate systemically and mitigate the adverse effects of wear particles.  


Understanding how relevant cell types respond to wear particles will reveal new avenues for treating osteolysis following joint replacements. In this study, we investigate the effects of ultrahigh molecular weight polyethylene (UHMWPE) particles on preosteoblast migration and function. We infused UHMWPE particles or saline into the left femur of mice and injected luciferase-expressing preosteoblasts (MC3T3 cells) into each left ventricle. Bioluminescence imaging (BLI) confirmed systemic administration of MC3T3 cells. BLI throughout the 28-day experiment showed greater MC3T3 migration to the site of particle infusion than to the site of saline infusion, with significant differences on days 0, 4, and 6 (p?0.055). Immunostaining revealed a greater number of osteoblasts and osteoclasts in the particle-infused femora, indicating greater bone turnover. The bone mineralization of the particle-infused femora increased significantly when compared to saline-infused femora (an increase of 146.4±27.9 vs. 12.8±8.7?mg/mL, p=0.008). These results show that infused preosteoblasts can migrate to the site of wear particles. Additionally, as the migrated cells were associated with increased bone mineralization in spite of the presence of particles, increasing osteoblast recruitment is a potential strategy for combating bone loss due to increased osteoclast/macrophage number and decreased osteoblast function. PMID:22741555

Fritton, Kate; Ren, Pei-Gen; Gibon, Emmanuel; Rao, Allison J; Ma, Ting; Biswal, Sandip; Gambhir, Sanjiv S; Goodman, Stuart B



Sciadopitysin protects osteoblast function via its antioxidant activity in MC3T3-E1 cells.  


Age-related osteoblast dysfunction is the main cause of age-related bone loss in both men and women. In the present study, the effect of sciadopitysin, a type of biflavonoids, on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. Sciadopitysin caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, mineralization, and glutathione content in the cells (P<0.05). Sciadopitysin also decreased the production of tumor necrosis factor-a (TNF-?) induced by antimycin A, a mitochondrial electron transport inhibitor. We investigated the protective effects of sciadopitysin on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused a significant reduction in osteoblast dysfunction. However, pretreatment with sciadopitysin prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, adenosine triphosphate (ATP) loss, reactive oxygen species (ROS) release, and nitrotyrosine increase, suggesting that sciadopitysin may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, sciadopitysin increased phosphorylation of cAMP-response element-binding protein (CREB) inhibited by antimycin A. Our results demonstrate that sciadopitysin may reduce or prevent osteoblasts degeneration. PMID:23624148

Suh, Kwang Sik; Lee, Young Soon; Kim, Young Seol; Choi, Eun Mi



Drinking water disinfection byproduct iodoacetic acid induces tumorigenic transformation of NIH3T3 cells.  


Iodoacetic acid (IAA) and iodoform (IF) are unregulated iodinated disinfection byproducts (DBPs) found in drinking water. Their presence in the drinking water of China has not been documented. Recently, the carcinogenic potential of IAA and IF has been a concern because of their mutagenicity in bacteria and genotoxicity in mammalian cells. Therefore, we measured their concentrations in Shanghai drinking water and assessed their cytotoxicity, genotoxicity, and ability to transform NIH3T3 cells to tumorigenic lines. The concentrations of IAA and IF in Shanghai drinking water varied between summer and winter with maximum winter levels of 2.18 ?g/L IAA and 0.86 ?g/L IF. IAA with a lethal concentration 50 (LC50) of 2.77 ?M exhibited more potent cytotoxicity in NIH3T3 cells than IF (LC50 = 83.37 ?M). IAA, but not IF, induced a concentration-dependent DNA damage measured by ?-H2AX staining and increased tail moment in single-cell gel electrophoresis. Neither IAA nor IF increased micronucleus frequency. Prolonged exposure of NIH3T3 cells to IAA increased the frequencies of transformed cells with anchorage-independent growth and agglutination with concanavalin A. IAA-transformed cells formed aggressive fibrosarcomas after inoculation into Balb/c nude mice. This study demonstrated that IAA has a biological activity that is consistent with a carcinogen and human exposure should be of concern. PMID:23641915

Wei, Xiao; Wang, Shu; Zheng, Weiwei; Wang, Xia; Liu, Xiaolin; Jiang, Songhui; Pi, Jingbo; Zheng, Yuxin; He, Gengsheng; Qu, Weidong



Characterization of miR-210 in 3T3-L1 adipogenesis.  


Although accumulating evidences indicate that miRNA emerge as a vital player in cell growth, development, and differentiation, how they contribute to the process of adipocyte differentiation remains elusive. In the present study, we revealed that the expression level of miR-210 was dramatically upregulated during 3T3-L1 adipogenesis. Ectopic introduction of miR-210 into 3T3-L1 cells promoted terminal differentiation as well as the expression of adipogenic markers. MTT assay showed that miR-210 significantly inhibited cell proliferation whereas the BrdU incorporation assay and flow cytometry analysis showed that miR-210 did not impair G1/S phase transition. Further experiments demonstrated that enhanced expression of miR-210 in 3T3-L1 cells provoked adipocyte differentiation via activation of PI3K/Akt pathway by targeting SHIP1, a negative regulator of PI3K/Akt pathway. Moreover, blockade of endogenous miR-210 during adipogenesis significantly repressed adipocyte differentiation. In summary, we have identified miR-210 as an important positive regulator in adipocyte differentiation through the activation of PI3K/Akt pathway. J. Cell. Biochem. 114: 2699-2707, 2013. © 2013 Wiley Periodicals, Inc. PMID:23798503

Liang, Wei-Cheng; Wang, Yan; Wan, David Chi-Cheong; Yeung, Venus Sai-Ying; Waye, Mary Miu-Yee



Resistin affects lipid metabolism during adipocyte maturation of 3T3-L1 cells.  


Resistin, an adipose-tissue-specific secretory factor, aggravates metabolic syndrome through impairment of glucose metabolism. Previously, we demonstrated that resistin expression was induced in both 3T3-L1 cells and primary pre-adipocytes derived from Zucker obese rats during the process of differentiation and maturation (Ikeda Y, Hama S, Kajimoto K, Okuno T, Tsuchiya H & Kogure K (2011) Biol Pharm Bull 34, 865-870). However, the biological function of resistin in adipocytes is poorly understood. In the present study, we examined the effects of resistin knockdown on the biological features of 3T3-L1 cells. We found that lipid content was significantly decreased in 3T3-L1 cells transfected with anti-resistin small interfering RNA (siRNA) after adipocyte differentiation. While expression of peroxisome proliferator activated receptor ? and CCAAT/enhancer-binding protein ? was not affected, protein expression and transcriptional activity levels of carbohydrate response element binding protein (ChREBP), which upregulates transcription of lipogenic genes, decreased after anti-resistin siRNA treatment. Moreover, gene expression of fatty acid synthase and acetyl-CoA carboxylase 2, which are known to be regulated by ChREBP, were also suppressed by resistin knockdown. In contrast, activity of the fatty acid ?-oxidation-regulating protein carnitine palmitoyltransferase 1 increased. These results suggest that resistin knockdown induces suppression of lipid production and activation of fatty acid ?-oxidation. Consequently, resistin may affect lipid metabolism during adipocyte maturation. PMID:24034627

Ikeda, Yoshito; Tsuchiya, Hiroyuki; Hama, Susumu; Kajimoto, Kazuaki; Kogure, Kentaro



Morphological transformation induced by glass fibers in BALB/c-3T3 cells.  


Studies were conducted to determine whether 1) glass fibers can induce morphological transformation in BALB/c-3T3 cells, 2) the transforming activity of glass fibers is related to fiber size, and 3) transformed cells induced by glass fibers possess neoplastic properties. In the transformation assay, BALB/c-3T3 cells were treated with three different types of glass fibers: Manville code 100 (JM-100, Manville Corp., Denver, CO), Owens-Corning AAA-10 (AAA-10, Owens-Corning Corp., Toledo, OH), and Owens-Corning general building insulation (ISL, Owens-Corning Corp.) fibers. The neoplastic properties were investigated using the soft agar cloning and gene transfection methods. All three different glass fibers were cytotoxic at high concentrations and induced dose-related increases in morphological transformation. The transforming activity was inversely related to fiber size, with AAA-10 showing higher activity than JM-100 and JM-100 showing higher activity than ISL fiber. Transformed cells induced by glass fibers exerted anchorage-independent growth (90%) and DNA transfection-mediated transformation (100%). These results indicate that glass fibers are capable of transforming mammalian (BALB/c-3T3) cells in vitro as a function of their physical properties and that glass fiber-induced transformed cells possess preneoplastic characteristics. PMID:8525469

Gao, H G; Whong, W Z; Jones, W G; Wallace, W E; Ong, T



Effect of Mangiferin and Mahanimbine on Glucose Utilization in 3T3-L1 cells  

PubMed Central

Background: Stem barks of Mangifera indica contain a rich content of mangiferin (xanthone glucoside), whereas Murraya koenigii leaves contain rich sources of mahanimbine (carbazole alkaloid) and used traditionally for the treatment of diabetes. Objective: To investigate the effects of mangiferin (xanthone glucoside) and mahanimbine (carbazole alkaloid) on glucose utilization in 3T3-L1 cells. Materials and Methods: Mangiferin was isolated from stem barks of Mangifera indica and mahanimbine was isolated from Murraya koenigii leaves. These isolated compounds were subjected to MTT assay and glucose utilization test with 3T3-L1 cells. Results: Treatment of the 3T3-L1 cells with mangiferin and mahanimbine increased the glucose utilization in a dose-dependent manner. At a concentration of 1 mM, mangniferin showed 2-fold increase in glucose utilization compared with untreated control. In case of mahanimbine, the observed effect at 1 mM was almost equivalent to positive control (insulin at 1 ?M). Moreover, MTT assay showed that both of these compounds were less toxic at a concentration of 1 mM (nearly 75% cells are viable). Conclusion: The present results indicated that these natural products (mangiferin and mahanimbine) exhibited potential ethnomedical uses in management of diabetes.

Kumar, B Dinesh; Krishnakumar, K; Jaganathan, Saravana Kumar; Mandal, Mahitosh



Fabrication and Properties of Ni-Base Self-Fluxing Alloy Layers by Diode Laser Cladding with Powder Feeder  

NASA Astrophysics Data System (ADS)

Laser cladding has many advantages compared to alternative surface treatment processes. However, the utilization of the laser energy should be improved in order to increase the efficiency of the laser cladding process. The Ni-base self-fusing alloys can produce layers by means of laser processing techniques. The diode laser is more compact and the electro-optical-efficiency is higher about one order of magnitude. This is an advantage in both the small-size manufacturing field and large-size construction out door field. Another advantage is the wavelength. The shorter wavelength output of the diode laser is better absorbed by cladding materials than the light of the YAG and especially the mid-infrared CO2 laser. For laser power exceeding 191W, the Vickers hardness of Ni-base self-fusing alloy layer increases with increasing laser power. The layer clad thickness decreased up to higher scan velocities. The layers formed at laser power of 262W and overlap rate of 66% had a high hardness of HV1177.

Kusuhara, Takayoshi; Morimoto, Junji; Abe, Nobuyuki; Tsukamoto, Masahiro; Ishimatsu, Jun


Detecting Low Levels of Cytochalasin B in 3T3 Fibroblast Cells by Analysis of Electrical Noise Obtained from Cellular Micromotion  

Microsoft Academic Search

We performed several micromotion experiments using the electric cell-substrate impedance sensing (ECIS) apparatus on a confluent layer of 3T3 fibroblast cells exposed to differing, low-level amounts of the toxin cytochalasin B. We previously developed a technique to distinguish cancerous from non-cancerous cultures. ootnotetextD.C. Lovelady et alia, Phys. Rev. E 76, 041908 (2007) Our goal here is to see if the

Douglas Lovelady; David Rabson; Chun-Min Lo



Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells  

SciTech Connect

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.

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



Involvement of matrix metalloproteinases in the adipose conversion of 3T3-L1 preadipocytes.  

PubMed Central

When mouse 3T3-L1 preadipocytes are induced to differentiate into adipocytes, they change from an extended fibroblast-like morphology to a rounded one. This change most likely occurs through extracellular matrix remodelling, a process known to be mediated in part by matrix metalloproteinases (MMPs). In this study, we have shown by semi-quantitative reverse transcriptase-PCR, zymographic and immunoblot analysis that MMP-2, MMP-9 and membrane type 1 (MT1)-MMP are regulated during adipose conversion. To assess the importance of MMPs for adipocytic differentiation we have used MMP-specific inhibitors as well as neutralizing antibodies. Treatment of 3T3-L1 preadipocytes with the broad MMP inhibitor Ilomastat or the more restricted MMP-2 Inhibitor I prevented their differentiation into adipocytes in a dose-dependent manner, as evidenced by absence of triglyceride accumulation. Inhibitor treatment prevented the fibronectin-network degradation, as well as the induction of the genes for peroxisome-proliferator-activated receptor gamma and adipsin, two adipocyte phenotype markers. Inhibitor treatment was effective when applied during the early stages of adipocytic conversion, whereas inhibitor treatment during later stages had little effect. Inhibitor treatment did not inhibit clonal mitotic expansion; nor did it affect the expression pattern of the adipogenic transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) or its nuclear translocation. It did, however, markedly reduce C/EBPbeta DNA-binding capacity. Taken together, these results suggest that MMPs, and notably MMP-2 and MMP-9, may be necessary mediators of adipocytic differentiation of 3T3-L1 cells.

Croissandeau, Gilles; Chretien, Michel; Mbikay, Majambu



Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells.  


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. PMID:16487757

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



Gsalpha signalling suppresses PPARgamma2 generation and inhibits 3T3L1 adipogenesis.  


Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that Gbetagamma signalling may be inhibitory but failed to induce adipogenesis using activated Gsalpha (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPARgamma (PPARG as listed in the HUGO database) by phosphorylation but expression of PPARgamma1 was reduced and PPARgamma2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gsalpha signalling impedes FOXO1 phosphorylation and thus inhibits PPARgamma transcription and the alternative promoter usage required to generate PPARgamma2, the fat-specific transcription factor necessary for adipogenesis. PMID:19460852

Zhang, Lei; Paddon, Carol; Lewis, Mark D; Grennan-Jones, Fiona; Ludgate, Marian



Gs? signalling suppresses PPAR?2 generation and inhibits 3T3L1 adipogenesis  

PubMed Central

Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that G?? signalling may be inhibitory but failed to induce adipogenesis using activated Gs? (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPAR? (PPARG as listed in the HUGO database) by phosphorylation but expression of PPAR?1 was reduced and PPAR?2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gs? signalling impedes FOXO1 phosphorylation and thus inhibits PPAR? transcription and the alternative promoter usage required to generate PPAR?2, the fat-specific transcription factor necessary for adipogenesis.

Zhang, Lei; Paddon, Carol; Lewis, Mark D; Grennan-Jones, Fiona; Ludgate, Marian



Lanthanides inhibit adipogenesis with promotion of cell proliferation in 3T3-L1 preadipocytes.  


Lanthanides are widely used in various fields for industrial, agricultural and medical purposes. They have also been used in Chinese agriculture either as fertilizers in plant production or as performance-enhancers in animal nutrition for many years. In view of their possible application for growth enhancing purposes and new medical applications, detailed information on how lanthanides influence physiological processes in biological systems is indispensable. In the present work, the effects of lanthanides (LaCl3, CeCl3 and GdCl3) on cell proliferation and adipogenesis in 3T3-L1 preadipocytes were evaluated. The results demonstrate that lanthanides inhibit adipogenesis in 3T3-L1 preadipocytes, evidenced by decreased triglyceride content and expression of C/EBP? and PPAR?. Simultaneously, the results show that lanthanides can promote cell proliferation during the different stages of differentiation. Firstly, prior to the addition of differentiation inducers (MDI), all the three types of lanthanides resulted in a significant increase of cell growth. Secondly, during the mitotic clonal expansion (MCE) process, GdCl3, as a representative compound, is able to promote cell-cycle entry into the S phase and levels of cell cycle-regulating proteins. Third, at the late stage of the terminal differentiation, on day 8, in the presence of GdCl3, cells exhibited higher levels of G1/S regulatory proteins and proliferating cell nuclear antigen (PCNA). In addition, GdCl3 caused stronger sustained ERK activation during the differentiation process of 3T3-L1 cells. The present study demonstrates that lanthanides may modulate lipid metabolism by inhibition of adipocyte differentiation. The sustained activation of the ERK pathway might be responsible for their inhibition of differentiation and a possible link between their pro-proliferative ability and inhibition of the differentiation process is indicated. PMID:23612852

Hou, Cong-Cong; Feng, Min; Wang, Kui; Yang, Xiao-Gai



Oxidative stress provokes atherogenic changes in adipokine gene expression in 3T3-L1 adipocytes  

Microsoft Academic Search

Increased oxidative stress has been associated with obesity-related disorders. In this study, we investigated how oxidative stress, in different ways of exposure, regulates gene expression of various adipokines in 3T3-L1 adipocytes. Exposure to 100–500?M H2O2 for 10min, as well as exposure to 5–25mU\\/ml glucose oxidase for 18h, similarly decreased adiponectin, leptin, and resistin mRNAs, and increased plasminogen activator inhibitor-1 mRNA.

Mitsunori Kamigaki; Shinji Sakaue; Ichizo Tsujino; Hiroshi Ohira; Daisuke Ikeda; Naofumi Itoh; Shinji Ishimaru; Yoshinori Ohtsuka; Masaharu Nishimura



Dexamethasone regulates beta adrenergic receptors in 3T3-L1 fibroblasts  

SciTech Connect

3T3-L1 fibroblasts, containing ..beta..-adrenergic receptors (..beta..AR) of predominantly the ..beta../sub 1/AR subtype, express only ..beta../sub 2/AR after 48 hr treatment with 250 nM dexamethasone (dex). A two-fold increase in ..beta..AR number was also observed upon dex treatment. ..beta..AR were assayed in membranes using the radioligand (/sup 125/I)-cyanopindolol and the ..beta../sub 2/AR selective antagonist ICI. The relative potency of agonists in stimulating whole cell adenylate cyclase activity was consistent with the subtypes determined by binding experiments. The ability of compounds to cause the subtype switch correlates with glucocorticoid activity since these compounds were more effective in facilitating the subtype conversion and the increase in ..beta..AR number than mineralocorticoids. Sex steroids and thyroid hormone were ineffective at concentrations up to in causing these changes. RNA and protein synthesis appear to be required for the ..beta..AR subtype switch and increase in ..beta..AR number since these changes were not observed with dex treatment in the presence of actinomycin D or cyclohexamide. This study suggests that glucocorticoids may induce gene activation in 3T3-L1 fibroblasts that results in an increase in ..beta..AR number and a shift in subtype.

Nakada, M.T.; Stadel, J.M.; Poksay, K.S.; Crooke, S.T.



Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells  

SciTech Connect

The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of M{sub r} 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of {sup 32}P{sub i}-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C{sub {gamma}}, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.

Zachary, I.; Gil, J.; Lehmann, W.; Sinnett-Smith, J.; Rozengurt, E. (Imperial Cancer Research Fund, London (England))



Persistent induction of cyclooxygenase in p60 sup v-src -transformed 3T3 fibroblasts  

SciTech Connect

A BALB/c 3T3 cell line infected with the temperature-sensitive Rous sarcoma virus strain LA90 has been used to investigate early, p60{sup v-src}-dependent changes in gene expression (protein synthesis). Giant two-dimensional electrophoresis, which can resolve >3,000 polypeptides from ({sup 35}S)methionine-labeled cell lysates, was used to detect the induction of a p72-74 (72-74 kDa) doublet (pI 7.5) after activation of p60{sup v-src} at 35{degree}C. Antiserum against cyclooxygenase (prostaglandin synthase or prostaglandin endoperoxide synthase) specifically immunoprecipitated the p72-74 doublet. The p72-74 doublet was also induced by platelet-derived growth factor and by phorbol 12-myristate 13-acetate and was elevated in an NIH 3T3 cell line transformed by wild-type src. Activation of p60{sup v-src} caused a persistant increase in p72-74, whereas the effect of the growth factor was transient. These dissimilar kinetics of induction were paralleled by changes in cyclooxygenase activity. Although induction of this enzyme may not be directly involved in transformation, the data support the view that oncogenic transformation may result, not from expression of transformation-specific genes, but from persistent changes in the expression of genes normally induced only transiently during passage from the G{sub 0} stage of the cell cycle.

Han, Jiawen; Sadowski, H.; Young, D.A.; Macara, I.G. (Univ. of Rochester Medical Center, NY (USA))



NMR studies of pH regulation in NIH 3T3 cells  

SciTech Connect

In order to understand the correlation of intracellular pH (pH/sub in/) and mitogenic stimulation, they have undertaken NMR studies of the regulation of the intracellular pH in perfused cultures of 3T3 cells anchored on microcarrier beads. Because these cells have very low levels of intracellular inorganic phosphate under well energized conditions, an added pH indicator is required in this system. The /sup 31/P NMR indicators, deoxyglucose 6P, and methyl phosphonate, have, for different reasons, also proven unsatisfactory. Of a series of phosphono-amino acids studied as possible alternative indicators, the best thus far have proven to be 2-amino-5-phosphonovaleric acid and 2-amino-6 phosphono-hexanoic acid. Their properties include: (a) physiological pKa, 7.1 and 7.5, respectively; (b) sensitivity greater than 1 ppm/1 pH unit; (c) resonant frequency far from the phosphate region; (d) low toxicity. In confluent cultures of 3T3 cells grown on 200 diameter polystyrene beads the pH/sub in/ remains approximately constant at 7.05 as the external pH is varied between 7.0 and 7.8. No pH shift is observed in this system upon addition of 10% FBS to cells previously incubated for 24 hours in 1% FBS. This finding is at variance with previous reports of an intracellular alkalinization following stimulation by serum or other mitogens.

Szwergold, B.S.; Brown, T.R.; Freed, J.J.



The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts  

PubMed Central

Background Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate. Principal Findings We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation. Conclusions Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

Markovic, Jelena; Mora, Nancy J.; Broseta, Ana M.; Gimeno, Amparo; de-la-Concepcion, Noelia; Vina, Jose; Pallardo, Federico V.



Tissue distribution of SNAP-23 and its subcellular localization in 3T3-L1 cells.  


The SNARE hypothesis of vesicular traffic proposes that three proteins, VAMP/synaptobrevin, syntaxin, and SNAP-25, constitute a complex that docks the vesicle at the target membrane. VAMP and syntaxin isoforms have been identified outside the nervous system, and a cDNA to a SNAP-25 related protein, SNAP-23, was recently identified in human lymphocytes. Here we report the generation of isoform-specific antibodies to SNAP-23 cloned from human melanoma cells, and their use in detecting the expression and localization of the endogenous SNAP-23 protein in several tissues and cell lines. SNAP-23 was readily detected in liver, lung, kidney, and spleen, to a lesser extent in muscle and heart, and was almost undetectable in brain. The protein was also abundant in fibroblast, muscle, and fat cell lines, but relatively less enriched in neuroendocrine PC12 cells. SNAP-23 abundance did not change during differentiation of 3T3-L1 fibroblasts into adipocytes. In both, SNAP-23 was membrane-bound and below detectable levels in the cytosolic fraction. Subcellular fractionation of 3T3-L1 adipocytes revealed that the majority of the protein was associated with plasma membranes. These findings support the conclusion that a tripartite SNARE complex exists outside of the nervous system, and suggest that SNAP-23 may play a role in vesicle traffic in most cell types. PMID:9020061

Wong, P P; Daneman, N; Volchuk, A; Lassam, N; Wilson, M C; Klip, A; Trimble, W S



RA induces the neural-like cells generated from epigenetic modified NIH/3T3 cells.  


Recently, differentiated somatic cells had been reprogrammed to pluripotential state in vitro, and various tissue cells had been elicited from those cells. Epigenetic modifications allow differentiated cells to perpetuate the molecular memory needed for the cells to retain their identity. DNA methylation and histone deacetylation are important patterns involved in epigenetic modification, which take critical roles in regulating DNA expression. In this study, we dedifferentiated NIH/3T3 fibroblasts by 5-aza-2-deoxycytidine (5-aza-dC) and Trichstatin A (TSA) combination, and detected gene expression pattern, DNA methylation level, and differentiation potential of reprogrammed cells. As the results, embryonic marker Sox2, klf4, c-Myc and Oct4 were expressed in reprogrammed NIH/3T3 fibroblasts. Total DNA methylation level was significant decreased after the treatment. Moreover, exposure of the reprogrammed cells to all trans-retinoic acid (RA) medium elicited the generation of neuronal class IIIbeta-tubulin-positive, neuron-specific enolase (NSE)-positive, nestin-positive, and neurofilament light chain (NF-L)-positive neural-like cells. PMID:19263240

Zhang, Xi-Mei; Li, Qiu-Ming; Su, Dong-Ju; Wang, Ning; Shan, Zhi-Yan; Jin, Lian-Hong; Lei, Lei



Biglycan Deletion Alters Adiponectin Expression in Murine Adipose Tissue and 3T3-L1 Adipocytes  

PubMed Central

Obesity promotes increased secretion of a number of inflammatory factors from adipose tissue. These factors include cytokines and very lately, extracellular matrix components (ECM). Biglycan, a small leucine rich proteoglycan ECM protein, is up-regulated in obesity and has recently been recognized as a pro-inflammatory molecule. However, it is unknown whether biglycan contributes to adipose tissue dysfunction. In the present study, we characterized biglycan expression in various adipose depots in wild-type mice fed a low fat diet (LFD) or obesity-inducing high fat diet (HFD). High fat feeding induced biglycan mRNA expression in multiple adipose depots. Adiponectin is an adipokine with anti-inflammatory and insulin sensitizing effects. Due to the importance of adiponectin, we examined the effect of biglycan on adiponectin expression. Comparison of adiponectin expression in biglycan knockout (bgn?/0) and wild-type (bgn+/0) reveals higher adiponectin mRNA and protein in epididymal white adipose tissue in bgn?/0 mice, as well higher serum concentration of adiponectin, and lower serum insulin concentration. On the contrary, knockdown of biglycan in 3T3-L1 adipocytes led to decreased expression and secretion of adiponectin. Furthermore, treatment of 3T3-L1 adipocytes with conditioned medium from biglycan treated macrophages resulted in an increase in adiponectin mRNA expression. These data suggest a link between biglycan and adiponectin expression. However, the difference in the pattern of regulation between in vivo and in vitro settings reveals the complexity of this relationship.

Ward, Meliza G.; Ajuwon, Kolapo M.



Up-regulation of VEGF by MC3T3-E1 cells treated with curculigoside.  


Empirical evidence has shown that curculigoside, the main active compound of the traditionally used Chinese herb, Curculigo orchioides (Amaryllidaceae, rhizome), affects bone formation and fracture healing. However, the mechanistic details of these processes remain unclear. Therefore, the effects of curculigoside on immortalized, pre-osteoblastic mouse MC3T3-E1 cells was investigated. Following treatment with curculigoside, MC3T3-E1 cells exhibited an increased rate of proliferation. Higher levels of vascular endothelial growth factor (VEGF), Fms-like tyrosine kinase-1 (Flt-1) and bone morphogenetic protein-2 (BMP-2) were also detected in cell supernatants and cell lysates by ELISA and western blot analysis, respectively. Furthermore, the stimulatory effect of curculigoside was observed at relatively low doses (i.e. 10-100 ?g/mL). In combination, these responses to treatment with curculigoside elucidate mechanistic details underlying the therapeutic effects of Curculigo orchioides on bone, and identifies these molecules as potential targets for the treatment of common metabolic bone diseases. PMID:21394809

Ma, Chengjian; Zhang, Junyu; Fu, Jiehai; Cheng, Linfang; Zhao, Guangshu; Gu, Yaping



Bisphenol A affects glucose transport in mouse 3T3-F442A adipocytes.  


Recently, environmental chemicals have appeared in daily human life, and these chemicals have been incidentally taken in by humans. The serum concentrations of some of these chemicals have been found to be associated with the onset and incidence rate of diabetes mellitus. It has been suggested that one of the environmental chemicals, bisphenol A (BPA), has hormone-like activity. It has also been demonstrated that some hormones affect insulin resistance and fat distribution in the body. To study the effects of these environmental chemicals on glucose metabolism, the effect of BPA on glucose transport in mouse 3T3-F442A adipocytes was investigated. The 3T3-F442A adipocytes were incubated with various concentrations of BPA in a medium. Deoxyglucose uptake assay was performed with and without insulin. Immunoblot analysis was performed with a glucose transporter (GLUT) 4-specific antibody and antiphosphotyrosine antibody. The BPA treatment enhanced basal and insulin-stimulated glucose uptake, and caused an increased amount of GLUT4 protein. Thus, the enhanced glucose uptake resulting from the BPA treatment was at least partially due to the increased amount of GLUT4. Tyrosine phosphorylation of insulin receptor substrate-1 with insulin stimulation was not significantly affected. In conclusion, it was demonstrated that BPA, one of the chemicals that we intake incidentally, affects the glucose transport in adipocytes, and also that the environmental chemicals may be identified as one of the environmental factors that affect diabetes and obesity. PMID:14707028

Sakurai, Kenichi; Kawazuma, Michiru; Adachi, Tetsuya; Harigaya, Toshio; Saito, Yasushi; Hashimoto, Naotake; Mori, Chisato



Phenolic compounds from maté (Ilex paraguariensis) inhibit adipogenesis in 3T3-L1 preadipocytes.  


Leaves of Ilex paraguariensis are used to prepare a tea known as maté which is a common beverage in several South American countries. The ethanol extract was fractionated to identify the compounds responsible for the anti-adipogenic activity in 3T3-L1 cells. Extracts of both fresh and dried maté leaves were subjected to column chromatography using molecular permeation to obtain the saponin (20 % yields) and the polyphenol extracts (40 % yields) from the fresh and dried leaves. The phenolic content was determined using high-performance liquid chromatography analysis and the Folin-Ciocalteau method. Also, maté extracts (50 ?g/ml to 1,000 ?g/ml) did not display citotoxicity using MTT. The polyphenol extract from the dried leaves was the most effective (50 ?g/ml) in the inhibition of triglyceride accumulation in 3T3-L1 adipocytes, and rutin (100 ?g/ml) likely accounted for a large portion of this activity. Additionally, maté extracts had a modulatory effect on the expression of genes related to the adipogenesis as PPAR?2, leptin, TNF-? and C/EBP?. PMID:22544347

Gosmann, Grace; Barlette, Adriana Gregory; Dhamer, Tabitha; Arçari, Demétrius P; Santos, Juliana Carvalho; de Camargo, Eloá Ramalho; Acedo, Simone; Gambero, Alessandra; Gnoatto, Simone Cristina Baggio; Ribeiro, Marcelo Lima



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

PubMed Central

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.



Radial distribution test feeders  

Microsoft Academic Search

Many computer programs are available for the analysis of radial distribution feeders. In 1992 a paper was published that presented the complete data for three four-wire wye and one three-wire delta radial distribution test feeders. The purpose of publishing the data was to make available a common set of data that could be used by program developers and users to

W. H. Kersting



Feeder cell cultures for zebrafish embryonal cells in vitro.  


Use of fibroblast cells derived from mouse embryos as feeder layers was one of the major steps leading to the establishment of pluripotential mouse embryonal stem (ES) cells in culture. In attempts to obtain a culture of pluripotential ES cells from zebrafish, a culture of fibroblastoid cells, designated zebrafish embryo fibroblast (ZEF), was established from early gastrula stage zebrafish embryos for use as feeder layer. In primary cultures initiated from early embryos of zebrafish without feeder layers, melanocytes appeared on the second day of culture. In contrast, melanogenesis was markedly suppressed in cocultures containing confluent monolayers of ZEF or Buffalo rat liver (BRL) cells. BRL cells are commonly used feeder layer cells for mouse ES cells. Suppression of melanogenesis was not observed in primary cultures initiated in medium containing human recombinant differentiation-inhibiting activity (DIA) or in medium conditioned by cultures of BRL feeder cells. Proliferation of zebrafish embryonal cells was enhanced significantly in cocultures with either feeder layer. Zebrafish embryonal cells cocultured short-term on ZEF and BRL feeder layers gave rise to melanocytes and formed embryoid body-like structures when removed from feeder layers and cultured in suspension, suggesting that the cells remained pluripotent in culture. PMID:7749465

Sun, L; Bradford, C S; Barnes, D W



Sensitivities of NIH/3T3-derived clonal cell lines to ionizing radiation: Significance for gene transfer studies  

SciTech Connect

Rodent cells are frequently used as recipients in experiments involving gene transfer, isolation, and characterization. The present studies were designed to investigate the clonal responses to ionizing radiation of NIH/3T3 cells subjected to DNA-mediated gene transfer. Radiation sensitivity (D0) values were determined for the parental NIH/3T3 cell strain, six clonal cell lines transfected with DNA from radiation-resistant human tumor cells, and six nontransfected clonal cell lines. The radiation sensitivities of four transfected and two nontransfected clonal cell lines differed significantly from parental NIH/3T3 cells (P less than 0.05). Detailed karyotype analysis of two nontransfected clonal cell lines with differing radiation sensitivities showed variation in chromosomal composition. Specifically, a minute chromosome was observed to segregate consistently (in 49 of 50 metaphases) with the genome of one NIH/3T3 clone (D0 2.07 Gy) and was completely absent (from 50 metaphases) in another NIH/3T3 clone (D0 1.06 Gy). In the parental NIH/3T3 strain (D0 2.02 Gy) 10% of cells (3 of 30 metaphases) had such minute chromosomes. These findings demonstrate that the clonal cellular heterogeneity of NIH/3T3 cells is characterized by genotypic and phenotypic variations which must be considered in the experimental design involving gene transfer and expression.

Kasid, U.N.; Weichselbaum, R.R.; Brennan, T.; Mark, G.E.; Dritschilo, A. (Georgetown Univ. School of Medicine, Washington, DC (USA))



Reduction of 3T3 Fibroblast Adhesion on SS316L by Methyl-Terminated SAMs.  


Inhibiting the non-specific adhesion of cells and proteins to biomaterials such as stents, catheters and guide wires is an important interfacial issue that needs to be addressed in order to reduce surface-related implant complications. Medical grade stainless steel 316L was used as a model system to address this issue. To alter the interfacial property of the implant, self assembled monolayers of long chain phosphonic acids with -CH(3), -COOH, -OH tail groups were formed on the native oxide surface of medical grade stainless steel 316L. The effect of varying the tail groups on 3T3 fibroblast adhesion was investigated. The methyl terminated phosphonic acid significantly prevented cell adhesion however presentation of hydrophilic tail groups at the interface did not significantly reduce cell adhesion when compared to the control stainless steel 316L. PMID:21461313

Raman, Aparna; Gawalt, Ellen S



Reduction of 3T3 Fibroblast Adhesion on SS316L by Methyl-Terminated SAMs  

PubMed Central

Inhibiting the non-specific adhesion of cells and proteins to biomaterials such as stents, catheters and guide wires is an important interfacial issue that needs to be addressed in order to reduce surface-related implant complications. Medical grade stainless steel 316L was used as a model system to address this issue. To alter the interfacial property of the implant, self assembled monolayers of long chain phosphonic acids with ?CH3, ?COOH, ?OH tail groups were formed on the native oxide surface of medical grade stainless steel 316L. The effect of varying the tail groups on 3T3 fibroblast adhesion was investigated. The methyl terminated phosphonic acid significantly prevented cell adhesion however presentation of hydrophilic tail groups at the interface did not significantly reduce cell adhesion when compared to the control stainless steel 316L.

Raman, Aparna; Gawalt, Ellen S.



Synthesis of Nerve Growth Factor by L and 3T3 Cells in Culture  

PubMed Central

Mouse submaxillary gland nerve growth factor (NGF) has been covalently joined to bacteriophage and the resulting phage conjugates remain biologically active in stimulating neurite extension from sensory ganglia. A sensitive bacteriophage immunoassay has been developed to measure concentrations of NGF as low as 1 ng/ml. With this method, we find that mouse L and 3T3 cells in culture produce a biologically active nerve growth factor that is immunologically similar if not identical to mouse submaxillary gland NGF. Since L cells are known to be a source of “conditioned medium” for tissue culture, it could be that one or more of the conditioning factor activities secreted by these cells are due to NGF itself. Images

Oger, Joel; Arnason, Barry G. W.; Pantazis, Nicholas; Lehrich, James; Young, Michael



Anti-adipogenic diarylheptanoids from Alnus hirsuta f. sibirica on 3T3-L1 cells.  


A new diarylheptanoid, (5S)-hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-hepta-1E-en-3-one (1), was isolated along with seventeen known diarylheptanoids (2-18) from the methanol extract of Alnus hirsuta f. sibirica leaves using bioactivity-guided fractionation. Among the isolated compounds, compounds 1 and 2 and 4-12 reduced lipid accumulation dose-dependently in 3T3-L1 preadipocytes. Of the compounds active in the present assay system, the most potent compound 7, platyphyllonol-5-O-?-d-xylopyranoside, significantly suppressed the induction of peroxisome proliferator activated receptor ? (PPAR? and CCAAT/enhancer binding protein ? (C/EBP?) protein expression, and inhibited adipocyte differentiation induced by troglitazone, a PPAR? agonist. It was demonstrated that compound 7 has anti-adipogenic activity mediated by the regulation of PPAR? dependent pathways. PMID:23465614

Lee, Mina; Song, Ji Yeon; Chin, Young-Won; Sung, Sang Hyun



Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes  

PubMed Central

Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.

Fleuren, Wilco W. M.; Linssen, Margot M. L.; Toonen, Erik J. M.; van der Zon, Gerard C. M.; Guigas, Bruno; de Vlieg, Jacob; Dokter, Wim H. A.; Ouwens, D. Margriet



(-)-Epigallocatechin-3-gallate enhances uncoupling protein 2 gene expression in 3T3-L1 adipocytes.  


In this study, we investigated the effects of green tea (-)-epigallocatechin-3-gallate (EGCG) on the mRNA level and promoter activity of uncoupling protein 2 (UCP2), a mitochondrial membrane transporter that regulates energy expenditure and thermogenesis in 3T3-L1 adipocytes. EGCG up-regulated the UCP2 mRNA level in a dose-dependent manner. UCP2 promoter activity was significantly stimulated by EGCG treatment, to an extent similar to that seen in mRNA expression. These results suggest that expression of UCP2 gene is directly regulated by green tea EGCG, which is mediated through the transcriptional activation of its proximal promoter. PMID:19202275

Lee, Mak-Soon; Kim, Yangha



Irradiation of Polystyrene and Polypropylene to study NIH 3T3 fibroblasts adhesion  

NASA Astrophysics Data System (ADS)

When polymers are irradiated with heavy ions new chemical groups are created in a few microns of the material. The irradiation changed the polarity and wettability on the surface so that could enhance the biocompatibility of the modified polymer. The study of chemistry and nanoscale topography of the biomaterial is important in determining its potential applications in medicine and biotechnology, because their strong influence on cell function, adhesion and proliferation. In this study, thin films of Polystyrene and Polypropylene samples were modified by irradiation with low energy ion beams (30-150 keV) and swift heavy ions both with various fluences and energies. The changes were evaluated with different methods. Adhesion of NIH 3T3 fibroblasts onto unirradiated and irradiated surfaces has been studied by in vitro techniques. The correlations between physicochemical properties as a function of different irradiations parameters were compared with cell adhesion on the modified polymer surface.

Arbeitman, C. R.; Del Grosso, M. F.; Ibańez, I.; Bermúdez, G. García; Durán, H.; Chappa, V. C.; Mazzei, R.; Behar, M.



Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA  

SciTech Connect

Protein kinase C (PKC), a multi-gene family of enzymes, plays key roles in the pathways of signal transduction, growth control and tumorigenesis. Variations in the intracellular localization of the individual isoforms are thought to be an important mechanism for the isoform-specific regulation of enzyme activity and substrate specificity. To provide a dynamic method of analyzing the localization of the specific isoforms of PKC in living cells, we generated fluorescent fusion proteins of the various PKC isoforms by using the green fluorescent protein (GFP) as a fluorescent marker at the carboxyl termini of these enzymes. The intracellular localization of the specific PKC isoforms was then examined by fluorescence microscopy after transient transfection of the respective PKC-GFP expression vector into NIH3T3 cells and subsequent TPA stimulation. We found that the specific isoforms of PKC display distinct localization patterns in untreated NIH3T3 cells. For example, PKC{alpha} is localized mainly in the cytoplasm while PKC{epsilon} is localized mainly in the Golgi apparatus. We also observed that PKC{alpha}, {beta}1, {beta}2, {gamma}, {delta}, {epsilon}, and {eta} translocate to the plasma membrane within 10 min of the start of TPA treatment, while the cellular localizations of PKC{zeta} and {iota} were not affected by TPA. Using a protein kinase inhibitor, we also showed that the kinase activity was not important for the translocation of PKC. These results suggest that specific PKC isoforms exert spatially distinct biological effects by virtue of their directed translocation to different intracellular sites.

Kazi, Julhash U. [Biomedical Research Center for Signal Transduction Networks, Department of Chemistry, Inha University, Incheon 402-751 (Korea, Republic of); Soh, Jae-Won [Biomedical Research Center for Signal Transduction Networks, Department of Chemistry, Inha University, Incheon 402-751 (Korea, Republic of)], E-mail:



Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells.  


A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation. PMID:17694516

Hirose, H; Suhara, T; Kaji, N; Sakuma, N; Sekijima, M; Nojima, T; Miyakoshi, J



Effect of blueberry polyphenols on 3T3-F442A preadipocyte differentiation.  


Today obesity is an epidemic, and its prevalence has increased significantly over the last few decades. To avoid excessive accumulation of fat, optimum energy intake along with regular exercise is mandatory. Polyphenols present in green tea, grape seeds, orange, and grapefruit combat adipogenesis at the molecular level and also induce lipolysis. However, very little is known regarding the role of blueberry polyphenols on adipocyte differentiation. Hence we tested the dose-dependent effects of blueberry polyphenols on mouse 3T3-F442A preadipocyte differentiation and lipolysis. 3T3-F442A preadipocytes were incubated with three doses of blueberry polyphenols (150, 200, and 250 ?g/mL [BB-150, BB-200, and BB-250, respectively]), and intracellular lipid content, cell proliferation, and lipolysis were assayed. Blueberry polyphenols suppressed adipocyte differentiation determined by Oil Red-O staining and AdipoRed assay. Intracellular lipid content in control (11,385.51±1,169.6 relative fluorescence units) was significantly higher (P<.05) than with the three doses of blueberry polyphenols (8336.86±503.57, 4235.67±323.17, and 3027.97±346.61, respectively). This corresponds to a reduction of 27%, 63%, and 74%, respectively. Cell proliferation was observed to be significantly higher in the control (0.744±0.035 optical density units) than with BB-150 (0.517±0.031), BB-200 (0.491±0.023), and BB-250 (0.455±0.012). However, when tested for lipolysis, there was no significant difference observed among the groups. We conclude that blueberry polyphenols may play an effective role in inhibiting adipogenesis and cell proliferation. PMID:22400911

Moghe, Shiwani S; Juma, Shanil; Imrhan, Victorine; Vijayagopal, Parakat




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



Human Ornithine Decarboxylase-overproducing NIH3T3 Cells Induce Rapidly Growing, Highly Vascularized Tumors in Nude Mice1  

Microsoft Academic Search

Overexpressionof human ornithine decarboxylase(ODC) under the control of strong promoters induces morphological transformation of immortalized NIH3T3 and Rat.! fibroblasts (M. Auvinen et aL, Nature (Lond.), 360: 355â€\\

Merja Auvinen; Aire Lame; Aino Paasinen-Sohns; Anneli Kangas; Lauri Kangas; Leif C. Andersson


Low molecular weight molecules of oyster nacre induce mineralization of the MC3T3-E1 cells.  


The nacre layer from the pearl oyster shell is considered as a promising osteoinductive biomaterial. Nacre contains one or more signal molecules capable of stimulating bone formation. The identity and the mode of action of these molecules on the osteoblast differentiation were analyzed. Water-soluble molecules from nacre were fractionated according to dialysis, solvent extraction, and reversed-phase HPLC. The activity of a fraction composed of low molecular weight molecules in the mineralization of the MC3T3-E1 extracellular matrix was investigated. Mineralization of the preosteoblast cells was monitored according to alizarin red staining, Raman spectroscopy, scanning electron microscopy, and quantitative RT-PCR. Molecules isolated from nacre, ranging from 50 to 235 Da, induced a red alizarin staining of the preosteoblasts extracellular matrix after 16 days of culture. Raman spectroscopy demonstrated the presence of hydroxyapatite (HA) in samples treated with these molecules. Scanning electron microscopy pictures showed at the surface of the treated cells the occurrence of clusters of spherical particles resembling to HA. The treatment of cells with nacre molecules accelerated expression of collagen I and increased the mRNA expression of Runx2 and osteopontin. This study indicated that the nacre molecules efficient in bone cell differentiation are certainly different from proteins, and could be useful for in vivo bone repair. PMID:17729263

Rousseau, Marthe; Boulzaguet, Hélčne; Biagianti, Julie; Duplat, Denis; Milet, Christian; Lopez, Evelyne; Bédouet, Laurent



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

PubMed Central

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.

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



Expression of human insulin gene wrapped with chitosan nanoparticles in NIH3T3 cells and diabetic rats  

Microsoft Academic Search

Aim:To study the expression of human insulin gene wrapped with chitosan nanoparticles in NIH3T3 cells and diabetic rats.Methods:pCMV.Ins, an expression plasmid of the human insulin gene, was constructed. In total, 100 ?g pCMV.Ins wrapped with chitosan nanoparticles (chitosan–pCMV.Ins) was transfected to NIH3T3 cells and diabetes rats through lav age and coloclysis, respectively. The transfected cells were grown in Dulbecco's modified

Li Niu; Yan-cheng Xu; Hai-ying Xie; Zhe Dai; Hui-qin Tang



Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts  

Microsoft Academic Search

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n\\/CH3 peak areas R=1.56±0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm

Amalia Ferretti; Arno Knijn; Egidio Iorio; Simonetta Pulciani; Massimo Giambenedetti; Agnese Molinari; Stefania Meschini; Annarita Stringaro; Annarica Calcabrini; Isabel Freitas; Roberto Strom; Giuseppe Arancia; Franca Podo



Adhesion of Metastatic, ras-transformed NIH 3T3 Cells to Osteopontin, Fibronectin, and Laminin1  

Microsoft Academic Search

We previously reported that H-ras-induced metastatic ability in murine NIH 3T3 cells is accompanied by increased expression of osteopontin (OPN). OPN is a secreted phosphoprotein that contains a GRGDS amino acid sequence, suggesting adhesive function, but the function of OPN in tumor cells remains poorly understood. Here we report that PAP2 cells (ras-transformed, metastatic NIH 3T3 cells) adhere and spread

Ann F. Chambers; Charulata Hota; Charles W. Prince



Mevalonic Acid Products as Mediators of Cell Proliferation in Simian Virus 40-transformed 3T3 Cells1  

Microsoft Academic Search

Effects of treatment with serum-free medium and 25-hydroxycholes- terol (2S-OH) on the cell cycle of simian virus 40-transformed 3T3 fibroblasts, designated SV-3T3 cells, were studied and compared with simultaneous effects on the activity of 3-hydroxy-3-methylglutaryl (HMG) CoA reducĂ­ase and incorporation of |3H|mevalonic acid into cholesterol, Coenzyme Q, and dolichol. The data confirm our previous finding (O. Larsson and A. Zetterberg,

Olle Larsson; Brht-Marie Johansson



Temporal and spatial assembly of lipid droplet-associated proteins in 3T3-L1 preadipocytes  

Microsoft Academic Search

This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination

Seu-Mei Wang; Ran-Der Hwang; Andrew S. Greenberg; Hui-Ling Yeo



Different substrate utilization between prostaglandin endoperoxide H synthase-1 and -2 in NIH3T3 fibroblasts  

Microsoft Academic Search

Recent studies suggested that prostaglandin endoperoxide H synthase-1 and prostaglandin endoperoxide H synthase-2 (PGHS-1 and PGHS-2) utilize different pools of arachidonic acid for synthesizing prostanoids. Using cultured murine NIH3T3 fibroblasts, we investigated the mechanism for the different utilization of arachidonic acid between PGHS-1 and -2. Histofluorescence staining for PGHS activity in intact cells demonstrated that quiescent 3T3 cells expressed only

Miki Shitashige; Ikuo Morita; Sei-itsu Murota



Growth-Regulatory Activity of the Growth Arrest-Specific Gene, GAS1, in NIH3T3 Fibroblasts  

Microsoft Academic Search

The growth arrest-specific gene, Gas-1, is preferentially expressed in quiescent NIH3T3 cells and inhibits DNA synthesis, suggesting that Gas-1 may be a tumor suppressor gene. When GAS1 cDNA, under the control of the strong constitutive CMV promoter, was transfected into NIH3T3 cells, no stable transfectant cell lines were produced, confirming that high levels of expression of GAS1 mRNA inhibit proliferation.

A. Evdokiou; P. A. Cowled



Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies  

Microsoft Academic Search

Background:Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells.Methods:Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras.

L Hampson; X T He; A W Oliver; J A Hadfield; T Kemp; J Butler; A McGown; H C Kitchener; I N Hampson



Protein kinase Ce promotes adipogenic commitment and isessential for terminal differentiation of 3T3-F442A preadipocytes  

Microsoft Academic Search

The role of protein kinase C (PKC) isoforms in the commitment of multipotent fibroblasts to the adipocyte lineage and in their terminal differentiation into mature adipocytes was investigated. Ectopic overexpression of PKC-e, but not other PKC isoforms, committed multipotent NIH-3T3 cells to adipogenic differentiation in the presence of hormonal inducers. In committed 3T3-F442A preadipocytes, PKC-e protein expression increased during the

P. R. Webb; C. Doyle; N. G. Anderson



The licorice root derived isoflavan glabridin increases the function of osteoblastic MC3T3-E1 cells  

Microsoft Academic Search

Glabridin, an isoflavan purified from licorice root, exhibits diverse biological activities, including estrogen-like activity. To investigate the bioactivities of glabridin, which act on bone metabolism, the effects of glabridin on the function of mouse osteoblastic cell line (MC3T3-E1) and the production of local factors in osteoblasts were studied. Glabridin (1–10?M) significantly increased the growth of MC3T3-E1 cells and caused a

Eun-Mi Choi



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

PubMed Central

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.

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



Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells  

SciTech Connect

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due n not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.

Kuzumaki, N.; Ogiso, Y.; Oda, A.; Fujita, H.; Suzuki, H.; Sato, C.; Mullauer, L.



Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells  

SciTech Connect

In mouse 3T3-L1 fibroblasts, the glucocorticoid dexamethasone (dex) affects a switch in ..beta..-adrenergic receptor (..beta..AR) subtype expression from ..beta../sub 1/AR to ..beta../sub 2/AR and increases total ..beta..AR number. They now demonstrate a similar effect by sodium butyrate (B) and find that the combined effect of these two gene-activating agents is greater than additive suggesting different mechanisms of action on the ..beta..AR. ..beta..AR are assayed in membranes prepared from 3T3-L1 cells using the radiolabeled ..beta..AR-specific antagonist (/sup 125/I)-cyanopindolol. ..beta..AR subtype is determined by competition binding of the ..beta../sub 2/AR-selective antagonist ICI 118.551 for the radioligand. B (2-10mM) causes a dose-dependent increase in total ..beta..AR number (up to 2-fold over control) and the proportion of ..beta../sub 2/AR. B (5mM) causes a time-dependent increase in total ..beta..AR number (2-fold) and the proportion of ..beta../sub 2/AR up to 24 hr. Dex maximally increases total ..beta..AR number (2-fold) when treated for 48 hr at concentrations greater than or equal to 100nM. B (2 or 5mM) together with dex (250nM) have a greater than additive effect on total ..beta..AR number at 24 hr (1.7-fold) and at 48 hr (1.4-2.4-fold, using 5 or 10mM B and dex greater than or equal to 10nM). The proportion of ..beta../sub 2/AR is also greater when both compounds are added together. In comparison with proprionate and valerate, B increases total ..beta..AR number and the proportion of ..beta../sub 2/AR to a greater extent and at lower concentrations. To determine a functional correlate to these findings, cells were pre-treated for 48 hr with B and/or dex, intracellular ATP labeled with /sup 3/H-adenine, followed by treatment with forskolin ( and ..beta..AR agonists. B caused a dramatic increase in /sup 3/H-cAMP produced compared to control and dex treatments and a greater than additive effect was again achieved when B and dex were added together.

Poksay, K.S.; Nakada, M.T.; Crooke, S.T.; Stadel, J.M.



Secreted Nucleobindin-2 Inhibits 3T3-L1 Adipocyte Differentiation  

PubMed Central

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPAR?, aP2, and adipsin). When Nucleobindin-2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.

Tagaya, Yuko; Osaki, Aya; Miura, Atsuko; Okada, Shuichi; Ohshima, Kihachi; Hashimoto, Koshi; Yamada, Masanobu; Satoh, Tetsurou; Shimizu, Hiroyuki; Mori, Masatomo



Glucagon-like Peptide-1 Upregulates Visfatin Expression in 3T3-L1 Adipocytes.  


The incretin hormone glucagon-like peptide-1 (GLP-1) exerts important functions in controlling glucose homeostasis. Many studies have revealed molecular targets of GLP-1, but its influence on adipokines has not been determined. Visfatin, a recently discovered adipokine, has been shown to attenuate insulin resistance by binding to insulin receptor. Our study shows that GLP-1 induced secretion of visfatin into the culture medium of 3T3-L1 adipocytes due to increased visfatin mRNA expression. Furthermore, the effect of GLP-1 on visfatin was dose- and time-dependent. H89, a protein kinase A inhibitor, prevented the induction of visfatin expression by GLP-1. Moreover, inhibition of NF-?B by PDTC reduced the basal visfatin release while having no effect on the transcription regulation by GLP-1. In addition, GLP-1 alleviated the decrease of visfatin mRNA expression under endoplasmic reticulum stress induced by thapsigargin. Taken together, our study suggests that GLP-1 promotes the novel insulin-mimetic adipocytokine visfatin expression via the PKA pathway and might influence glucose metabolism. PMID:23670349

Liu, R; Ding, X; Wang, Y; Wang, M; Peng, Y



Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes  

PubMed Central

Background Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear. Methodology/Principal Findings This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects. Conclusions/Significance The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism.

Si, Yaguang; Shi, Hai; Lee, Kyongbum



Modulation of intracellular glutathione affects adipogenesis in 3T3-L1 cells.  


Impairment of redox homeostasis has been extensively associated with obesity, as a consequence of the chronic inflammatory state present in overweight subjects. Deregulation of glutathione (GSH), the most important non-enzymatic intracellular anti-oxidant, induces insulin resistance in mature adipocytes, but data are lacking about its effects on adipogenesis. In this report we demonstrate that during adipogenesis of 3T3-L1 cells the GSH/GSSG ratio decreases, shifting redox status towards oxidizing conditions. Moreover, we demonstrate that inhibition of GSH synthesis, obtained by treatment with L-buthionine-sulfoximine (BSO), enhances C/EBP? LAP/LIP ratio and PPAR? expression during mitotic clonal expansion (MCE) stimulating adipogenesis. On the contrary, GSH ethyl ester (GSHest) supplementation completely abrogates this process also in the presence of BSO. GSH decrement during the first 24?h of adipogenesis is sufficient to induce higher triglyceride accumulation in differentiated adipocytes with respect to control, whereas GSHest treatment inhibits lipid droplets formation. We further demonstrate that Resveratrol (RV) could exert anti-adipogenic properties also by increasing GSH content through ?-glutamyl-cysteine ligase (GCL) induction. Overall data indicate that in pre-adipocytes the decrease of GSH accelerates adipogenesis, suggesting that the use of agents able to maintain GSH redox status in adipose tissue, such as RV, could be promising in stopping the lipogenic loop of obesity. PMID:21520053

Vigilanza, Paola; Aquilano, Katia; Baldelli, Sara; Rotilio, Giuseppe; Ciriolo, Maria Rosa



Dietary xenoestrogens differentially impair 3T3-L1 preadipocyte differentiation and persistently affect leptin synthesis.  


Recent observations have highlighted adipogenesis alterations under exposure to several xenoestrogens at critical stages, and pointed at their possible involvement in the pathogenesis of obesity. However, it remains unclear whether these effects are mediated by classical estrogen receptor (ER) binding and subsequent transcriptional modulation. The aim of this study was to determine the (anti-)adipogenic impact of apigenin, bisphenol A, genistein and 17beta-estradiol at the onset of adipose cell maturation, and to correlate it to their estrogenic potential. In steroid-free conditions, 3T3-L1 preadipocytes were induced to differentiate in the presence of xenoestrogens for 2 days. DNA and triglyceride levels, leptin secretion and expression of Pref-1, C/EBPbeta, PPARgamma2, FAS, leptin and ERs were measured on days 0, 3 and 8 of differentiation. Genistein potently blocked mitotic clonal expansion and all markers of maturation. Bisphenol A and estradiol did not modify triglyceride accumulation but increased the expression of differentiation genes. Apigenin caused a weak but reversible delay in adipogenesis although it unexpectedly enhanced leptin synthesis. However, the expression of steroid hormone receptors was not associated with these differential effects. In conclusion, we could not put a clear estrogen-dependent mechanism forward, but early exposure to xenoestrogens persistently disrupted adipocyte gene expression and leptin synthesis. PMID:18359623

Phrakonkham, Pascal; Viengchareun, Say; Belloir, Christine; Lombčs, Marc; Artur, Yves; Canivenc-Lavier, Marie-Chantal



Quantification of Electroporation-Mediated Propidium Iodide Delivery into 3T3 Cells  

NASA Astrophysics Data System (ADS)

Electroporation is an effective means to deliver exogenous molecules into the cellular cytoplasm, while simultaneously maintaining cell viability and functionality. In this technique, an applied electric field transiently permeabilizes the cellular membrane to enable molecular exchange. The main objective of the current work is to identify the transport mechanisms involved during electroporation, and to quantify the amount of molecules delivered into the cellular cytoplasm. An optical diagnostic system is developed to examine the transport of Propidium Iodide (PI) into 3T3 mouse fibroblast cells. Upon entering the permeabilized cell, PI binds to DNA/RNA within the cytoplasm to emit fluorescence, which is measured to track the dynamic accumulation of the dye within the cell. The results show that the total fluorescence intensity increases with a decreasing buffer electrical conductivity. The data are compared with numerical simulations, which reveals good agreement. The experimental observations and numerical analysis demonstrate that: 1) Electrophoresis plays a dominant role in mediating the transport. 2) An electrokinetic mechanism, field-amplified sample stacking, controls the achievable delivery efficiency. The study in this work is an important step toward the quantification as well as the eventual improvement of this useful technique.

Sadik, Mohamed M.; Li, Jianbo; Shan, Jerry W.; Shreiber, David I.; Lin, Hao



Hindered Diffusion of Inert Tracer Particles in the Cytoplasm of Mouse 3T3 Cells  

NASA Astrophysics Data System (ADS)

Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size-fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 angstrom in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 angstrom, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.

Luby-Phelps, Katherine; Castle, Philip E.; Lansing Taylor, D.; Lanni, Frederick



Differentiation of the insulin-sensitive glucose transporter in 3T3-L1 adipocytes  

SciTech Connect

3T3-L1 fibroblasts differentiate in culture to resemble adipocytes both morphologically and biochemically. Insulin-sensitive glucose transport, as measured by 2-deoxy-(1-/sup 14/C)- glucose uptake in the undifferentiated cell is small (2X). In contrast, the rate of glucose transport in fully differentiated cells is elevated 15-fold over basal in the presence of insulin. To determine if this is due to an increase in the number of transporters/cell or accessibility to the transporters, the number of transporters was measured in subcellular fractions over differentiation using a /sup 3/H-cytochalasin B binding assay. The increase in the rate of insulin-sensitive glucose transport directly parallels an increase in the number of transporters which reside in an insulin-responsive intracellular compartment. This observation was confirmed by identifying the transporters by immunoblotting using an antibody generated against the human erythrocyte transporter. The molecular weight of this transporter increases over differentiation from a single band of 40kDa to a heterogeneous triplet of 40, 44 and 48kDa. These data suggest that the transporter undergoes differential processing and that the functional, insulin-responsive transporter may be different from the insulin-insensitive (basal) transporter.

Frost, S.C.; Baly, D.L.; Cushman, S.W.; Lane, M.D.; Simpson, I.A.



SPR analysis of ricin binding to plasma membranes isolated from NIH 3T3 cells  

PubMed Central

As the potential for bioterrorism has appeared to increase, the need for simple systems for identifying potential inhibitors of the binding of such agents to cell membranes has increased. In this work, surface plasmon resonance (SPR) was used to monitor binding of ricin, a ribosome inactivating protein, to the plasma membranes of NIH 3T3 cells. Once conditions were established, efficacy of the system for monitoring effectiveness of compounds at inhibiting ricin binding was ascertained by determining the IC50s for asialofetuin and for bovine serum albumin derivatized with an average of 34 lactosyl moieties (BSA-Lac34). Results indicated that SPR is an efficient method for measuring adherence of a toxin to isolated cell plasma membranes. SPR can also indicate whether a compound that is an effective inhibitor of binding when a single ligand such as ASF is used will be as effective when used in studies with cells that may express multiple cell surface ligands for ricin and/or the inhibitor.

Blome, Matthew C.; Petro, Kimberly A.; Schengrund, Cara-Lynne



Cyclic restricted feeding enhances lipid storage in 3 T3-L1 adipocytes  

PubMed Central

Background People who skip breakfast have more visceral fat than those who eat breakfast; however, the mechanism underlying this difference is unclear. In this study, we examined 3 T3-L1 adipocytes and assessed 1) whether restricted feeding (i.e., “breakfast skipping”) alters the cyclic expression of brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) and lipogenic proteins and 2) whether repeated exposure to growth media at the time-points with enhanced lipogenic regulatory signals increases de novo lipogenesis and lipid storage. Methods Differentiated adipocytes were divided into two groups: a control group and a restricted feeding group, for which incubation with growth medium from ZT 9 to ZT 12 was withheld. Results A bout of restricted feeding disrupted the cyclic expression of BMAL1 protein and increased the expression of lipogenic proteins, such as fatty acid synthase and peroxisome proliferator-activated receptor gamma in adipocytes. Furthermore, the repeated exposure to growth media at the time-points with enhanced lipogenic regulatory signals increased de novo lipogenesis and lipid storage. Conclusion These findings suggest that direct disruption of intracellular molecular clock systems by breakfast skipping and the concurrent changes in the daily cycle of lipogenic proteins in adipocytes, as a consequence of repeated nutrition at the time-points with enhanced lipogenic regulatory signals, would result in increased lipogenesis and lipid storage. These alterations are important molecular mechanisms underlying augmented adiposity induced by breakfast skipping.



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


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

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



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


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?M. 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 PPAR? 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:23583842

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



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


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

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



Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes.  

PubMed Central

Previous studies indicated that the erythroidtype (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labelling (with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. Intrinsic catalytic activities specifically associated with heterologously expressed human GLUT1 protein in transfected CHO-K1 versus 3T3-L1 cells were determined by subtracting appropriate control cell values for hexose transport and delta-antibody binding from those determined in the transfected cells expressing high levels of human GLUT1. The results confirmed a greater than 90% inhibition of the intrinsic catalytic activity of human GLUT1 transporters on the surface of mouse 3T3-L1 adipocytes relative to CHO-K1 fibroblasts. We conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes. Images

Harrison, S A; Buxton, J M; Czech, M P



Mitogenic stimuli and phosphatidylinositol (PI) turnover in cultured 3T3 fibroblasts  

SciTech Connect

The hydrolysis of PI and polyphosphoinositides by phopholipase C is an early and rapid response to cell activation by a variety of neurotransmitters, hormones, growth factors and pharmacological agonists. The authors have examined the role of PI turnover and the generation of second messengers (diacylglycerol and inositol trisphosphate) in the mitogenic response of cultured Balb/c and Swiss 3T3 cells to polypeptide growth factors. Cells were prelabelled with /sup 3/H inositol for 18-20 hours, washed and suspended in Herpes + Li/sup +/ buffer, and stimulated with platelet-derived growth factor (PDGF), vasopressin, insulin, and other growth factors. PI turnover was measured as the increase in total inositol phosphate (IP) production. IP1, IP2, and IP3 were characterized by sequential elution from a Dowex column. Partially purified PDGF produced a 2-4 fold stimulation of total IP production. This was seen as early as 30 seconds after stimulation and increased for up to 1-2 hours. Balb/c cells were more sensitive than Swiss cells to the mitogenic and PI effects of PDGF. Other mitogenic stimuli had differential effects on PI turnover. Vasopressin (4-400 ng/ml) markedly stimulated PI turnover (3-6 fold) in Swiss, but not Balb/c cells. Insulin (100 ng/ml - 10 increased total IP to a greater degree in Balb/c cells. Epidermal growth factor (10 ng/ml - 10 had no effect on PI turnover and fibroblast growth factor (10 ng/ml - 10 only stimulated at the higher concentrations in Swiss cells. Thrombin (1U/ml - 10 U/ml) produced a 1.5 - 2 fold stimulation in Balb/c cells. Thus, various polypeptide growth factors have differential effects on PI turnover depending on their mitogenic potential and the effector cell type.

Kohler, C.; Petersen, R.



Lithium differentially affects clock gene expression in serum-shocked NIH-3T3 cells.  


Bipolar disorder has been associated with disturbances in circadian rhythms. Lithium is frequently used in the long-term treatment of bipolar disorder, and has been shown to prolong such rhythms in animals and humans. To examine whether lithium affects the expression of genes regulating the circadian clock, cultured NIH-3T3 cells were synchronized by serum-shocking, and the relative expression of the clock genes Period1 (Per1), Period2 (Per2), Period3 (Per3), Cryptochrome1 (Cry1), Cryptochrome2 (Cry2), Brain and muscle aryl hydrocarbon nuclear translocator-like 1 (Bmal1), Circadian locomotor output cycles kaput (Clock), Rev-Erb-? (Nr1d1), RAR-related orphan receptor ? (Ror-?), Glycogen synthase kinase-3? (Gsk-3?), Casein kinase 1-? (CK1-?; Csnk1?), E4 binding protein 4 (E4BP4; Nfil-3) and albumin D-binding protein (Dbp) was examined for three consecutive days in the presence of lithium (20?mM) or vehicle (20?mM NaCl). We found that lithium significantly increased the expression of Per2 and Cry1, whereas Per3, Cry2, Bmal1, E4BP4 and Rev-Erb-? expression was reduced. We also found that lithium prolonged the period of Per2. Taken together, these effects on clock gene expression may be relevant for the effects of lithium on biological rhythms and could also give new leads to further explore its mood-stabilizing actions in the treatment of bipolar disorder. PMID:20837565

Osland, Teresa M; Fernř, Johan; Hĺvik, Bjarte; Heuch, Ivar; Ruoff, Peter; Lćrum, Ole Didrik; Steen, Vidar M



Mango fruit peel and flesh extracts affect adipogenesis in 3T3-L1 cells.  


Obesity is associated with many chronic disease states, such as diabetes mellitus, coronary disease and certain cancers, including those of the breast and colon. There is a growing body of evidence that links phytochemicals with the inhibition of adipogenesis and protection against obesity. Mangoes (Mangifera indica L.) are tropical fruits that are rich in a diverse array of bioactive phytochemicals. In this study, methanol extracts of peel and flesh from three archetypal mango cultivars; Irwin, Nam Doc Mai and Kensington Pride, were assessed for their effects on a 3T3-L1 pre-adipocyte cell line model of adipogenesis. High content imaging was used to assess: lipid droplets per cell, lipid droplet area per cell, lipid droplet integrated intensity, nuclei count and nuclear area per cell. Mango flesh extracts from the three cultivars did not inhibit adipogenesis; peel extracts from both Irwin and Nam Doc Mai, however, did so with the Nam Doc Mai extract most potent at inhibiting adipogenesis. Peel extract from Kensington Pride promoted adipogenesis. The inhibition of adipogenesis by Irwin (100 ?g mL(-1)) and Nam Doc Mai peel extracts (50 and 100 ?g mL(-1)) was associated with an increase in the average nuclear area per cell; similar effects were seen with resveratrol, suggesting that these extracts may act through pathways similar to resveratrol. These results suggest that differences in the phytochemical composition between mango cultivars may influence their effectiveness in inhibiting adipogenesis, and points to mango fruit peel as a potential source of nutraceuticals. PMID:22699857

Taing, Meng-Wong; Pierson, Jean-Thomas; Hoang, Van L T; Shaw, Paul N; Dietzgen, Ralf G; Gidley, Michael J; Roberts-Thomson, Sarah J; Monteith, Gregory R



Substrate selectivity of diacylglycerol kinase in PDGF-stimulated 3T3 cells  

SciTech Connect

The authors investigated the properties of Diacylglycerol (DAG) Kinase in 3T3 cells. PDGF treatment caused an increase in DAG mass, an increase in incorporation of /sup 32/P into phosphatidic acid (PA) and phosphatidylinositol (PI), and an increase in the rate of phosphorylation of membrane DAG in vitro. The mechanism of enhanced phosphorylation of DAG was studied with dicaprylin (diC/sub 10/) as a probe. Cells were prelabeled with /sup 32/P and treated with PDGF or carrier. DiC/sub 10/ was added to the cell medium before harvesting. With PDGF treatment, the radioactivity in endogenous PA increased fourfold, whereas the radioactivity in PA/sub 10/ and PI/sub 10/ was consistently decreased. To verify that the PDGF effect on PA/sub 10/ formation in intact cells was due to reduced phosphorylation of diC/sub 10/ by DAG kinase, cells were treated with PDGF and/or diC/sub 10/, freeze-thawed, and then incubated with Mg(/sup 32/P)ATP. The rate of phosphorylation of cell-associated diC/sub 10/ was decreased 50% by PDGF treatment. This effect could not be explained by decreased intracellular levels of diC/sub 10/, or by saturation of DAG kinase with endogenous DAGs. Therefore, it seemed that endogenous DAGs, derived from PI, might be better substrates for DAG kinase than is diC/sub 10/. In studies of the properties of DAG kinase with pure DAGs in mixed detergent micelles, they found that the enzyme phosphorylated arachidonoyl-DAG more readily than diC/sub 10/. The selectivity of DAG kinase may play a key role in the formation of arachidonoyl species of PI.

MacDonald, M.L.; Mack, K.F.; Glomset, J.A.



Effects of lipoic acid on lipolysis in 3T3-L1 adipocytes[S  

PubMed Central

Lipoic acid (LA) is a naturally occurring compound with beneficial effects on obesity. The aim of this study was to evaluate its effects on lipolysis in 3T3-L1 adipocytes and the mechanisms involved. Our results revealed that LA induced a dose- and time-dependent lipolytic action, which was reversed by pretreatment with the c-Jun N-terminal kinase inhibitor SP600125, the PKA inhibitor H89, and the AMP-activated protein kinase activator AICAR. In contrast, the PI3K/Akt inhibitor LY294002 and the PDE3B antagonist cilostamide enhanced LA-induced lipolysis. LA treatment for 1 h did not modify total protein content of hormone-sensitive lipase (HSL) but significantly increased the phosphorylation of HSL at Ser563 and at Ser660, which was reversed by H89. LA treatment also induced a marked increase in PKA-mediated perilipin phosphorylation. LA did not significantly modify the protein levels of adipose triglyceride lipase or its activator comparative gene identification 58 (CGI-58) and inhibitor G(0)/G(1) switch gene 2 (G0S2). Furthermore, LA caused a significant inhibition of adipose-specific phospholipase A2 (AdPLA) protein and mRNA levels in parallel with a decrease in the amount of prostaglandin E2 released and an increase in cAMP content. Together, these data suggest that the lipolytic actions of LA are mainly mediated by phosphorylation of HSL through cAMP-mediated activation of protein kinase A probably through the inhibition of AdPLA and prostaglandin E2.

Fernandez-Galilea, Marta; Perez-Matute, Patricia; Prieto-Hontoria, Pedro L; Martinez, J Alfredo; Moreno-Aliaga, Maria J



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


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. PMID:9783541

Yamaguchi, T; Chattopadhyay, N; Kifor, O; Butters, R R; Sugimoto, T; Brown, E M



Wnt signaling protects 3T3-L1 preadipocytes from apoptosis through induction of insulin-like growth factors.  


Ectopic expression of Wnt-1 in 3T3-L1 preadipocytes stabilizes beta-catenin, activates TCF-dependent gene transcription, and blocks adipogenesis. Here we report that upon serum withdrawal, Wnt-1 causes 3T3-L1 cells to resist apoptosis through a mechanism that is partially dependent on phosphatidylinositol 3-kinase. Although activation of Wnt signaling by inhibition of GSK-3 activity or ectopic expression of dominant stable beta-catenin blocks apoptosis, inhibition of Wnt signaling through expression of dominant negative TCF-4 increases apoptosis. Wnt-1 stimulates 3T3-L1 preadipocytes to secrete factors that increase PKB/Akt phosphorylation at levels comparable with treatment with 10% serum. With DNA microarrays, we identified several secreted antiapoptotic genes that are induced by Wnt-1, notably insulin-like growth factor I (IGF-I) and IGF-II. Consistent with IGFs mediating the antiapoptotic effects of Wnt-1 in preadipocytes, conditioned medium from Wnt-1 expressing 3T3-L1 cells was unable to promote protein kinase B phosphorylation after the addition of recombinant IGFBP-4. Thus, we demonstrated that Wnt-1 induces expression of antiapoptotic genes in 3T3-L1 preadipocytes such as IGF-I and IGF-II, which allows these cells to resist apoptosis in response to serum deprivation. PMID:12154096

Longo, Kenneth A; Kennell, Jennifer A; Ochocinska, Margaret J; Ross, Sarah E; Wright, Wendy S; MacDougald, Ormond A



Endogenous lectins from cultured cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts  

PubMed Central

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured 3T3 fibroblasts. In the present study, rabbit antibodies directed against CBP35 were used to analyze the subcellular distribution of CBP35 in 3T3 cells. Several lines of evidence indicate that CBP35 is found externally exposed at the cell surface: immunofluorescent staining of live 3T3 cells; agglutination of suspension of 3T3 fibroblasts by specific antibodies; and isolation, by immunoaffinity chromatography, of a Mr 35,000 component from cells surface-labeled with 125I. In addition to the plasma membrane, CBP35 could also be found intracellularly, as revealed by immunofluorescence studies of fixed and permeabilized 3T3 cells. The staining pattern showed the presence of CBP35 on the nucleus and in the cytoplasm. These results are consistent with the finding that among several subcellular fractions, CBP35 can be found by immunoblotting procedures in the nuclear pellet, the soluble fraction, and the plasma membrane fraction of the postnuclear supernatant.



Cadmium inhibits the differentiation of 3T3-L1 preadipocyte through the C/EBP? and PPAR? pathways.  


Cadmium, a well-known toxic heavy metal, affects cellular physiology by disturbing cellular signaling pathways. We investigate the effect of cadmium on cellular differentiation using 3T3-L1 preadipocyte cell lines as an in vitro model. Here, it was shown that cadmium (3 ?M) significantly decreased both glycerol-3-phosphate dehydrogenase (GPDH) activity and lipid accumulation of differentiating 3T3-L1 cells in a dose-dependent manner. In addition, inhibitory action of cadmium on differentiating 3T3-L1 cells was effective only at the initial stage of 3T3-L1 preadipocyte differentiation. Western blot analysis revealed that cadmium suppressed the expression of CCAAT/enhancer-binding protein alpha (C/EBP?) and peroxisome proliferator-activator receptor gamma (PPAR?) proteins, key transcriptional activators for adipogenesis, in a dose-dependent manner. These results suggest that the inhibitory effects of cadmium on 3T3-L1 preadipocyte differentiation, as indicated by a decrease in GPDH activity and lipid accumulation, a marker of adipogenesis, appeared to be mediated through the downregulated expression of C/EBP? and PPAR? proteins. PMID:21848503

Lee, Eun Jee; Moon, Jun Yeon; Yoo, Byung Sun



Mechanisms of Resveratrol-Induced Inhibition of Clonal Expansion and Terminal Adipogenic Differentiation in 3T3-L1 Preadipocytes.  


We show that resveratrol prevents clonal expansion and terminal adipogenesis in 3T3-L1 preadipocytes. An early resveratrol effect was the inhibition of AKT and mitogen-activated protein kinase signaling, accompanied by down regulation of cyclin D1 expression, abrogation of retinoblastoma protein hyperphosphorylation, and subsequent inhibition of cell cycle reentry and clonal expansion, as indicated by cyclin A2 repression. Resveratrol inhibited terminal adipogenesis at the level of peroxisome proliferator-activated receptor-?2 expression and activity. This was independent from the preceding inhibition of clonal expansion. Peroxisome proliferator-activated receptor-?2 overexpression and activation partially restored fatty acid-binding protein 4 induction in resveratrol-treated 3T3-L1. Resveratrol activated AMP-activated protein kinase (AMPK) but did not induce PPAR-? co-activator 1? (PGC1?) and mitochondrial biogenesis in 3T3-L1. Treatment with the Sirt1 inhibitor splitomicin augmented downregulation of peroxisome proliferator-activated receptor-?2 and fatty acid-binding protein 4 expressions in resveratrol-treated 3T3-L1 and did not prevent the inhibition of terminal adipogenesis. Moreover, splitomicin could not obviate resveratrol-induced cyclin D1 repression, retinoblastoma protein hypophosphorylation, and inhibition of clonal expansion. Our data suggest that resveratrol inhibits clonal expansion and terminal adipogenesis in 3T3-L1 by several mechanisms. PMID:23525482

Mitterberger, Maria C; Zwerschke, Werner



Lipid Droplets Characterization in Adipocyte Differentiated 3T3-L1 Cells: Size and Optical Density Distribution  

PubMed Central

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.

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



A Swiss 3T3 variant cell line resistant to the effects of tumor promoters cannot be transformed by src.  

PubMed Central

To study the relationship between oncogenesis by v-src and normal cellular signalling pathways, we determined the effects of v-src on 3T3-TNR9 cells, a Swiss 3T3 variant which does not respond mitogenically to tumor promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We found that src was unable to transform these variant cells, whether the oncogene was introduced by infection with a murine retrovirus vector or by transfection with plasmid DNA. 3T3-TNR9 cells were not inherently resistant to transformation, since infection with similar recombinant retroviruses containing either v-ras or v-abl did induce transformation. Further analysis of Swiss 3T3 and 3T3-TNR9 cell populations infected with the v-src-containing retrovirus revealed that although the amount of v-src DNA in each was approximately the same, the level of the v-src message and protein and the overall level of phosphotyrosine expressed in the infected variants was much less than in infected parental cells. Cotransfection experiments using separate v-src and neo plasmids revealed a decrease in the number of G418-resistant colonies when transfections of TNR9 cells occurred in the presence of the src-containing plasmid, suggesting a growth inhibitory effect of v-src on 3T3-TNR9 cells, as has also been found for TPA itself. Since v-src cannot transform this variant cell line, which does not respond mitogenically to the protein kinase C agonist TPA, we suggest that src makes use of the protein kinase C pathway as part of its signalling activities. Images

Nori, M; Shawver, L K; Weber, M J



Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts  

PubMed Central

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4- 6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid- phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.



Differential effects of homocysteine and beta aminopropionitrile on preosteoblastic MC3T3-E1 cells.  


Compounds, like beta-aminopropionitrile (bAPN) and homocysteine (hcys), are known to inhibit a stable matrix formation. Osteoblast-synthesized collagen matrix regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase, an enzyme involved in the collagen cross-linking process. Lately, plasma hcys levels have recently been strongly correlated with fracture in humans. We have previously shown that bAPN not only disturbs collagen cross-links but also affects osteoblastic differentiation in a cell culture system. The aim of the present study was to investigate the effects of bAPN and hcys on collagen cross-links and gene expression at the mRNA level by FTIR and quantitative RT-PCR, respectively. We found that bAPN and hcys down-regulated cell multiplication. While bAPN also down-regulated the metabolic activity of MC3T3-E1 cells, hcys down-regulated it by lower concentrations but up-regulated it by higher; both substances up-regulated alkaline phosphatase activity. The substances increased the ratio of pyr/divalent cross-links of collagen, and down-regulated mRNA expression of lysyl hydroxylase (Plod2) and lysyl oxidase (Lox), genes which play an important role in the formation of a stable matrix. Furthermore, we demonstrate that both substances stimulated the expression of Runx2, an indispensable regulator of osteoblastic differentiation. However, analysis of genome wide mRNA expression suggests that hcys and bAPN have differential effects on genes involved in osteoblastic differentiation and phenotype regulation. The results indicate that although both bAPN and hcys affect collagen cross-link post-translational modifications in a similar manner as far as pyr and divalent cross-links are concerned, they have differential effects on the monitored genes expression at the mRNA level, with hcys exerting a broader effect on the genome wide mRNA expression. PMID:19895920

Thaler, Roman; Spitzer, Silvia; Rumpler, Monika; Fratzl-Zelman, Nadja; Klaushofer, Klaus; Paschalis, Eleftherios P; Varga, Franz



Bird Feeder Project  

NSDL National Science Digital Library

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!

Zoo, Sacramento



Induction of Differentiation of 3T3-L1 Fibroblasts to Adipocytes by 3-Deazaadenosine and Insulin,  

National Technical Information Service (NTIS)

Because of the hypothesis that DNA hypomethylation may lead to gene expression and cellular differentiation, we examined the effect of 3-deazaadenosine on the differentiation of 3T3-L1 fibroblasts to adipocytes (fat cells). This subline of fibroblasts, 3T...

P. K. Chiang N. D. Brown F. N. Padilla R. K. Gordon



Evidence for the Progressive and Adaptive Nature of Spontaneous Transformation in the NIH 3T3 Cell Line  

Microsoft Academic Search

The NIH 3T3 mouse cell line is widely used as a recipient of DNA from tumors to demonstrate the presence of transforming oncogenes. We show that these cells produce transformed foci spontaneously if kept in the confluent state for more than 10 days. The formation of foci depends on the type and concentration of bovine serum used in the medium

H. Rubin; K. Xu



Effects of in vitro-digested ginsenosides on lipid accumulation in 3T3-L1 adipocytes.  


Ginseng, the root of Panax ginseng C. A. Meyer, is frequently used in traditional oriental medicines. The major active components of ginseng are the saponins, which are also called ginsenosides and are known for their pharmacological and biological activities. In this study, the effects of ginsenosides on lipid accumulation in 3T3-L1 adipocytes were investigated after the ginsenosides were in vitro-digested with artificial gastric and intestinal fluids. Ginseng extract was incubated with an artificial digestive fluid, and the changes were analyzed by HPLC, after which the effects of the digest on 3T3-L1 adipocytes were observed. Polar ginsenosides were transformed into less-polar ginsenosides at the low pH of the gastric acid, without any influence from the digestive enzymes. Additionally, the artificially digested ginsenosides showed inhibitory effects on lipid accumulation in 3T3-L1 adipocytes. When the 3T3-L1 adipocytes were treated with various ginseng samples that possessed different polarities, the less polar ginsenosides were more effective in reducing lipid accumulation. Furthermore, when the Rg3, Rk1, and Rg5 ginsenosides were used to treat the cells individually, Rg3 ginsenoside was the most effective at inhibiting lipid accumulation. These results suggest that the less polar ginsenosides, particularly ginsenoside Rg3, effectively reduce lipid accumulation in adipocytes. Accordingly, our results suggest that ginsenoside Rg3 should be developed as an antiobesity treatment. PMID:19204893

Kim, Su Na; Lee, Ju Hyeon; Shin, Heungsop; Son, Sung Ho; Kim, Yeong Shik



High throughput screening (HTS) for phototoxicity hazard using the in vitro 3T3 neutral red uptake assay  

Microsoft Academic Search

Testing for phototoxic hazard is usually carried out for product ingredients intended for use on skin, which may be exposed to sunlight. Unilever currently uses the validated in vitro 3T3 Neutral Red Uptake phototoxicity test (NRU PT). This protocol involves 2–3 experiments, each taking 3 days to perform. One person can test up to seven test materials plus positive control

P. A. Jones; A. V. King



Making It Better - Our Faculty and Ourselves: A P3T3 Case Study of Web Design and Video Integration  

Microsoft Academic Search

The staff of the P3T3 project have a primary mission to train and mentor faculty members in the School of Education as they assimilate technology in instruction. This study investigates the evolution of select faculty members; how did they 1) integrate technology, 2) perceive technology, and 3) implement technology effectively? In addition, existing problems in faculty training and inefficient processes

Pamela A. White; Chaoyan Dong; James D. Lehman


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

Microsoft Academic Search

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

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



Iodothyronine Interactions with the System L1 Amino Acid Exchanger in 3T3-L1 Adipocytes  

PubMed Central

Thyroid hormones enter isolated white adipocytes largely by a System L1-type amino acid transporter en route to exerting genomic actions. Differentiated 3T3-L1 mouse adipocytes in culture express mRNA for LAT1 (the catalytic subunit of high-affinity System L1). L-[125I]-T3 uptake into 3T3-L1 adipocytes included a substantial saturable component inhibited by leucine. L-[3H]phenylalanine uptake into 3T3-L1 cells was saturable (Km of 31??M), competitively inhibited by T3 (Ki of 1.2??M) and blocked by leucine, BCH, and rT3 as expected for substrate interactions of System L1. Efflux of preloaded L-[3H]phenylalanine from 3T3-L1 adipocytes was trans stimulated by external leucine, demonstrating the obligatory exchange mechanism of System L1 transport. T3 (10??M) did not significantly trans stimulate L-[3H]phenylalanine efflux, but did competitively inhibit the trans stimulatory effect of 10 ?M leucine. The results highlight strong competitive interactions between iodothyronines (T3, rT3) and amino acids for transport by System L1 in adipocytes, which may impact cellular iodothyronine exchanges during altered states of protein nutrition.

Mitchell, Fiona E.; Roy, Lisa A.; Taylor, Peter M.



[The effect of microencapsulated NGF-expressing NIH-3T3 cells on bioengineered dermis function in vitro].  


Nerve growth factor (NGF) can promote the regeneration of peripheral nerve as well as contraction and reepithelization of wound. We constructed a bioengineered dermis containing microencapsulated NGF-expressing NIH-3T3 cells and study the effect of the microencapsule to the bioengineered dermis and seed cells. NGF gene was transfected into NIH-3T3 cells and enclosed into alginate-poly-L-lysine-alginate (APA) microencapsulation and cultivated in vitro. Content of NGF in microencapsules culturing supernatant was measured by enzyme linked immunosorbent assay (ELISA) method. These microencapsules were co-cultured with epidermic cells and fibroblast cells. Bioengineered dermis was constructed with NGF-expressing micorencapsules as seed cells using tissue engineering method. NIH-3T3 microencapsules, empty microencapsules, normal culture media were control groups. After one week culture, the characteristics of the dermis were described by MTT test, the content of hydroxyproline (HP), HE staining and ultrastructure photograph. We found the NGF-expressing microencapsulates can secret NGF steadly after cultured 8w in vitro, promot the proliferation of epidermic cells and secret collagen of fibroblast cells. These functions can maintaine in bioengineered dermis. So NGF-expressing NIH-3T3 microencapsulates can promote the quality of bioengineered dermis. PMID:17333893

Hu, Zhaohua; Chen, Shaozong; Jin, Yan; Xiong, Ying; Wang, Wei; Ma, Xiaojun; Zhao, Yu; Li, Wangzhou; Lei, Zhanjun



Effects of low energy beta-irradiation from tritiated water on the morphology of 3T3 fibroblasts  

SciTech Connect

Cellular alterations of cultured 3T3 cells irradiated with beta-rays from tritiated water were studied by scanning and transmission electron microscopy. We observed decreased negative surface charges, vacuolization of rough endoplasmic reticulum and Golgi-complex, degeneration of mitochondria, increase of lysosomal activity and changes in distribution and amount of microfilaments in the irradiated cells, that parallelled changes in cell shape.

Somosy, Z.; Kubasova, T.; Kovacs, J.; Koeteles, G.J. (Frederic Joliot-Curie, National Research Institute for Radiobiology and Radiohygiene, Budapest, (Hungary))



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


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. PMID:23921150

Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing; Liao, Er-Yuan



Detecting Low Levels of Cytochalasin B in 3T3 Fibroblast Cells by Analysis of Electrical Noise Obtained from Cellular Micromotion  

NASA Astrophysics Data System (ADS)

We performed several micromotion experiments using the electric cell-substrate impedance sensing (ECIS) apparatus on a confluent layer of 3T3 fibroblast cells exposed to differing, low-level amounts of the toxin cytochalasin B. We previously developed a technique to distinguish cancerous from non-cancerous cultures.ootnotetextD.C. Lovelady et alia, Phys. Rev. E 76, 041908 (2007) Our goal here is to see if the same technique can be used to distinguish toxin levels in a single cell type. The noise of the time series extracted from these experiments is characterized by the power spectrum, Hurst exponent, DFA (detrended fluctuation analysis) exponent, first zero and first 1/e crossing of the autocorrelation function. These measures describe the long- and short-term correlations in the signal, which tell us something about the average behavior of these cells in culture. A change in the behavior of these cells is clearly revealed by an examination of these measures. A principal-component analysis shows a separation of the different toxin levels in the multidimensional space. To our knowledge, this is the most sensitive technique for detecting such a low level of cytochalasin B in 3T3 fibroblast cells.

Lovelady, Douglas; Rabson, David; Lo, Chun-Min



Feeder-free growth of undifferentiated human embryonic stem cells.  


Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production. PMID:11581665

Xu, C; Inokuma, M S; Denham, J; Golds, K; Kundu, P; Gold, J D; Carpenter, M K



Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor  

PubMed Central

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN- induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell- binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.



Role of versican and hyaluronan in the differentiation of 3T3-L1 cells into preadipocytes and mature adipocytes.  


We have previously shown that during the adipose conversion of these cells the culture medium changed its viscoelastic properties due to the presence of hyaluronan and a chondroitin sulfate proteoglycan [Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M., 1991. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. J. Biol. Chem. 266, 11237-11244., Calvo, J.C., Gandjbakhche, A.H., Nossal, R., Hascall, V.C., and Yanagishita, M., 1993. Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures. Arch. Biochem. Biophys. 302, 468-475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a "versican-like" molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from non-differentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100-94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation. PMID:17513099

Zizola, Cynthia F; Julianelli, Vanina; Bertolesi, Gabriel; Yanagishita, Masaki; Calvo, Juan Carlos



Pigeons discriminate between human feeders.  


Considered as plague in many cities, pigeons in urban areas live close to human activities and exploit this proximity to find food which is often directly delivered by people. In this study, we explored the capacity of feral pigeons to take advantage of this human-based food resource and discriminate between friendly and hostile people. Our study was conducted in an urban park. Pigeons were fed by two experimenters of approximately the same age and skin colour but wearing coats of different colours. During the training sessions, the two human feeders displayed different attitudes: one of the feeders was neutral and the second was hostile and chased away the pigeons. During the two test phases subsequent to the training phase, both feeders became neutral. Two experiments were conducted, one with one male and one female feeder and the second with two female feeders. In both experiments, the pigeons learned to quickly (six to nine sessions) discriminate between the feeders and maintained this discrimination during the test phases. The pigeons avoided the hostile feeder even when the two feeders exchanged their coats, suggesting that they used stable individual characteristics to differentiate between the experimenter feeders. Thus, pigeons are able to learn quickly from their interactions with human feeders and use this knowledge to maximize the profitability of the urban environment. This study provides the first experimental evidence in feral pigeons for this level of human discrimination. PMID:21647649

Belguermi, Ahmed; Bovet, Dalila; Pascal, Anouck; Prévot-Julliard, Anne-Caroline; Saint Jalme, Michel; Rat-Fischer, Lauriane; Leboucher, Gérard



Hydroxyframoside B, a secoiridoid of Fraxinus rhynchophylla, inhibits adipocyte differentiation in 3T3-L1 cells.  


Fraxinus rhynchophylla showed significant inhibitory activity on adipocyte differentiation in the 3T3-L1 preadipocyte cell line as assessed by measuring fat accumulation using Oil Red O staining. Further fractionation led to the isolation of two secoiridoids, oleuropein and hydroxyframoside B. Hydroxyframoside B significantly reduced fat accumulation and triglyceride content in differentiated 3T3-L1 cells without affecting cell viability, whereas oleuropein showed little effect. Further studies with interval treatment demonstrated that hydroxyframoside B exerted inhibitory activity on adipocyte differentiation when treated within 2 days (days 0-2) after differentiation induction. In addition, hydroxyframoside B significantly blocked the induction of adipogenic transcription factors such as C/EBP ?, C/EBP ?, and PPAR ?. Taken together, these results suggest that hydroxyframoside B inhibited early/middle stage of adipogenic differentiation, in part, via inhibition of C/EBP ?, C/EBP ?, and PPAR ?-dependent pathways. PMID:21294077

Choi, Kyeong-Mi; Shin, Eunjin; Liu, Qing; Yoo, Hwan-Soo; Kim, Young Choong; Sung, Sang Hyun; Hwang, Bang Yeon; Lee, Mi Kyeong



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

PubMed Central

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.

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



Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.  


Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. PMID:22180388

Choi, Eun Mi



Arachidonic acid inhibits lipogenic gene expression in 3T3-L1 adipocytes through a prostanoid pathway  

Microsoft Academic Search

This report examines the effect of polyunsatu- rated fatty acids (PUFA) on lipogenic gene expression in cultured 3T3-L1 adipocytes. Arachidonic acid (20:4, n-6) and eicosapentaenoic acid (20:5, n-3) suppressed mRNAs encoding fatty acid synthase (FAS) and S14, but had no ef- fect on b -actin. Using a clonal adipocyte cell line containing a stably integrated S14CAT fusion gene, oleic acid

Michelle K. Mater; David Pan; W. G. Bergen; Donald B. Jump


Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line.  


It has been known for many years that adipocytes express high affinity ACTH and alpha-melanocyte stimulating hormone (MSH) binding sites, and that ACTH, alpha-MSH, and beta-lipotropin are potent lipolytic hormones. We show here that the adipocyte response to the melanocortin peptides results from the expression of both the MC2 (ACTH) receptor as well as the newly discovered MC5 receptor. Using RT-PCR and Northern blot hybridization, high levels of MC2 receptor messenger RNA (mRNA) were found in all adipose tissues examined in the mouse, whereas MC5 receptor mRNA was found in a subset of these. Both receptors mRNAs were also found in the 3T3-L1 cell line but only after the cells had been induced to differentiate into adipocytes. This cell line was then used to characterize the pharmacological properties of the MC2 and MC5 receptor sites in situ. The MC2 receptor exhibits properties similar to the ACTH receptor characterized in adrenocortical cells, coupling to activation of adenylyl cyclase with an EC50 of approximately 1 nM. An MSH binding site characterized in these cells is presumably the MC5 receptor, based on the observation that this is the only other melanocortin receptor mRNA detected in these cells. The MC5 receptor in the 3T3-L1 adipocyte activated adenylyl cyclase in response to alpha-MSH stimulation. Interestingly, Nle4, D-Phe7-alpha-MSH (NDP-MSH), a commonly used synthetic alpha-MSH agonist, was a potent antagonist of the MC5 receptor expressed in the 3T3-L1 cell line. Although the agouti signaling peptide is a potent antagonist of NDP-MSH binding to the MC1 and MC4 melanocortin receptors, agouti was unable to block NDP-MSH binding in the 3T3-L1 adipocyte. PMID:8612546

Boston, B A; Cone, R D



Potential Role of the src Gene Product in Inhibition of Gap-Junctional Communication in NIH\\/3T3 Cells  

Microsoft Academic Search

The effects of the src gene on the activity of protein kinase C and intercellular communication have been studied in transformed NIH\\/3T3 clones isolated from soft agar following transfection with the plasmid carrying the v-src gene (psrc-11). Six transformed clones that were studied contained newly incorporated v-src genes in the genome, had an increased amount of pp60src, and showed enhanced

Chia-Cheng Chang; James E. Trosko; Hsing-Jien Kung; David Bombick; Fumio Matsumura



Expression of Gene rrg is Associated with Reversion of NIH 3T3 Transformed by LTR-c-H-ras  

Microsoft Academic Search

A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH 3T3 cells, but is down-regulated after cellular transformation by long terminal repeat (LTR)-activated c-H-ras (LTR-c-H-ras). This gene was reexpressed in a nontumorigenic persistent revertant cell line created by prolonged treatment of the transformed cells with mouse interferon alpha\\/beta. Persistent revertants stably transfected with

Sara Contente; Kaylene Kenyon; Donata Rimoldi; Robert M. Friedman



Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells  

Microsoft Academic Search

We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In con- trol cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were ap- parently unstained.

Karen L. Leach; Elaine A. Powers; Valerie A. Ruff; Susan Jaken; Scott Kaufmann



Induction of Cell Proliferation in Quiescent NIH 3T3 Cells by Oncogenic cRaf1  

Microsoft Academic Search

The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (DRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion




[Antisense TIF3 reverses the oncogenic potential of CdCl2-transformed BALB/c-3T3 cells].  


TIF3 (GenBank Accession Number AF 271072) is identified as a novel cadmium- responsive proto-oncogene. In order to determine whether the antisense TIF3 reverses the oncogenic potential of Cd-transformed BALB/c-3T3 cells or not, a stable expression system of CdCl2-transformed BALB/c-3T3 cells with the expression vector containing TIF3 cDNA in the antisense orientation using calcium phosphate and G418 selection protocols is firstly established. Then, the reversal of the oncogenic potential of these cells is tested by soft agar and nude mouse tumorigenicity assay. The results demonstrated that expression of the antisense TIF3 in the CdCl2-transformed BALB/c-3T3 cells results in reversal of the transformed phenotype of the cells. This is evidenced by a 25%-70% decrease in the number of anchorage-independent colonies growing on soft agar and the significant reduced tumorigenic potential of cells in nude mice compared with the corresponding controls. In addition to a significant delay in the onset of appearance of tumors, a significant reduction in size and a 50.8%-55.1% decrease in weight of the tumors are also observed in the mice injected with the TIF3 antisense expressing cells compared with the corresponding controls. The results indicate that antisense TIF3 mRNA expression reverses its oncogenic potential of Cd-transformed BALB/c-3T3 cells and may have therapeutic potential to cancer induced by cadmium. PMID:12600033

Lei, Yixiong; Pius, Joseph; Wu, Zhongliang; Ong, Tong-man



Alignment and proliferation of MC3T3-E1 osteoblasts in microgrooved silicone substrata subjected to cyclic stretching  

Microsoft Academic Search

Previous studies have shown that many types of cells align in microgrooves in static cultures. However, whether cells remain aligned and also proliferate in microgrooves under stretching conditions has not been determined. We grew MC3T3-E1 osteoblasts in deformable silicone dishes containing microgrooves oriented in the stretch direction. We found that with or without 4% stretching, cells aligned in microgrooves of

James H.-C Wang; Edward S Grood; Jane Florer; Richard Wenstrup



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


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

Mukherjee, Rajib; Yun, Jong Won



Role of Epidermal Growth Factor and ErbB2 Receptors in 3T3-L1 Adipogenesis  

Microsoft Academic Search

Objective: Epidermal growth factor (EGF) stimulates proliferation in 3T3-L1 preadipocytes, but EGF action in differentiation is less clear. EGF promotes differentiation at concentrations <1 nM but inhibits differentiation at higher concentrations, suggesting a dual role in adipogenesis. We hypothesized that differences in EGF receptor activation and downstream signaling mediate distinct biological effects of EGF at low vs. high abundance.Research Methods

Molly Harrington; Sunthorn Pond-Tor; Charlotte M. Boney



Adhesion to fibronectin's EDbdomain induces tyrosine phosphorylation of focal adhesion proteins in Balb\\/c 3T3 cells  

Microsoft Academic Search

Balb\\/c 3T3 cell adhesion on substrata coated with fibronectin's (FN) alternatively-spliced EDb, implicated in some tumor cell systems, and its neighboring type III repeats (III7 and III8) induced intracellular signaling coincident with morphological responses. These events were analysed using minigene-expressed proteins containing various permutations of type III repeats of FN. Cells adherent to the tri-repeat protein 7-EDb-8 were compared to

Wensheng Chen; Lloyd A. Culp



Antigenic Properties of Endogenous Type-C Viruses from Spontaneously Transformed Clones of BALB\\/3T3  

Microsoft Academic Search

During long-term tissue culture of spontaneously transformed clones derived from BALB\\/c 3T3 mouse-embryo cells, some clones spontaneously begin to produce high titers of endogenous murine type-C viruses. The antigenic properties of these viruses have been analyzed by indirect immunoelectronmicroscopy and can be classified into two distinguishable populations: (a) BALB\\/c murine myeloma-associated extracellular viruses that carry a specific envelope antigen, xVEA,

Tadao Aoki; George J. Todaro



Monensin-resistant mouse Balb\\/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus  

Microsoft Academic Search

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb\\/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles




C-reactive protein inhibits adiponectin gene expression and secretion in 3T3-L1 adipocytes  

Microsoft Academic Search

C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. The aim of the present study is to investigate the effects of CRPon the production of adiponectin in 3T3-L1 adipocytes. Northern and western blot analysis revealed that CRP treatment inhibited adiponectin mRNA expression and secretion in a dose- and time-dependent manner. Co-incubation of adipocytes with rosiglitazone

Guoyue Yuan; Xia Chen; Qinyun Ma; Jie Qiao; Rongying Li; Xuesong Li; Shengxian Li; Jinfeng Tang; Libin Zhou; Huaidong Song; Mingdao Chen



Wortmannin, a Specific Phosphatidylinositol 3Kinase Inhibitor, Inhibits Adipocytic Differentiation of 3T3-L1 Cells  

Microsoft Academic Search

The effect of wortmannin, which inhibits phosphatidylinositol 3-kinase (PI 3-kinase), on adipocytic differentiation of 3T3-L1 cells was examined. The extent of differentiation was evaluated by the staining of adipocytes with Oil Red O and measurement of glycerol 3-phosphate dehydrogenase (GPDH) activity. Wortmannin, at over 100 nM, significantly inhibited adipogenesis of cells treated with isobutyl-methylxanthine, dexamethasone, and insulin with an IC50

K. Tomiyama; H. Nakata; H. Sasa; S. Arimura; E. Nishio; Y. Watanabe



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

Microsoft Academic Search

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

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



Contribution of enhanced transcriptional activation by ER to [12Val] K-Ras mediated NIH3T3 cell transformation  

Microsoft Academic Search

We investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation by the [12Val] K-Ras mutant. This mutant enhanced the steady state level of ER. Cells expressing mutant K-Ras (K12V cell) were tumorigenic. To determine the role of ER accumulation in Ras-transformed cells, we developed cells (KwtER cells) that overexpressed both wild-type (wt) K-Ras and ER, and found

Kiyoko Kato; Yousuke Ueoka; Keiji Kato; Toshiro Hachiya; Jun-ichi Nishida; Norio Wake; N Wake



DJ1, a Novel Oncogene Which Transforms Mouse NIH3T3 Cells in Cooperation with ras  

Microsoft Academic Search

We have isolated and characterized the cDNA encoding a novel protein designated DJ-1. DJ-1, sharing no significant homology with the sequences so far reported, did not show transactivation activity in the Gal4 recombinant system, but transformed mouse NIH3T3 cells by itself. Furthermore, DJ-1 showed a cooperative transforming activity with H-Ras, more than 3 times as strong as the activity ofras\\/myccombination.

Daisuke Nagakubo; Takahiro Taira; Hirotake Kitaura; Masako Ikeda; Katsuyuki Tamai; Sanae M. M. Iguchi-Ariga; Hiroyoshi Ariga



Gas6-mediated survival in NIH3T3 cells activates stress signalling cascade and is independent of Ras  

Microsoft Academic Search

Gas6 is a growth factor membrane of the vitamin K-dependent family of proteins which is preferentially expressed in quiescent cells. Gas6 was identified as the ligand for Axl tyrosine kinase receptor family. Consistent with this, Gas6 was previously reported to induce cell cycle re-entry of serum-starved NIH3T3 cells and to prevent cell death after complete growth factor withdrawal, the survival

Sandro Goruppi; Elisabetta Ruaro; Brian Varnum; Claudio Schneider



Effects of G6PD Overexpression in NIH3T3 Cells Treated with TertButyl Hydroperoxide or Paraquat  

Microsoft Academic Search

The major physiological role of glucose-6-phosphate dehydrogenase (G6PD) is to provide NADPH, which is required for reductive biosynthesis and for detoxification of free radicals and peroxides in mature red blood cells. To study the function of G6PD in non-erythroid cells, we examined the sensitivity of NIH3T3 cells transfected with a plasmid containing human G6PD cDNA to tert-butyl hydroperoxide (TBH) and

Wei-Ying Kuo; Tang K Tang



Characterization of Wnt1 and Wnt2 induced growth alterations and signaling pathways in NIH3T3 fibroblasts  

Microsoft Academic Search

Members of the Wnt family induce mouse mammary tumors and partially transform mammary epithelial cells in culture. However, their mechanism of transformation remains to be elucidated. In NIH3T3 mouse embryo fibroblasts, a standard transformation model, Wnt-1 and Wnt-2 were shown to induce altered properties including increased saturation density and growth in soft agar. Such cells also exhibited increased cell-cell adhesiveness.

Anna Bafico; Arnona Gazit; Sidney S Wu-Morgan; Abraham Yaniv; Stuart A Aaronson



Repression of thrombospondin-1 expression, a natural inhibitor of angiogenesis, in polyoma middle T transformed NIH3T3 cells  

Microsoft Academic Search

Thrombospondin-1 (TS1) is a modular extracellular matrix glycoprotein and expressed by many cell types in culture. Thrombospondin-1 inhibits angiogenesis and its expression inversely correlates with the degree of invasiveness and metastasis in tumor cell lines. Here, we demonstrate that expression of Polyoma middle T oncogene in NIH3T3 cells results not only in transformation but also represses expression of three thrombospondin

Nader Sheibani; William A. Frazier



Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts  

Microsoft Academic Search

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK?

Georgina Caruana; Antony C Cambareri; Leonie K Ashman



Overexpression of phospholipase C?-1 protects NIH3T3 cells from oxidative stress-induced cell death  

Microsoft Academic Search

Oxidative stress has been implicated in a wide range of cellular damage which includes DNA oxidation, membrane lipid peroxidation, and apoptosis. In our study, we found that overexpression of PLC-?1 in NIH3T3 fibroblasts protected them from cell death occuring in response to oxidative stress. Cell death caused by treatment with prooxidant tert-butylhydroperoxide (TBH), H2O2, or CdCl2 was considerably suppressed in

Young Han Lee; Seong-Yong Kim; Jae-Ryong Kim; Kyu-Tong Yoh; Suk-Hwan Baek; Myong Jong Kim; Sung Ho Ryu; Pann-Ghill Suh; Jung-Hye Kim



FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes  

Microsoft Academic Search

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and

Brian M. Wiczer; Sandra Lobo; G. Luke Machen; Lee M. Graves; David A. Bernlohr



The Effect of Secoisolariciresinol on 3t3-L1 Adipocytes and the Relationship Between Molecular Structure and the Activity  

Microsoft Academic Search

\\u000a Secoisolariciresinol (SECO) is one of the lignans contained in flaxseed. SECO has some stereoisomeric forms, and they exist\\u000a as the racemic form in flaxseed. Therefore, we stereoselectively synthesized (+)-SECO, (–)-SECO, and meso-SECO forms. In this study, we focused on the effects of SECOs on the regulation of 3T3-L1 adipocytes, and revealed the structure-activity\\u000a relationships. As a result, (–)-SECO accelerated adiponectin

Shiori Tominaga; Takuya Sugahara; Sogo Nishimoto; Manami Yamawaki; Yuki Nakashima; Taro Kishida; Koichi Akiyama; Masafumi Maruyama; Satoshi Yamauchi


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

Microsoft Academic Search

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.

A. Zetterberg; Olle Larsson



Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells  

Microsoft Academic Search

Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to




Establishment and use of 3t3 NRU assay for assessment of phototoxic hazard of cosmetic products  

Microsoft Academic Search

Objective: To establish an alternative 3T3 mouse fibroblast neutral red uptake assay and investigate the feasibility of utilizing an alternative in vitro method to replace the animal testing, in particular, in phototoxicity assessment of cosmetics. Method: Fifteen phototoxic and nine non-phototoxic chemicals were tested and 20 functional cosmetic products were assessed. The mean photo effect (MPE) and the photo-irritation factor

Ying Yanga; Xikun Xiong; Xinfen Yang; Junming Huan; Xiaohua Tan; Qing Li; Xiwen He


The Differentiation and Proliferation-Inhibitory Effects of Sporamin from Sweet Potato in 3T3-L1 Preadipocytes  

Microsoft Academic Search

The aim of this study was to investigate the effect of different concentrations of sporamin on the differentiation and proliferation of 3T3-L1 preadipocytes, providing the theoretical basis for the development of food to treat obesity and diabetes. The isolation and purification of sporamin from sweet potato species 55-2 were performed by ammonium sulphate precipitation in combination with ion-exchange and gel

Zhi-dong XIONG; Peng-gao LI; Tai-hua MU



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

PubMed Central

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.

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



Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines  

Microsoft Academic Search

Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of

P. Wang; E. Mariman; J. Keijer; F. Bouwman; J.-P. Noben; J. Robben; J. Renes



Dietary Fatty Acids Differentially Regulate Production of TNF-? and IL10 by Murine 3T3-L1 Adipocytes  

Microsoft Academic Search

Objective:Obesity correlates with increased production of adipocyte-derived cytokines, which may contribute to a chronic subclinical inflammation seen in obese individuals. This study evaluated the ability of specific fatty acids to modulate production of the proinflammatory cytokine, tumor necrosis factor-? (TNF-?), and the anti-inflammatory cytokine, interleukin-10 (IL-10), in murine 3T3-L1 adipocytes. Effects on nuclear factor-?B (NF-?B), a key transcriptional activator of

Richard L. Bradley; FFolliott M. Fisher; Eleftheria Maratos-Flier



Bisphenol A Accelerates Terminal Differentiation of 3T3-L1 Cells into Adipocytes through the Phosphatidylinositol 3Kinase Pathway  

Microsoft Academic Search

In order to identify whether bisphenol A (BPA) acts as an adipogenic agent, following the hormonal induction of differen- tiation into adipocytes, 3T3-L1 cells were treated for six days with BPA alone. Treatment with BPA increased the triacylglycerol (TG) content of the cultures, increased the percentage of Oil Red O-staining cells in the cultures, and increased the levels of lipoprotein

Hiroshi Masuno; Jun Iwanami; Teruki Kidani; Kenshi Sakayama; Katsuhisa Honda



Bisphenol A in combination with insulin can accelerate the conversion of 3T3-L1 fibroblasts to adipocytes  

Microsoft Academic Search

The confluent cultures of 3T3-L1 fibroblasts were treated with or without bisphenol A (BPA) for 2 days and subsequently treated with insulin (INS) alone for 9 days. When BPA was absent during the first 2-day treatment period, the cultures contained 1.6 ? g\\/ ? g DNA of triacyl- glycerol (TG), 202 mU\\/mg DNA of lipoprotein lipase (LPL) activity, and 462

Hiroshi Masuno; Teruki Kidani; Keizo Sekiya; Kenshi Sakayama; Takahiko Shiosaka; Haruyasu Yamamoto; Katsuhisa Honda


Dimethylfumarate suppresses adipogenic differentiation in 3T3-L1 preadipocytes through inhibition of STAT3 activity.  


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

Kang, Hyeon-Ji; Seo, Hyun-Ae; Go, Younghoon; Oh, Chang Joo; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu



Expression of tissue transglutaminase in Balb-C 3T3 fibroblasts: effects on cellular morphology and adhesion  

Microsoft Academic Search

Tissue transglutaminase is a cytosolic en- zyme whose primary function is to catalyze the cova- lent cross-linking of proteins. To investigate the func- tions of this enzyme in physiological systems, we have established lines of Balb-C 3T3 fibroblasts stably transfected with a constitutive tissue transglutaminase expression plasmid. Several cell lines expressing high levels of catalytically active tissue transglutaminase have been

Vittorio Gentile; Vilmos Thomazy; Mauro Piacentini; Laszlo Fesus; Peter J. A. Davies



The effect of heat shock on amino acid transport and cell volume in 3T3 cells  

Microsoft Academic Search

Summary.   In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up\\u000a to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The\\u000a activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased\\u000a temperature. The maintenance

P. G. Petronini; A. E. Caccamo; R. R. Alfieri; M. A. Bonelli; A. F. Borghetti



S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C  

SciTech Connect

Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by ({sup 3}H)thymidine incorporation into nuclear DNA. In addition, {approx} 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC to override the cellular G{sub 0} block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.

Smith, M.R.; Ryu, Sungho; Suh, Panghill; Rhee, Suegoo; Kung, Hsiangfu (National Cancer Institute, Frederick, MD (USA))



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

SciTech Connect

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.

Lubin, M.



Critical role of leukotriene B4 receptor signaling in mouse 3T3-L1 preadipocyte differentiation  

PubMed Central

Background Various inflammatory mediators related to obesity might be closely related to insulin resistance. Leukotrienes (LTs) are involved in inflammatory reactions. However, there are few reports regarding the role of LTs in adipocyte differentiation. Therefore, we investigated the role of leukotriene B4 (LTB4)-leukotriene receptor (BLT) signaling in mouse 3T3-L1 fibroblastic preadipocyte differentiation to mature adipocytes. Methods Mouse 3T3-L1 preadipocytes were treated with lipoxygenase (LOX) inhibitors, BLT antagonist, and small interfering RNA (siRNA) for BLT1 and BLT2 to block the LTB4-BLT signaling pathway, then the adipocyte differentiation such as lipid accumulation and the increase in triglyceride was evaluated. Results Blockade of BLT signaling by treatment with a LOX inhibitor or a BLT antagonist suppressed preadipocyte differentiation into mature adipocytes. In addition, knockdown of BLT1 and BLT2 by siRNAs dramatically inhibited differentiation. These results indicate the LTB4-BLT signaling pathway may positively regulate preadipocyte differentiation and be a rate-limiting system to control adipocyte differentiation. Conclusions The LTB4-BLT signaling pathway provides a potent regulatory signal that accelerates the differentiation of mouse 3T3-L1 preadipocytes. Further investigations are necessary to confirm the exact role of LTB4 and BLTs signaling pathways in preadipocyte differentiation.



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

PubMed Central

?-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 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.

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



Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates  

SciTech Connect

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10{sup {minus}9} and 10{sup {minus}7} M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.

Kato, Shigemi; Ben, T.L.; De Luca, L.M. (National Institutes of Health, Bethesda, MD (USA))



Recombinant tissue metalloproteinase inhibitor-3 protein induces apoptosis of murine osteoblast MC3T3-E1.  


Tissue inhibitor of metalloproteinases (TIMPs) plays an essential role in the regulation of bone metabolism. Here we report that recombinant tissue metalloproteinase inhibitor-3 (TIMP-3) protein induces the apoptosis of MC3T3-E1 osteoblasts. Cell apoptosis was detected by sandwich-enzyme-immunoassay. Fas and Fasl protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-8. The phosphorylation of JNK, p38 and ERK1/2 was examined by Western blot analysis. The ELISA suggested that TIMP-3 promoted MC3T3-E1 cell apoptosis. TIMP-3 treatment induced the expression of Fas and Fasl proteins, and the activation of caspase-8 and caspase-3. TIMP-3 treatment induced p38 and ERK phosphorylation. SB203580 and PD98059, the inhibitor of p38 and ERK, respectively, abolished the TIMP-3 effect on osteoblast apoptosis. In conclusion, the signal pathway through which TIMP-3 induces MC3T3-E1 cell apoptosis, mediated by Fas and involves the p38 and ERK signal transduction pathways. PMID:18157585

Yuan, L-Q; Liu, Y-S; Luo, X-H; Guo, L-J; Xie, H; Lu, Y; Wu, X-P; Liao, E-Y



A long-range attraction between aggregating 3T3 cells mediated by near-infrared light scattering  

PubMed Central

At what range can a mammalian cell sense the presence of another cell and through what medium? To approach these questions, the formation of aggregates of a 3T3 cell variant (3T3x cells) grown on solid substrates was studied. Each of the aggregates consisted of cells that, at the time of their seeding, were single and located randomly. Yet somehow they seemed to detect each other within a certain range (Ra) and move together to form aggregates. The article describes a simple assay to measure the value of Ra. When applied to 3T3x cells with altered intensities of near-infrared light scattering (Isc) the assay showed that (i) Ra was much larger than one cell diameter, and (ii) Ra was directly related to Isc. The results suggest that near-infrared light scattering by the cells mediate a long-range attraction between them, which does not require physical contact and enables them to detect each other's presence.

Albrecht-Buehler, Guenter



Geometry and emplacement of basaltic feeder dykes  

NASA Astrophysics Data System (ADS)

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.

Galindo, I.; Gudmundsson, A.



895. The differential effect of BMP4 on C2C12 and NIH\\/3T3 cells: Implications for gene therapy for bone and cartilage repair  

Microsoft Academic Search

The present study was designed to compare the osteogenic potential of NIH\\/3T3 cells and C2C12 cells following BMP4 stimulation and their potential for bone and cartilage repair after genetic engineering to express BMP4. Using different concentrations of BMP4 protein, we observed that NIH\\/3T3 cells were able to undergo osteogenic differentiation in vitro. Compared to the myogenic C2C12 cell line, NIH\\/3T3

Guangheng Li; Hairong Peng; Karin Corsi; Arvydas Usas; Johnny Huard



GH induced lipolysis stimulation in 3T3-L1 adipocytes stably expressing hGHR: analysis on signaling pathway and activity of 20K hGH  

Microsoft Academic Search

We have constructed a cell line of 3T3-L1 which can efficiently express human GHR (3T3-L1-hGHR) after differentiation to adipocytes. The expressed hGHR was detected as two bands with approximate molecular sizes of 120K by Western analysis using hGHR specific monoclonal antibody. Maximum lipolytic activity induced by hGH in the 3T3-L1-hGHR was enhanced 10-fold as compared to that in 3T3-L1, suggesting

N Asada; Y Takahashi; M Wada; N Naito; H Uchida; M Ikeda; M Honjo




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



Distribution feeder voltage regulation control  

Microsoft Academic Search

Step voltage regulators are the work horse of distribution feeders for maintaining the voltage at every customer's meter to be within the ANSI standards. A step voltage regulator can be viewed as a tap changing autotransformer. This paper will apply a model of the step voltage regulator. The IEEE 13 Node Test Feeders will be used to demonstrate how the

W. H. Kersting



Distribution Feeder Voltage Regulation Control  

Microsoft Academic Search

Step voltage regulators are the workhorse of distribution feeders for maintaining the voltage at every customer's meter to be within the ANSI standards. A step voltage regulator can be viewed as a tap-changing autotransformer. This paper will apply a model of the step voltage regulator. The IEEE 13 Node Test Feeders will be used to demonstrate how the regulator is

W. H. Kersting



New Insights into Cytosolic Glucose Levels during Differentiation of 3T3-L1 Fibroblasts into Adipocytes*  

PubMed Central

Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolic free glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600?. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only 56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with the one recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (?R) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p < 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose.

Kovacic, Petra Brina; Chowdhury, Helena H.; Velebit, Jelena; Kreft, Marko; Jensen, J?rgen; Zorec, Robert



Effect of TAT-obestatin on proliferation, differentiation, apoptosis and lipolysis in 3T3-L1 preadipocytes.  


It has been reported that obestatin regulates adipocyte metabolism via receptors on the cell surface. We wondered whether obestatin can interact with intracellular components that activated signalling pathways in adipocytes. Because obestatin (human) only presents one lysine (at position 10), which cannot penetrate the cell membrane, therefore, we used a cell-permeable peptide TAT (49-57) as a vector to carry obestatin across the cell membrane. The goal of this study was to further understand the function of obestatin after penetrating the cell membrane. Our results showed that TAT-obestatin could cross the 3T3-L1 cell membrane in the absence of cytotoxicity. TAT-obestatin showed no effect on the proliferation of 3T3-L1 preadipocytes. In contrast, obestatin significantly stimulated proliferation at a dose of 10(-11) ?M and 10(-13) ?M. In addition, TAT-obestatin demonstrated a more potent inhibitory effect on cell apoptosis induced by serum starvation than that of obestatin. During the progress of adipocyte differentiation, TAT-obestatin and obestatin had no effect on adipogenesis. In the lipolysis assay, TAT-obestatin significantly increased glycerol and free fatty acid release from 3T3-L1 adipocytes after 3?h treatment but showed no significant effect on lipolysis after 24?h and 48?h of treatment. In contrast, obestatin (10(-7) ?M) had no effect on glycerol release after 3, 24 and 48?h of treatment. The difference between the effect of TAT-obestatin and obestatin on adipocytes metabolism indicated that TAT-obestatin may trigger intracellular signalling as well as signalling at the cell membrane. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. PMID:24106000

Ren, Guangcai; He, Zuyong; Cong, Peiqing; Yu, Jingwei; Qin, Yufeng; Chen, Yaosheng; Liu, Xiaohong



MC3T3-E1 Cell Adhesion to Hydroxyapatite with Adsorbed Bone Sialoprotein, Bone Osteopontin, and Bovine Serum Albumin  

PubMed Central

Native bone tissue is composed of a complex matrix of collagen, non-collagenous proteins, and hydroxyapatite (HAP). Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the non-collagenous protein family termed the SIBLING (small integrin-binding ligand, N-linked glycoproteins) proteins, which are primarily found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen, while the MC3T3-E1 cell adhesion was shown to be dependant on the conformational flexibility of BSP specifically bound to collagen. Additionally, OPN was shown to play a greater role than BSP for cell binding to collagen. In this work, the orientations and conformations of BSP and OPN specifically bound to HAP are probed under similar conditions. Radiolabeled adsorption isotherms were obtained for BSP and OPN on HAP formed from a simulated body fluid, and the results show that HAP has the capacity to bind significantly more BSP than OPN. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of adsorbed BSP and OPN specifically bound to HAP. It was found that there is a preference for cell binding to HAP with adsorbed BSP as compared to OPN, but not to a statistically significant level. However, the maximum cell binding was observed on HAP substrates with adsorbed heat denatured bovine serum albumin (BSA). The influence of BSA on cell binding was shown to be concentration dependant and it is believed that the adsorbed BSA modulates the proliferation state of the bound cells.

Bernards, Matthew T.; Qin, Chunlin; Jiang, Shaoyi



SiRNA against Fabp5 induces 3T3-L1 cells apoptosis during adipocytic induction  

Microsoft Academic Search

Fatty acid-binding protein 5 (Fabp5), exhibits an important role in binding free fatty acids, as well as regulating lipid\\u000a metabolism and transport. The purpose of this study was to evaluate the role of Fabp5 during adipogenesis. 3T3-L1 preadipocytes\\u000a were selected as cell differentiation model and short interfering RNAs (siRNA) against Fabp5 (siFabp5) were prepared. Our\\u000a results showed that two potent

Xi MaXia; Xia Ren; Pengfei Han; Shengdi Hu; Junjun Wang; Jingdong Yin



Effects of local anesthetics on cell morphology and membrane-associated cytoskeletal organization in BALB/3T3 cells  

PubMed Central

Tertiary amine local anesthetics (dibucaine, tetracaine, procaine) reversibly affect the morphology of untransformed BALB/3T3 cells and the organization of membrane-associated cytoskeletal elements. In the presence of these drugs cells contract and become rounded in shape with the appearance of numerous surface "blebs." Electron microscope examination of anesthetic-treated cells revealed significant reductions in plasma membrane-associated microtubules and microfilaments and/or their plasma membrane attachment. The relationship of the findings on local anesthetic-induced changes in cellular cytoskeletal systems is discussed in relation to previous proposals on plasma membrane organization and control of cell surface receptor topography and mobility.



Inhibitory effects of flavonoids from Abelmoschus manihot flowers on triglyceride accumulation in 3T3-L1 adipocytes.  


The 95% EtOH extract from the flowers of Abelmoschus manihot (L.) Medic showed inhibitory activity on TG accumulation in 3T3-L1 preadipocyte. Chemical studies on the active fraction led to the isolation of 14 flavonoids (1-14). To clarify the multi-mechanism of the isolates on preadipocyte differentiation, the levels of TG and FFA and the related role transcription factors (PPAR?, CEBP/?, and ap2) expression were evaluated. At the concentration of 30 ?M, compounds 1-6 and 10-14 showed inhibitory activity on TG accumulation significantly in mature 3T3-L1 cells. 1, 2, 4-7, 9, 10, 13, and 14 reduced the level of FFA. At the molecular level, the mRNA expressions of PPAR?, CEBP/?, and ap2 were down-regulated by compounds 1, 5, 9, 12, 13; 1-8, 10-14; and 1-4, 6, 8-12, 14, respectively. The structure-activity relationships of the 14 flavonoids were also discussed. PMID:21281705

An, Yating; Zhang, Yi; Li, Chunmei; Qian, Qian; He, Wei; Wang, Tao



Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts.  


The present work was initiated to investigate regulation of the taurine transporter TauT by reactive oxygen species (ROS) and the tonicity-responsive enhancer binding protein (TonEBP) in NIH3T3 mouse fibroblasts during acute and long-term (4 h) exposure to low-sodium/hypo-osmotic stress. Taurine influx is reduced following reduction in osmolarity, keeping the extracellular Na(+) concentration constant. TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions. In contrast, TonEBP activity and TauT transcription are significantly increased following hyperosmotic exposure. Swelling-induced ROS production in NIH3T3 fibroblasts is generated by NOX4 and by increasing total ROS, by either exogenous application of H(2)O(2) or overexpressing NOX4, we demonstrate that TonEBP activity and taurine influx are regulated negatively by ROS under hypo-osmotic, low-sodium conditions, whereas the TauT mRNA level is unaffected. Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics. Thus, swelling-induced ROS production could account for the reduced taurine uptake under low-sodium/hypo-osmotic conditions by direct modulation of TauT. PMID:22383044

Hansen, Daniel Bloch; Friis, Martin Barfred; Hoffmann, Else Kay; Lambert, Ian Henry



Overexpression of EMS1/cortactin in NIH3T3 fibroblasts causes increased cell motility and invasion in vitro.  


Cortactin, a p80/85 protein first identified as a src kinase substrate, is thought to be involved in the signaling pathway of mitogenic receptors and adhesion molecules mediating cytoskeletal reorganization. The cortactin gene, EMS1, maps to chromosome 11q13, a region amplified in head and neck squamous cell carcinomas (HNSCC) and breast cancer, which display lymph node metastasis and an unfavorable clinical outcome. To further address the role of cortactin in the malignant phenotype of cells, we stably overexpressed cortactin in NIH3T3 fibroblasts and evaluated the effects of elevated cortactin on cellular proliferation, motility and invasiveness. Cortactin overexpressing cells did not display any striking morphological changes, nor any significant differences in cell proliferation or saturation density as compared to control NIH3T3 cells. Furthermore, the cortactin overexpressing cells were anchorage dependent for growth. Interestingly, cortactin overexpressing cells were more motile and invasive in modified Boyden chamber assays. These results suggest that overexpression of cortactin may play a role in tumor progression by influencing tumor cell migration and invasion. PMID:9681820

Patel, A S; Schechter, G L; Wasilenko, W J; Somers, K D



The anti-obesity effect of Lethariella cladonioides in 3T3-L1 cells and obese mice  

PubMed Central

The aim of this study was to investigate whether a water extract of L. cladonioides (LC) has an anti-obesity effect in 3T3-L1 cells and obese mice. Treatment of differentiated 3T3-L1 adipocytes with LC caused a significant increase in glycerol release and reduced the protein expression of the adipogenic transcription factors, PPAR? and C/EBP?. In an animal model, obese mice were artificially induced by a high fat diet for 10 weeks. Experimental groups were treated with LC (100 mg/kg/day) by gavage for the next 10 weeks. At the end of experiment, the body weight of the LC group mice was reduced by 14.2% compared to the high fat diet (HFD) group. The treatment also decreased liver (31.0%), epididymal (18.0%) and retroperitoneal (19.3%) adipose tissue, and kidney (6.7%) weights, respectively, compared with those of the HFD group. LC prevented diet-induced increases in the serum level of TC (22.6%), TG (11.6%), and glucose (35.0%), respectively, compared with the HFD group. However, the HDL-C level was higher in the LC group (26.1%) than the HFD group. The results of this study thus suggest that LC suppressed lipid accumulation and expression of adipogenic transcription factors, and increased the amount of glycerol release. LC also indicated an anti-obese and anti-hyperlipidemic effect.

Sung, Ju-Hyun; Chon, Jeong-Woo; Lee, Mi-Ae; Park, Jin-Kyung; Woo, Jeong-Taek



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


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

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



Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes  

SciTech Connect

This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n = 3; p < 0.001), and BrdU incorporation was impaired by 55% (n = 3; p < 0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27{sup kip1}, a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n = 3; p < 0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBP{beta} were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.

Yarmo, Michelle N.; Landry, Anne; Molgat, Andre S.D.; Gagnon, AnneMarie [Department of Medicine, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Sorisky, Alexander [Department of Medicine, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada)], E-mail:



Pancreatic lipase inhibitory gallotannins from Galla Rhois with inhibitory effects on adipocyte differentiation in 3T3-L1 cells.  


Activity-guided isolation of a methanolic extract of Galla Rhois using pancreatic lipase and 3T3-L1 adipocytes led to the isolation of seven phenolic compounds: protoaphin-fb (1), 2-O-digalloyl-1,3,4,6-tetra-O-galloyl-?-D-glucose (2), 1,2,3,4,6-penta-O-galloyl-?-D-glucose (3), 1,2,4,6-tetra-O-galloyl-?-D-glucose (4), 3-hydroxy-5-methoxy-phenol 1-O-?-D-glucoside (5), methylgallate (6), and gallic acid (7). Their structures were established on the basis of NMR and MS spectroscopic data interpretation. All isolates were evaluated for their inhibitory effects on pancreatic lipase, and compounds 1-5 exhibited potent inhibitory effects on this enzyme, with IC?? values ranging from 30.6 ± 2.4 to 3.5 ± 0.5 mM. In addition, the highly galloylated compound 2 was also found to induce potent inhibition of adipocyte differentiation in 3T3-L1 cells. PMID:24002138

Kwon, O Jun; Bae, Jong-Sup; Lee, Ha Yeong; Hwang, Ju-Young; Lee, Eun-Woo; Ito, Hideyuki; Kim, Tae Hoon



Environmental endocrine disruptors promote adipogenesis in the 3T3-L1 cell line through glucocorticoid receptor activation.  


The burgeoning obesity and diabetes epidemics threaten health worldwide, yet the molecular mechanisms underlying these phenomena are incompletely understood. Recently, attention has focused on the potential contributions of environmental pollutants that act as endocrine disrupting chemicals (EDCs) in the pathogenesis of metabolic diseases. Because glucocorticoid signaling is central to adipocyte differentiation, the ability of EDCs to stimulate the glucocorticoid receptor (GR) and drive adipogenesis was assessed in the 3T3-L1 cell line. Various EDCs were screened for glucocorticoid-like activity using a luciferase reporter construct, and four (bisphenol A (BPA), dicyclohexyl phthalate (DCHP), endrin, and tolylfluanid (TF)) were shown to significantly stimulate GR without significant activation of the peroxisome proliferator-activated receptor-gamma. 3T3-L1 preadipocytes were then treated with EDCs and a weak differentiation cocktail containing dehydrocorticosterone (DHC) in place of the synthetic dexamethasone. The capacity of these compounds to promote adipogenesis was assessed by quantitative oil red O staining and immunoblotting for adipocyte-specific proteins. The four EDCs increased lipid accumulation in the differentiating adipocytes and also upregulated the expression of adipocytic proteins. Interestingly, proadipogenic effects were observed at picomolar concentrations for several of the EDCs. Because there was no detectable adipogenesis when the preadipocytes were treated with compounds alone, the EDCs are likely promoting adipocyte differentiation by synergizing with agents present in the differentiation cocktail. Thus, EDCs are able to promote adipogenesis through the activation of the GR, further implicating these compounds in the rising rates of obesity and diabetes. PMID:19927138

Sargis, Robert M; Johnson, Daniel N; Choudhury, Rashikh A; Brady, Matthew J



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

PubMed Central

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.

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



Ethanolic extract of Actaea racemosa (black cohosh) potentiates bone nodule formation in MC3T3-E1 preosteoblast cells.  


Aceaea racemosa (formerly Cimicifuga racemosa, black cohosh, AR) extracts have been widely used as an alternative to hormonal replacement therapy for menopausal symptoms. Recent evidences suggest AR extracts are also effective in protecting against postmenopausal bone loss. To determine whether AR has any direct anabolic effect on osteoblasts, we investigated the ethanolic extract of AR on bone nodule formation in mouse MC3T3-E1 preosteoblast cells. AR did not stimulate osteoblast proliferation. Rather, at high doses of 1000 ng/mL for 48 h, AR suppressed (7.2+/-0.9% vs. control) osteoblast proliferation. At 500 ng/mL, a significant increase in bone nodule formation was seen with Von Kossa staining. Using quantitative PCR analysis, AR was shown to enhance the gene expression of runx2 and osteocalcin. Co-treatment with ICI 182,780, the selective estrogen receptor antagonist, abolished the stimulatory effect of AR on runx2 and osteocalcin gene induction, as well as on bone nodule formation in MC3T3-E1 cells. This is a first report of the direct effect of AR on enhancement of bone nodule formation in osteoblasts, and this action was mediated via an estrogen receptor-dependent mechanism. The results provide a scientific rationale at the molecular level for the claim that AR can offer effective prevention of postmenopausal bone loss. PMID:18555764

Chan, B Y; Lau, K S; Jiang, B; Kennelly, E J; Kronenberg, F; Kung, A W C



Characterization of microRNA expression profiles in 3T3-L1 adipocytes overexpressing C10orf116.  


Our data in the previous report demonstrated that C10orf116 (AFRO) is an adipocyte lineage-specific nuclear factor that can modulate the master adipogenesis transcription factors early during differentiation. However, more precise functional properties of this gene need to be clarified and await further investigation. Therefore, in this study, we performed an expression profile of cellular MicroRNAs (miRNAs) in the C10orf116 overexpression 3T3-L1 adipocytes and performed target prediction and functional enrichment of the differentially expressed miRNAs. Our study identified 34 miRNAs up-regulated in the 3T3-L1 adipocytes stably overexpressing C10orf116, whereas 43 miRNAs up-regulated in the control cells. The target genes of differentially expressed miRNAs were found to be involved in multiple signalling pathways, such as Wnt, TGF-beta, MAPK, Jak-STAT, insulin signalling pathway, et al. Our data provided novel information for the identification of C10orf116. PMID:24052233

Qiu, Jie; Zhou, Xiao-Guang; Zhou, Xiao-Yu; Zhu, Chun; Shi, Chun-Mei; Ji, Chen-Bo; Cheng, Rui; Li, Yong; Guo, Xi-Rong



Characterization of RNA from Noninfectious Virions Produced by Sarcoma Positive-Leukemia Negative Transformed 3T3 Cells  

PubMed Central

RNA from noninfectious virions produced by two established clonal lines of sarcoma positive-leukemia negative (S+L-)-transformed 3T3 cells has been characterized. RNA from virions or nucleoids of S+L--(C243) cells consisted of three to four sizes: ±44 S (6%), 28 S (17%), 18 S (38%), and <18 S (39%). 28S virion RNA contained some virus-specific information demonstrable by RNA·DNA hybridization with a DNA probe derived from the RNA-directed DNA polymerase product of murine sarcoma-leukemia virus, while parallel studies indicated that 28S ribosomal RNA from ribosomal subunits of transformed and nontransformed 3T3 cells did not contain virus-specific information. In contrast to the S+L-(C243) virions, RNA from virions or nucleoids of S+L-(D56) cells consisted of five sizes: 80 S (6%), 68 S (8%), 56 S (5%), 28 S (28%), and <28 S (53%). Thermal dissociation studies suggested that this S+L- genome is comprised of 28S RNA subunits. From these studies we postulate that the 28S viral RNA is most probably the monomeric genome of S+L- virions.

Phillips, Leo A.; Hollis, Vincent W.; Bassin, Robert H.; Fischinger, Peter J.



1'-acetoxychavicol acetate inhibits adipogenesis in 3T3-L1 adipocytes and in high fat-fed rats.  


Alpinia galanga and Languas galanga, which are plants belonging to the ginger family, are frequently used for cooking, especially in Thai and Indonesian cuisine. The compound 1'-acetoxychavicol acetate (ACA), which is naturally obtained from the rhizomes and seeds of these gingers, has antioxidant and anti-inflammatory properties. We investigated the anti-obesity effects of ACA in 3T3-L1 adipocytes and in high fat diet (HFD)-induced rat models of obesity. ACA caused a significant decrease in the activity of GPDH in 3T3-L1 adipocytes without eliciting cell cytotoxicity, and it inhibited cellular lipid accumulation through the down-regulation of transcription factors such as PPAR? and C/EBP?. ACA also induced a dose-dependent phosphorylation of AMP-activated protein kinase (AMPK). In the animal model, rats fed an HFD containing 0.05% ACA gained less weight than rats fed an HFD alone. The visceral fat mass in rats fed an HFD containing 0.05% ACA tended to be lower than that in rats fed an HFD alone. Furthermore, a histological examination of livers from rats fed an HFD showed steatohepatitis. However, rats fed an HFD containing 0.05% ACA showed no histopathological changes in the liver tissue. Our results show that ACA exerts anti-obesity activities both in vitro and in vivo and suggests that ACA may have a novel preventive activity against obesity and possibly other metabolic diseases. PMID:23227791

Ohnishi, Rie; Matsui-Yuasa, Isao; Deguchi, Yohei; Yaku, Keisuke; Tabuchi, Masaki; Munakata, Hiroshi; Akahoshi, Yasumitsu; Kojima-Yuasa, Akiko



Effect of sterols and fatty acids on growth and triglyceride accumulation in 3T3-L1 cells.  


Epidemiologic studies suggest a role of dietary fat in the development of obesity. Populations that consume Western diets have a higher incidence of obesity than do those that consume a vegetarian type diet such as Asians. Because dietary fats are made up mostly of triglyceride with minor lipids such as sterols, the objective of this study was to examine the effect of different fatty acids, the main component of triglycerides, and sterols on cell growth and triglyceride accumulation in 3T3-L1 cells. These cells are being used as an in vitro model for studying obesity because upon differentiation in culture they accumulate triglycerides. Cells were seeded at 5,000 cells/cm(2) and supplemented with 0, 3, 10, or 30 microM of oleic acid, elaidic acid, or docosahexaenoic acid (DHA). Similarly, cells were supplemented with 0, 2, 8, or 16 microM of cholesterol, beta-sitosterol (SIT), or campesterol. Cell growth was measured by cell counting. Cellular triglycerides were measured by the Oil Red O method. In some experiments, fatty acids were combined with sterols and growth and triglyceride content were assessed as described. Both DHA and SIT had inhibitory effects on 3T3-L1 cell growth. However, SIT was more potent than DHA in this regard. The combination of SIT and oleic acid was the most potent in inhibiting cell growth and increasing cellular triglyceride content. It is concluded that cell growth and triglyceride accumulation in 3T3-L1 cells is influenced by fatty acid and sterols. When used alone, DHA and SIT inhibit cell growth. SIT was more effective in this process than was DHA. There was an interaction between fatty acids and sterols. The most effective combination inhibiting cell growth and triglyceride concentration was the combination of SIT and oleic acid. This combination reduced cell growth and increased triglyceride accumulation. These data suggest that diets rich in both monounsaturated fatty acids and phytosterols may play a role in controlling obesity. PMID:10742660

Awad, A B; Begdache, L A; Fink, C S



Regulation of Myosin Light Chain Kinase during Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes  

PubMed Central

Myosin II (MyoII) is required for insulin-responsive glucose transporter 4 (GLUT4)-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC) of MyoIIA via myosin light chain kinase (MLCK). The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid, (BAPTA) (in the presence of insulin) impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKII? did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

Woody, Shelly; Stall, Richard; Ramos, Joseph; Patel, Yashomati M.



Regulation of Myosin Light Chain Kinase during Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes.  


Myosin II (MyoII) is required for insulin-responsive glucose transporter 4 (GLUT4)-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC) of MyoIIA via myosin light chain kinase (MLCK). The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid, (BAPTA) (in the presence of insulin) impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKII? did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes. PMID:24116218

Woody, Shelly; Stall, Richard; Ramos, Joseph; Patel, Yashomati M



Advanced Feeder Design for Distributed Generation  

Microsoft Academic Search

Advanced distribution automation functions enable feeder design with high penetration levels of distributed generation, up to 75% of the feeder's peak load. An example design is presented, based on an IEEE radial test feeder. The distributed generation in this example requires use of directional elements in the feeder relay, along with adaptive settings for line voltage regulators and capacitor switching

T. M. Smith; B. A. Muschlitz; F. R. Goodman; T. E. McDermott



Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.  


Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells. PMID:7527532

Kane, M A; Portanova, L B; Kelley, K; Holley, M; Ross, S E; Boose, D; Escobedo-Morse, A; Alvarado, B



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

PubMed Central

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.

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



Liquiritigenin isolated from Glycyrrhiza uralensis stimulates osteoblast function in osteoblastic MC3T3-E1 cells.  


Liquiritigenin is one of the flavonoids present in Glycyrrhizae radix. In the present study, the effects of liquiritigenin on the function of osteoblastic MC3T3-E1 cells were studied. Liquiritigenin caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, mineralization, and glutathione content in the cells (P<0.05). Moreover, liquiritigenin significantly decreased the production of reactive oxygen species (ROS) and osteoclast differentiation inducing factors such as tumor necrosis factor ? (TNF-?), interleukin-6 (IL-6), and receptor activator of nuclear factor-?B ligand (RANKL) in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as a ROS generator. These results demonstrate that liquiritigenin may have positive effects on skeletal structure. PMID:22116056

Choi, Eun Mi



NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.  


The nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway responds to oxidative stress via control of several antioxidant defense gene expressions. Recent efforts demonstrate that Nrf2 modulates development of adiposity and adipogenesis. One of the major Nrf2-regulated proteins, NAD(P)H:quinone oxidoreductase 1 (NQO1), is implicated in the development of adipose tissue and obesity. However, little is known about in situ disposition of Nrf2, Keap1, and NQO1 during adipogenesis in isolated adipocytes. Based on literature data, we hypothesized that adipocyte differentiation would increase expression of the Nrf2/Keap1 pathway and NQO1. Using murine 3T3-L1 preadipocytes, we mapped an increase in NQO1 protein at limited clonal expansion and postmitotic growth arrest (Days 1-3) stages and a decrease in terminally differentiated (Day 8) adipocytes that lasted for several days afterward. Conversely, NQO1, Nrf2, and Keap1 mRNA expressions were all increased in differentiated adipocytes (Days 11-14), indicating a discrepancy between steady-state mRNA levels and resulting protein. Treatment of differentiated 3T3-L1 adipocytes with glycogen synthase kinase-3? (GSK-3?) inhibitor, LiCl, led to 1.9-fold increase in NQO1 protein. Sulforaphane enhanced NQO1 protein (10.5-fold) and blunted triglyceride and FABP4 accumulation. The decrement in triglyceride content was partially reversed when NQO1 activity was pharmacologically inhibited. These data demonstrate a biphasic response of Nrf2 and NQO1 during adipocyte differentiation that is regulated by Keap1- and GSK-3?-dependent mechanisms, and that hypertrophy is negatively regulated by NQO1 activity. PMID:22683604

Vomhof-DeKrey, Emilie E; Picklo, Matthew J



Inhibition by chondroitin sulfate E can specify functional Wnt/?-catenin signaling thresholds in NIH3T3 fibroblasts.  


Aberrant activation of the Wnt/?-catenin signaling pathway is frequently associated with human disease, including cancer, and thus represents a key therapeutic target. However, Wnt/?-catenin signaling also plays critical roles in many aspects of normal adult tissue homeostasis. The identification of mechanisms and strategies to selectively inhibit the disease-related functions of Wnt signaling, while preserving normal physiological functions, is in its infancy. Here, we report the identification of exogenous chondroitin sulfate-E (CS-E) as an inhibitor of specific molecular and biological outcomes of Wnt3a signaling in NIH3T3 fibroblasts. We demonstrate that CS-E can decrease Wnt3a signaling through the negative regulation of LRP6 receptor activation. However, this inhibitory effect of CS-E only affected Wnt3a-mediated induction, but not repression, of target gene expression. We went on to identify a critical Wnt3a signaling threshold that differentially affects target gene induction versus repression. This signaling threshold also controlled the effects of Wnt3a on proliferation and serum starvation-induced apoptosis. Limiting Wnt3a signaling to this critical threshold, either by CS-E treatment or by ligand dilution, interfered with Wnt3a-mediated stimulation of proliferation but did not impair Wnt3a-mediated reduction of serum starvation-induced apoptosis. Treatment with pharmacological inhibitors demonstrated that both induction and repression of Wnt3a target genes in NIH3T3 cells require the canonical Wnt/?-catenin signaling cascade. Our data establish the feasibility of selective inhibition of Wnt/?-catenin transcriptional programs and biological outcomes through the exploitation of intrinsic signaling thresholds. PMID:22915582

Willis, Catherine M; Klüppel, Michael



Reduction of O-GlcNAc protein modification does not prevent insulin resistance in 3T3-L1 adipocytes  

PubMed Central

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high (25 mM) glucose, provided that insulin (0.6 nM) is included, Akt activation is impaired, and high glucose and insulin act synergistically. Considerable evidence suggests that increased glucose flux via the hexosamine biosynthesis pathway enhances the O-GlcNAc modification (O-GlcNAcylation) of some critical protein(s) that may contribute to insulin resistance. However, whether enhanced protein O-GlcNAcylation is necessary for the development of insulin resistance is unknown. We used two strategies to test this hypothesis. The first strategy was the overexpression of O-GlcNAcase, which removes O-GlcNAc from Ser/Thr of proteins. Cells were infected with O-GlcNAcase-expressing adenovirus (or empty virus) 5 days before they were submitted to protocols that elicit (or not) insulin resistance. O-GlcNAcase was highly expressed and functional as assessed by Western blot, O-GlcNAcase assay, and marked reduction of O-GlcNAcylated proteins. The activity was mainly cytosolic. The second strategy was the expression of O-GlcNAc transferase (OGT) being markedly reduced by transfection of OGT siRNA, resulting in an approximately 90% decrease of nuclear and cytosolic OGT protein expression and similar reduction in O-GlcNAcylated proteins. Non-targeting siRNA had no effect. Preincubation in high glucose with low-dose insulin decreased the acute insulin response of glucose transport by at least 50% and impaired Akt activation. None of these parameters were affected by overexpression of O-GlcNAcase or by OGT knockout. Excess O-GlcNAcylation is one of many factors that can cause insulin resistance. It does not seem to be required for the development of glucose/insulin-induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes.

Robinson, Katherine A.; Ball, Lauren E.; Buse, Maria G.



Protective effect of ?-lipoic acid on islet cells co-cultured with 3T3L1 adipocytes  

PubMed Central

Obesity and ?-cell dysfunction due to oxidative stress impact the pathogenesis of type 2 diabetes mellitus. We co-cultured 3T3L1 adipocytes and islet cells in the presence or absence of the antioxidant ?-lipoic acid (LA) and assayed the effects of the adipocytes and LA on the secretion of insulin by the islet cells and on the activities of factors involved in secretion and oxidative stress. At low glucose concentrations (2.8 mmol/l), the presence of adipocytes (co-culture) increased insulin secretion compared with islet cells cultured alone (control) and this increase was diminished by LA (co-culture plus LA). At high glucose concentrations (22 mmol/l), insulin secretion levels were similar for all islet groups, resulting in a restoration of the stimulation index in the presence of LA. The mRNA levels of the glucose-stimulated insulin secretion (GSIS) genes glucokinase, glucose transporter 2 and Kir6.2 were downregulated under co-culture and co-culture plus LA conditions. Protein and tyrosine phosphorylation levels of insulin receptor-? and insulin receptor substrate-1 were decreased under co-culture conditions and were restored by LA treatment. Cellular malondialdehyde levels increased in the co-cultured islets and this increase was blocked by LA. The mRNA levels of superoxide dismutase and catalase were reduced under co-culture conditions and these reductions were eliminated by the addition of LA. In conclusion, 3T3L1 adipocytes disturb insulin secretion and induce islet dysfunction. The effects may be mediated by multiple pathways, which include downregulation of GSIS gene expression, suppression of islet cell insulin signaling and the induction of oxidative stress. LA may protect islet cells via activation of islet cell insulin signaling and the mRNA expression of antioxidant enzymes.




Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts.  


Although the activation of phospholipase A2 (PLA2) in ras-transformed cells has been well documented, the mechanisms underlying this activation are poorly understood. In this study we tried to elucidate whether the membrane phospholipid composition and physical state influence the activity of membrane-associated PLA2 in ras-transformed fibroblasts. For this purpose membranes from non-transfected and ras-transfected NIH 3T3 fibroblasts were enriched with different phospholipids by the aid of partially purified lipid transfer protein. The results showed that of all tested phospholipids only phosphatidylcholine (PC) increased PLA2 activity in the control cells, whereas in their transformed counterparts both PC and phosphatidic acid (PA) induced such effect. Further we investigated whether the activatory effect was due only to the polar head of these phospholipids, or if it was also related to their acyl chain composition. The results demonstrated that the arachidonic acid-containing PC and PA molecules induced a more pronounced increase of membrane-associated PLA2 activity in ras-transformed cells compared to the corresponding palmitate-stearate- or oleate- containing molecular species. However, we did not observe any specific effect of the phospholipid fatty acid composition in non-transformed NIH 3T3 fibroblasts. In ras-transformed cells incubated with increasing concentrations of arachidonic acid, PLA2 activity was altered in parallel with the changes of the cellular content of this fatty acid. The role of phosphatidic and arachidonic acids as specific activators of PLA2 in ras-transformed cells is discussed with respect to their possible role in the signal transduction pathways as well as in the processes of malignant transformation of cells. PMID:9924985

Momchilova, A; Markovska, T; Pankov, R



DMSO is a strong inducer of DNA hydroxymethylation in pre-osteoblastic MC3T3-E1 cells.  


Artificial induction of active DNA demethylation appears to be a possible and useful strategy in molecular biology research and therapy development. Dimethyl sulfoxide (DMSO) was shown to cause phenotypic changes in embryonic stem cells altering the genome-wide DNA methylation profiles. Here we report that DMSO increases global and gene-specific DNA hydroxymethylation levels in pre-osteoblastic MC3T3-E1 cells. After 1 day, DMSO increased the expression of genes involved in DNA hydroxymethylation (TET) and nucleotide excision repair (GADD45) and decreased the expression of genes related to DNA methylation (Dnmt1, Dnmt3b, Hells). Already 12 hours after seeding, before first replication, DMSO increased the expression of the pro-apoptotic gene Fas and of the early osteoblastic factor Dlx5, which proved to be Tet1 dependent. At this time an increase of 5-methyl-cytosine hydroxylation (5-hmC) with a concomitant loss of methyl-cytosines on Fas and Dlx5 promoters as well as an increase in global 5-hmC and loss in global DNA methylation was observed. Time course-staining of nuclei suggested euchromatic localization of DMSO induced 5-hmC. As consequence of induced Fas expression, caspase 3/7 and 8 activities were increased indicating apoptosis. After 5 days, the effect of DMSO on promoter- and global methylation as well as on gene expression of Fas and Dlx5 and on caspases activities was reduced or reversed indicating down-regulation of apoptosis. At this time, up regulation of genes important for matrix synthesis suggests that DMSO via hydroxymethylation of the Fas promoter initially stimulates apoptosis in a subpopulation of the heterogeneous MC3T3-E1 cell line, leaving a cell population of extra-cellular matrix producing osteoblasts.  PMID:22507896

Thaler, Roman; Spitzer, Silvia; Karlic, Heidrun; Klaushofer, Klaus; Varga, Franz



The effects of low dose X-irradiation on osteoblastic MC3T3-E1 cells in vitro  

PubMed Central

Background It has been indicated that moderate or high dose of X-irradiation could delay fracture union and cause osteoradionecrosis, in part, mediated by its effect on proliferation and differentiation of osteoblasts. However, whether low dose irradiation (LDI) has similar roles on osteoblasts is still unknown. In this study, we investigated whether and to what extent LDI could affect the proliferation, differentiation and mineralization of osteoblasts in vitro. Methods The MC3T3-E1 cells were exposed to single dose of X-irradiation with 0, 0.1, 0.5, 1.0?Gy respectively. Cell proliferation, apoptosis, alkaline phosphatase (ALP) activity, and mineralization was evaluated by methylthiazoletetrazolium (MTT) and bromodeoxyuridine (BrdU) assay, flow cytometry, ALP viability kit and von Kossa staining, respectively. Osteocalcin (OCN) and core-binding factor ?1 (Cbf?1) expressions were measured by real time-PCR and western blot, respectively. Results The proliferation of the cells exposed to 2.0?Gy was significantly lower than those exposed to ?1.0?Gy (p?3T3-E1 cells. In addition, mRNA and protein expressions of OCN and Cbf?1 were also markedly increased after treatment with LDI at 0.5 and 1?Gy. Conclusions LDI have different effects on proliferation and differentiation of osteoblasts from those of high dose of X-irradiation, which might suggest that LDI could lead to promotion of frature healing through enhancing the differentiation and mineralization of osteoblasts.



Co-culture of C2C12 and 3T3-L1 preadipocyte cells alters the gene expression of calpains, caspases and heat shock proteins.  


The present study was carried out to understand the co-culture effect of C2C12 and 3T3-L1 preadipocyte cells on calpain, caspase, and heat shock protein (Hsp) systems. Calpains, caspases, and heat shock proteins play critical roles in the growth and development of mammalian cells. Cells were co-cultured 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 and inserts containing C2C12 transferred to 3T3-L1 plates. Following co-culture for 24 and 48 h, the cells in the lower well were harvested for analysis. Calpains include ?-calpain, m-calpain, and their specific inhibitor calpastatin. The expression pattern of ?-calpain did not change in the co-cultured C2C12 and 3T3-L1 cells, whereas m-capain mRNA expression significantly reduced in the 48-h co-cultured 3T3-L1 cells. Calpastatin mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Caspase-7 mRNA expression did not change in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells. Caspase-3 mRNA expression significantly reduced in the 24- and 48-h co-cultured 3T3-L1 cells; caspase-9 mRNA had a significant reduction only at 48 h, whereas caspase-9 mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Hsp27 and Hsp90 mRNA expressions are significantly reduced in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells, whereas Hsp70 mRNA expression significantly increased in the 48-h co-cultured 3T3-L1 cells. The co-culture reflects three-dimensional views of C2C12 and 3T3-L1 cell types as in vivo, which is quite distinct from the one-dimensional monocultured C2C12 and 3T3-L1 cells. PMID:23054441

Pandurangan, Muthuraman; Jeong, Dawoon; Amna, Touseef; Van Ba, Hoa; Hwang, Inho



The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein ?  

Microsoft Academic Search

Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein ? (PI-TP?, SPI? cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPI?S262A cells overexpressing a mutant PI-TP? that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable with wtNIH3T3 cells. This suggests that the phosphorylation of

Martijn Schenning; Claudia M. van Tiel; Karel W. A. Wirtz; Gerry T. Snoek



No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus  

SciTech Connect

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were stimated by the method of fiber-autoradiography and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcome virus-transformed BALB/c 3T3 cells.

Oppenheim, A.; Horowitz, A.T.



[Determination of the activity of semicarbazide-sensitive amine oxidase in the differentiated 3T3-F442A cells by enzyme-linked spectrophotometric assay].  


In order to probe into the patho-physiology semicarbazide-sensitive amine oxidase (SSAO), we determined the variation of its activity in the days of 3T3-F442A cells' differentiation into adipocytes in vitro. The differentiated 3T3-F442A cells were collected in various days and disrupted by ultrasonication. Then we studied the SSAO activity of the collected cells by oxidase-linked spectrophotometric assay. In the course of 3T3-F442A cells' differentiation, SSAO expression was shown by a curve graph. There was no SSAO detected in 3T3-F442A preadipocyte, and only a little of SSAO was detected in the cells on the 3rd day of differentiation. Since then, SSAO significantly increased and reached to a maximum level six or seven days after the cells' differentiation. PMID:19024467

Zhu, Sha; Wang, Yan; Du, Ying; Li, Sanqiang; Xuan, Xiaoyan; Tang, Yue



Leptin-mediated inhibition of the insulin-stimulated increase in fatty acid uptake in differentiated 3T3-L1 adipocytes.  


The effects of insulin and leptin on fatty acid uptake in differentiated (adipocytes) and undifferentiated 3T3-L1 cells were investigated. It was demonstrated that in undifferentiated 3T3-L1 cells, insulin and leptin have no effect on fatty acid uptake. In differentiated 3T3-L1 adipocytes, insulin had a concentration-dependent stimulatory effect on fatty acid uptake, whereas leptin on its own had no effect. Leptin, when coincubated with 10 nmol/L insulin, resulted in a concentration-dependent inhibition of the insulin-stimulated fatty acid uptake in differentiated 3T3-L1 cells. These results indicate that leptin has a direct inhibitory effect on the stimulation of fatty acid uptake by insulin in differentiated murine adipocytes. PMID:16324913

Ho, Michael; Foxall, Susan; Higginbottom, Michael; Donofrio, David M; Liao, Jinfang; Richardson, Peter J; Maneuf, Yannick P



Detecting effects of low levels of cytochalasin B in 3T3 fibroblast cultures by analysis of electrical noise obtained from cellular micromotion.  


We performed micromotion experiments using electric cell-substrate impedance sensing (ECIS) on a confluent layer of 3T3 fibroblasts exposed to different low levels of the toxin cytochalasin B. This toxin is know to affect actin polymerization and to disrupt cytoskeletal structure and function in cells, changing the morphology of confluent cell cultures and altering the nature of the cellular micromotion, which is measured by ECIS as changes in impedance. By looking at several measures to characterize the long- and short-term correlations in the noise of the impedance time series, we are able to detect the effects of the toxin at concentrations down to 1 microM; there are intriguing hints that the effects may be discernible at levels as low as 0.1 microM. These measures include the power spectrum, the Hurst and detrended-fluctuation-analysis exponents, and the first zero and first 1/e crossings of the autocorrelation function. While most published work with ECIS uses only average impedance values, we demonstrate that noise analysis provides a more sensitive probe. PMID:19026529

Lovelady, Douglas C; Friedman, Jennifer; Patel, Sonali; Rabson, David A; Lo, Chun-Min



Detecting effects of low levels of cytochalasin B in 3T3 fibroblast cultures by analysis of electrical noise obtained from cellular micromotion  

PubMed Central

We performed micromotion experiments using electric cell-substrate impedance sensing (ECIS) on a confluent layer of 3T3 fibroblasts exposed to different low levels of the toxin cytochalasin B. This toxin is know to affect actin polymerization and to disrupt cytoskeletal structure and function in cells, changing the morphology of confluent cell cultures and altering the nature of the cellular micromotion, which is measured by ECIS as changes in impedance. By looking at several measures to characterize the long- and short-term correlations in the noise of the impedance time series, we are able to detect the effects of the toxin at concentrations down to 1 ?M; there are intriguing hints that the effects may be discernible at levels as low as 0.1 ?M. These measures include the power spectrum, the Hurst and detrended-fluctuation-analysis exponents, and the first zero and first 1/e crossings of the autocorrelation function. While most published work with ECIS uses only average impedance values, we demonstrate that noise analysis provides a more sensitive probe.

Lovelady, Douglas C.; Friedman, Jennifer; Patel, Sonali; Rabson, David A.; Lo, Chun-Min



Human Immunodeficiency Virus Type 1 VprGene Product Prevents Cell Proliferation on Mouse NIH3T3 Cells without the G 2Arrest of the Cell Cycle  

Microsoft Academic Search

Human immunodeficiency virus type 1 Vpr is a 96-amino-acid virion-associated protein that arrests cells in the G2phase of the cell cycle in peripheral blood lymphocytes, HeLa, 293, 293T, A549, Jurkat, CEM, SupT1, CV-1 and COS1 cells. When we transfected Vpr expression vector into mouse NIH3T3 and then cultured it in the presence of G418, NIH3T3 cells were the drug resistant

Yoshii Nishino; Tetsuya Myojin; Masakazu Kamata; Yoko Aida



Calcium-sensing receptor-mediated activation of phospholipase C-?1 is downstream of phospholipase C-? and protein kinase C in MC3T3-E1 osteoblasts  

Microsoft Academic Search

Elevated extracellular calcium (Cae) stimulates both chemotaxis and mitogenesis of MC3T3-E1 osteoblasts via a calcium-sensing receptor (CasR). Cae-mediated chemotaxis of these bone-forming cells is dependent on phospholipase C (PLC) and blocked by the Gi-protein inhibitor pertussis toxin. In this study, we examine the signaling mechanisms by which the CasR stimulates PLC activity in MC3T3-E1 osteoblasts. We found that elevated Cae

S. L Godwin; S. P Soltoff



Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of Swiss 3T3 cells  

Microsoft Academic Search

Summary  Improved culture conditions have been developed that will support clonal growth of Swiss mouse embryo 3T3 cells at concentrations\\u000a of serum protein as low as 125?g\\/ml. Survival of the cells under completely protein-free conditions also is enhanced greatly.\\u000a The improvements that made these results possible include: (a) use of medium MCDB 402, which was developed specifically for\\u000a Swiss 3T3 cells

Gary D. Shipley; Richard G. Ham



A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene.  


Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants. PMID:2153549

Fujita, H; Suzuki, H; Kuzumaki, N; Müllauer, L; Ogiso, Y; Oda, A; Ebisawa, K; Sakurai, T; Nonomura, Y; Kijimoto-Ochiai, S



A Novel Cell-Based Therapy for Contusion Spinal Cord Injury Using GDNF-Delivering NIH3T3 Cells with Dual Reporter Genes Monitored by Molecular Imaging  

Microsoft Academic Search

This aim of our study was to evaluate a novel cell-based therapy for contusion spinal cord injury (SCI) using embryonic-derived NIH3T3 cells, which endogenously express glial cell line-derived neurotrophic factor (GDNF).Methods: Proliferation and differen- tiation of transplanted NIH3T3 cells and their anti-apoptotic ef- fects were examined after their engraftment into the spinal cords of Long-Evans rats subjected to acute SCI

Wen-Cheng Lo; Chung-Huei Hsu; Alexander T. H. Wu; Liang-Yo Yang; Wei-Hong Chen; Wen-Ta Chiu; Wen-Fu Lai; Chih-Hsiung Wu; Juri G. Gelovani; Win-Ping Deng


Variants of BALB\\/c 3T3 cells lacking complex gangliosides retain a fibronectin matrix and spread normally on fibronectin-coated substrates  

Microsoft Academic Search

Evidence has accumulated that di- and trisi- alogangliosides are involved in the interaction of cells with fibronectin. We have therefore tested the ability of variants of BALB\\/c 3T3 deficient in such ganglio- sides to organize a fibronectin matrix and to spread on fibronectin-coated substrates. Whereas BALB\\/c 3T3 cells contained gangliosides GM3, GM 1, and GD 1 a, direct chemical analysis

Susanne L. Griffiths; Robert M. Perkins; Charles H. Streuli; David R. Critchley



The benefits of the 3T3 NRU test in the safety assessment of cosmetics: long-term experience from pre-marketing testing in the Czech Republic  

Microsoft Academic Search

We have introduced the 3T3 NRU cytotoxicity test for methodological, economical and ethical reasons as a regular part of tier pre-marketing testing to assess local tolerance of raw materials for cosmetics, household chemicals and final cosmetic products. Using the 3T3 cell line according to the standard INVITTOX protocol No.64 (NRU Assay) the borderline concentration, relevant to the highest tolerated dose,

D J??rová; K Kejlová; M Brabec; H Bendová; H Kolá?ová



A Role for Sp1 in Transcriptional Regulation of Phosphatidylethanolamine N-Methyltransferase in Liver and 3T3-L1 Adipocytes*  

PubMed Central

Phosphatidylcholine is made in all nucleated mammalian cells via the CDP-choline pathway. Another major pathway for phosphatidylcholine biosynthesis in liver is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We have now identified 3T3-L1 adipocytes as a cell culture model that expresses PEMT endogenously. We have found that PEMT mRNA and protein levels increased dramatically in 3T3-L1 cells upon differentiation to adipocytes. 5?-Deletion analysis of the PEMT promoter-luciferase constructs stably expressed in 3T3-L1 adipocytes identified a regulatory region between ?471 and ?371 bp (relative to the transcriptional start site). Competitive and supershift assays demonstrated binding sites for transcription factors Sp1, Sp3 (?408 to ?413), and YY1 (?417 to ?420). During differentiation of 3T3-L1 cells to adipocytes, the amount of Sp1 protein decreased by ?50% just prior to activation of PEMT. Transduction of 3T3-L1 adipocytes with retrovirus containing Sp1 cDNA demonstrated that Sp1 inhibited PEMT transcriptional activity. Similarly, short hairpin RNA directed against Sp1 in 3T3-L1 adipocytes enhanced PEMT transcriptional activation. Chromatin immunoprecipitation assays confirmed that Sp1 binds to the PEMT promoter, and this interaction decreases upon differentiation to adipocytes. These experiments directly link increased PEMT expression in adipocytes to decreased transcriptional expression of Sp1. In addition, our data established that Sp1 binding was required for tamoxifen-mediated inhibition of Pemt promoter activity.

Cole, Laura K.; Vance, Dennis E.



High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes.  


Sustained hyperglycemia induces insulin resistance, but the mechanism is still incompletely understood. Glucosamine (GlcN) has been extensively used to model the role of the hexosamine synthesis pathway (HSP) in glucose-induced insulin resistance. 3T3-L1 adipocytes were preincubated for 18 h in media +/- 0.6 nmol/l insulin containing either low glucose (5 mmol/l), low glucose plus GlcN (0.1-2.5 mmol/l), or high glucose (25 mmol/l). Basal and acute insulin-stimulated (100 nmol/l) glucose transport was measured after re-equilibration in serum and insulin-free media. Preincubation with high glucose or GlcN (1-2.5 mmol/l) inhibited basal and acute insulin-stimulated glucose transport only if insulin was present during preincubation. However, only preincubation with GlcN plus insulin inhibited insulin-stimulated GLUT4 translocation. GLUT4 and GLUT1 protein expression were not affected. GlcN (2.5 mmol/l) increased cellular UDP-N-acetylhexosamines (UDP-HexNAc) by 400 and 900% without or with insulin, respectively. High glucose plus insulin increased UDP-HexNAc by 30%. GlcN depleted UDP-hexoses, whereas high glucose plus insulin increased them. Preincubation with 0.5 mmol/l GlcN plus insulin maximally increased UDP-HexNAc without affecting insulin-stimulated or basal glucose transport. GlcN plus insulin (but not high glucose plus insulin) caused marked GlcN dose-dependent accumulation of GlcN-6-phosphate, which correlated with insulin resistance of glucose transport (r = 0.935). GlcN plus insulin (but not high glucose plus insulin) decreased ATP (10-30%) and UTP (>50%). GTP was not measured, but GDP increased. Neither high glucose plus insulin nor GlcN plus insulin prevented acute insulin stimulation (approximately 20-fold) of insulin receptor substrate 1-associated phosphatidylinositol (PI)-3 kinase. We have come to the following conclusions. 1) Chronic exposure to high glucose or GlcN in the presence of low insulin caused insulin resistance of glucose transport by different mechanisms. 2) GlcN inhibited GLUT4 translocation, whereas high glucose impaired GLUT4 "intrinsic activity" or membrane intercalation. 3) Both agents may act distally to PI-3 kinase. 4) GlcN has metabolic effects not shared by high glucose. GlcN may not model HSP appropriately, at least in 3T3-L1 adipocytes. PMID:10866051

Nelson, B A; Robinson, K A; Buse, M G



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


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

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



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


The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/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

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



Evaluation of the wound healing potentials of two subspecies of Hypericum perforatum on cultured NIH3T3 fibroblasts.  


For centuries, Hypericum perforatum has been used in folk medicine to treat wounds. In the present study, the wound healing activities of extracts of H. perforatum ssp. perforatum (HPP) and H. perforatum ssp. veronense (HPV) were evaluated by comparing with a titrated extract of Centella asiatica (TECA) on NIH3T3 fibroblasts. The cells were incubated with the extracts. Using microscopical methods by staining cells, mitotic ability, morphological changes and collagen production in the fibroblasts were evaluated as parameters to approach possible wound repair mechanism(s). The wound healing activity caused an increase in the percentage of polygonal fibroblasts and in collagen granule numbers in fibroblasts of HPP and HPV. At 1?µg/mL, HPP caused a greater increase in mitotic cell numbers than HPV. The most increases in polygonal cell numbers were observed with HPV at 1 and 10?µg/mL. The number of collagen granules was increased with 1?µg/mL HPV being higher than HPP. As an indication of cytotoxic effects, round cells in response to HPV and HPP extracts, were found to be increased in concentrations higher than 10-50?µg/mL. The results indicated that the two subspecies of H. perforatum have different wound healing profiles as a result of the fibroblast migration and stimulation of collagen synthesis. PMID:20632305

Dikmen, Miri?; Oztürk, Yusuf; Sagratini, Gianni; Ricciutelli, Massimo; Vittori, Sauro; Maggi, Filippo



Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells  

SciTech Connect

Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into Psi2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected Psi2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylaline hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normal synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.

Ledley, F.D.; Grenett, H.E.; McGinnis-Shelnutt, M.; Woo, S.L.C.



Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells  

SciTech Connect

Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ZnCl/sub 2/. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approx. = 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with /sup 125/I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.

Mroczkowski, B.; Reich, M.; Whittaker, J.; Bell, G.I.; Cohen, S.



S6 kinase in quiescent Swiss mouse 3T3 cells is activated by phosphorylation in response to serum treatment  

SciTech Connect

To investigate the role of phosphorylation in the activation of S6 kinase, the enzyme was isolated from {sup 32}P-labeled Swiss mouse 3T3 cells before and after stimulation with serum. The kinase activity was followed through several purification steps, and a radioactive protein of M{sub r} 70,000 was obtained from the stimulated cells. This band was not detected in resting cells. The M{sub r} 70,000 protein exhibited the same size upon NaDodSO{sub 4}/PAGE as the homogeneous kinase, and it comigrated with the in vitro autophosphorylated form of the enzyme. Treatment of the in vivo-labeled material with phosphatase 2A led to a loss of kinase activity concomitant with a release of {sup 32}P{sub i} from the M{sub r} 70,000 protein. The partially dephosphorylated protein migrated faster during PAGE, displaying distinct species of M{sub r} 69,000 and 68,000. Most importantly, phospho amino acid analysis of the labeled S6 kinase showed only phosphoserine and phosphothreonine. These results argue that the S6 kinase is phosphorylated at multiple sites in vivo and that it is activated by serine/threonine phosphorylation.

Ballou, L.M.; Siegmann, M.; Thomas, G. (Friedrich Miescher-Institut, Basel (Switzerland))



The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside.  


The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 ?M SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels. PMID:23220610

Bittencourt, L S; Machado, D C; Machado, M M; Dos Santos, G F F; Algarve, T D; Marinowic, D R; Ribeiro, E E; Soares, F A A; Barbisan, F; Athayde, M L; Cruz, I B M



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

SciTech Connect

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.

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:



Radish phospholipid hydroperoxide glutathione peroxidase provides protection against hydroperoxide-mediated injury in mouse 3T3 fibroblasts.  


Overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) genes has been reported to play an important role in protecting host cells from oxidative injury in several model systems. A radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) known to have high catalytic activity was applied to mouse 3T3 fibroblasts to determine the protective effects of PHGPx against oxidative injury triggered by hydroperoxides such as hydrogen peroxide (H(2)O(2)), tert-butyl hydroperoxide (t-BHP) and phosphatidylcholine hydroperoxide (PCOOH). We observed that preincubation of cells with RsPHGPx significantly increased cell viability, reduced levels of malondialdehyde (MDA), inhibited generation of reactive oxygen species (ROS), and maintained natural cell shapes after treatment with H(2)O(2), t-BHP or PCOOH, indicating that the exogenous RsPHGPx can act as an effective hydroperoxide-scavenger and may also protect target cells from oxidative damage. These results suggest the possibility for use of RsPHGPx as a therapeutic protectant. PMID:19874709

Li, Tian; Liu, Guan-Lan; Duan, Ming-Xing; Liu, Jin-Yuan



Inhibition of Adipogenesis and Induction of Apoptosis and Lipolysis by Stem Bromelain in 3T3-L1 Adipocytes  

PubMed Central

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 Gi?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.

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



Gene expression changes in BALB/3T3 transformants induced by poly(L-lactic acid) or polyurethane films.  


We performed DNA microarray analysis on two BALB/3T3 transformants (A5 and A6) induced by polyurethane (PU) film, two (L11 and L21) induced by biodegradable poly(L-lactic acid) (PLLA) film, and the parental cells. The transforming ability of the cells was in the order A5 < A6 < L21 < L11. In all, 1176 cancer-related genes were up- or down-regulated in at least one transformant. Those that were markedly up-regulated were c-fos protooncogene, FBJ osteosarcoma oncogene B, and Jun oncogene; those markedly down-regulated were pleiotrophin, histidine triad nucleotide-binding protein, protein kinase C iota, and large multifunctional protease 7. A common function of proteins encoded by genes that underwent marked expression changes was bone formation. The genes were c-fos, FBJ osteosarcoma, Jun, pleiotrophin, a disintegrin-like and metalloprotease with TS-1 motif protein 1. This finding was consistent with the tumor formation in the 2-year PLLA or PU subcutaneous implantation into rats. The number of genes that underwent marked expression change in each transformant was consistent with its malignancy. PLLA induced more malignant transformants than PU, especially in relation to osteosarcoma-like gene expression. PMID:14704980

Matsuoka, Atsuko; Tsuchiya, Toshie



Glycine suppresses TNF-?-induced activation of NF-?B in differentiated 3T3-L1 adipocytes.  


Glycine strongly reduces the serum levels of pro-inflammatory cytokines and increases the levels of anti-inflammatory cytokines. Recently, glycine has been shown to decrease the expression and secretion of pro-inflammatory adipokines in monosodium glutamate-induced obese (MSG/Ob) mice. It has been postulated that these effects may be explained by a reduction in nuclear factor kappa B (NF-?B) activation. NF-?B is a transcription factor, which is crucial to the inflammatory response. Hasegawa et al. (2011 and 2012) recently reported a glycine-dependent reduction in NF-?B levels. Here, we have investigated the role of glycine in the regulation of NF-?B in differentiated 3T3-L1 adipocytes. The results revealed that pretreatment with glycine interfered with the activation of NF-?B, which has been shown to be stimulated by tumor necrosis factor-alpha (TNF-?). Glycine alone stimulated NF-?B activation in an unusual way such that the inhibitor ?B-? (I?B-?) degradation was more significant than that of the inhibitor ?B-? (I?B-?) and led to NF-?B complexes comprised of p50 and p65 subunits; I?B-? degradation did not affect by glycine. These findings suggest that glycine could be used as an alternative treatment for chronic inflammation, which is a hallmark of obesity and other comorbidities, and is characterized by an elevated production of pro-inflammatory cytokines. PMID:22732655

Blancas-Flores, Gerardo; Alarcón-Aguilar, Francisco J; García-Macedo, Rebeca; Almanza-Pérez, Julio C; Flores-Sáenz, José L; Román-Ramos, Rubén; Ventura-Gallegos, José L; Kumate, Jesús; Zentella-Dehesa, Alejandro; Cruz, Miguel



Behaviors of NIH-3T3 fibroblasts on graphene/carbon nanotubes: proliferation, focal adhesion, and gene transfection studies.  


Carbon-based materials, including graphene and carbon nanotubes, have been considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for stem cell differentiation, and components of implant devices. Despite the potential biomedical applications of these materials, only limited information is available regarding the cellular events, including cell viability, adhesion, and spreading, that occur when mammalian cells interface with carbon-based nanomaterials. Here, we report behaviors of mammalian cells, specifically NIH-3T3 fibroblast cells, grown on supported thin films of graphene and carbon nanotubes to investigate biocompatibility of the artificial surface. Proliferation assay, cell shape analysis, focal adhesion study, and quantitative measurements of cell adhesion-related gene expression levels by RT-PCR reveal that the fibroblast cells grow well, with different numbers and sizes of focal adhesions, on graphene- and carbon nanotube-coated substrates. Interestingly, the gene transfection efficiency of cells grown on the substrates was improved up to 250% that of cells grown on a cover glass. The present study suggests that these nanomaterials hold high potential for bioapplications showing high biocompatibility, especially as surface coating materials for implants, without inducing notable deleterious effects while enhancing some cellular functions (i.e., gene transfection and expression). PMID:20979372

Ryoo, Soo-Ryoon; Kim, Young-Kwan; Kim, Mi-Hee; Min, Dal-Hee



Effect of Zinc Oxide Nanoparticles on the Function of MC3T3-E1 Osteoblastic Cells.  


Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 ?g/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01?1 ?g/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 ?g/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells (P?

Suh, Kwang Sik; Lee, Young Soon; Seo, Seung Hwan; Kim, Young Seol; Choi, Eun Mi



Effect of ginsenosides Rg3 and Re on glucose transport in mature 3T3-L1 adipocytes.  


Ginsenosides, the active component of Panax ginseng, have been shown to evidence a variety of biological activities associated with hyperglycemia, obesity and type 2 diabetes mellitus. This study evaluated the effects of the ginsenosides, Rg3 and Re, on glucose uptake and the glucose transport system in mature 3T3-L1 cells. The results demonstrated that the glucose uptake of ginsenosides Rg3 and Re at concentrations of 1-10?µM significantly increased by approximately ?10% and ?12%, respectively. Furthermore, the glucose transporter 4 (GLUT4) mRNA expression of ginsenosides Rg3 and Re at 10?µM was increased by approximately ?1.73 and 1.43 fold, respectively. It was further confirmed in a series of experiments that ginsenosides Rg3 and Re stimulated the mRNA expression of insulin receptor substrate (IRS-1) and the expression of phosphatidylinositol 3-kinase (PI3K)-110? protein, which is involved in downstream events in the insulin signaling pathway. These findings demonstrate that ginsenosides Rg3 and Re may stimulate glucose uptake via the PI3K pathways involving IRS-1. Further, our results suggest that both of these ginsenosides might prove useful as effective antidiabetic and antihyperglycemic agents. PMID:21520470

Lee, Ok-Hwan; Lee, Hee-Hyun; Kim, Ji-Hyup; Lee, Boo-Yong



Ha-ras-transformation alters the metabolism of phosphatidylethanolamine and phosphatidylcholine in NIH 3T3 fibroblasts.  


Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha-ras transformation. All phospholipid fractions were reduced in ras-transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras-cells. Acyl-CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine-cytidylyltransferase and CTP:phosphoethanolamine-chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline- and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP-choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP-ethanolamine pathway. PMID:10728571

Momchilova, A; Markovska, T; Pankov, R



Deoxyactein stimulates osteoblast function and inhibits bone-resorbing mediators in MC3T3-E1 cells.  


Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In order to improve the treatment of osteoporosis, identification of anabolic agents with minimal side effects is highly desirable. Cimicifuga racemosa has a long and diverse history of medicinal use and deoxyactein isolated from this species is one of the major constituents. In the present study, the effect of deoxyactein on the function of osteoblastic MC3T3-E1 cells was studied. Deoxyactein caused a significant elevation of cell growth, alkaline phosphatase activity, collagen content, and mineralization in the cells (P?

Choi, Eun Mi



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

PubMed Central

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.

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



Diversity of roles of protein kinase C alpha in the proliferation of Swiss 3T3 cells.  

PubMed Central

We examined the role of protein kinase C alpha (PKC alpha ) in the stimulation of DNA synthesis of Swiss 3T3 cells induced by bombesin, platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA). We found that cells in which this kinase had been down-regulated showed a partially abrogated mitogenic response to bombesin. The response to PDGF was unaltered; however, the response to PMA was completely suppressed. The mitogenic effect of maximal doses of bombesin and PMA combined was greater than that of either agent alone, suggesting that bombesin does not fully activate the PKC pathway. Accordingly, bombesin-induced PKC alpha translocation from cytosol to membranes was partial, while that observed with PMA was essentially complete. Moreover, exposure to Ro-31-8220, a PKC inhibitor, had significantly greater effects on the response to PMA than on that to bombesin. Our findings point out different roles that PKC alpha may play in diversely activated cells: while, in the case of PMA, stimulation of this kinase may be necessary and sufficient to induce proliferation, it appears to be necessary only for a full response to bombesin, and redundant among the mechanisms triggered by PDGF.

Florin-Christensen, J; Florin-Christensen, M; Meinardi, E; Calle, R



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

SciTech Connect

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.

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



NYGGF4 (PID1) effects on insulin resistance are reversed by metformin in 3T3-L1 adipocytes.  


NYGGF4 (also called PID1) is a recently discovered gene that is involved in obesity-related insulin resistance (IR). We aimed in the present study to further elucidate the effects of NYGGF4 on IR and the underlying mechanisms through using metformin treatment in 3T3-L1 adipocytes. Our data showed that the metformin pretreatment strikingly enhanced insulin-stimulated glucose uptake through increasing GLUT4 translocation to the PM in NYGGF4 overexpression adipocytes. NYGGF4 overexpression resulted in significant inhibition of tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, whereas incubation with metformin strongly activated IRS-1 and Akt phosphorylation in NYGGF4 overexpression adipocytes. The reactive oxygen species (ROS) levels in NYGGF4 overexpression adipocytes were strikingly enhanced, which could be decreased by the metformin pretreatment. Our data also showed that metformin increased the expressions of PGC1-?, NRF-1, and TFAM, which were reduced in the NYGGF4 overexpression adipocytes. These results suggest that NYGGF4 plays a role in IR and its effects on IR could be reversed by metformin through activating IRS-1/PI3K/Akt and AMPK-PGC1-? pathways. PMID:22968630

Qiu, Jie; Wang, Yu-Mei; Shi, Chun-Mei; Yue, Hong-Ni; Qin, Zhen-Ying; Zhu, Guan-Zhong; Cao, Xin-Guo; Ji, Chen-Bo; Cui, Yan; Guo, Xi-Rong



Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors.  


In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide adoption of the assay partially depend on a fair degree of subjectivity in foci scoring. An objective evaluation may be obtained after separating foci from background monolayer in the digital image, and quantifying values of statistical descriptors which are selected to capture eye-scored morphological features. The aim of this study was to develop statistical descriptors to be applied to transformed foci of BALB/c 3T3, which cover foci size, multilayering and invasive cell growth into the background monolayer. Proposed descriptors were applied to a database of 407 foci images to explore the numerical features, and to illustrate open problems and potential solutions. PMID:23820182

Urani, C; Corvi, R; Callegaro, G; Stefanini, F M



Phenylenediamine derivatives induce GDF-15/MIC-1 and inhibit adipocyte differentiation of mouse 3T3-L1 cells.  


Phenylenediamine derivatives can function as a hydrogen donor and reportedly exert various biological actions including cytoprotective effects against oxidative stress, possibly by acting as an antioxidant. Previous studies showed that feeding of such compounds to mice reduced their body weight, but the precise mechanism remains unknown at present. Here, we found that these compounds inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes, suggesting that, at least in part, reduced generation of adipocytes might contribute to the observed weight loss in mice. Next, we performed array analysis and found that the expression of GDF-15/MIC-1, which is a TGF? superfamily cytokine, and Trib 3, an intracellular downstream effector of the cytokines, was up-regulated by these derivatives. Thus, we identified the compounds as inducers of GDF-15/MIC-1 and suggest that such induction may have led to inhibition of adipocyte differentiation, which could account for the weight-loss effect of these compounds. PMID:22155240

Yanagitai, Mika; Kitagawa, Tomomi; Okawa, Kyouji; Koyama, Hiroki; Satoh, Takumi



Flavanone exhibits PPAR{gamma} ligand activity and enhances differentiation of 3T3-L1 adipocytes  

SciTech Connect

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} (PPAR{gamma}) ligand in a dose-dependent manner. Together, our results suggest that flavanone enhances adipogenesis, at least in part, through its PPAR{gamma} ligand activity.

Saito, Takeshi; 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:



Friedelin and lanosterol from Garcinia prainiana stimulated glucose uptake and adipocytes differentiation in 3T3-L1 adipocytes.  


Friedelin and lanosterol have been isolated from twigs of Garcinia prainiana. Their structures were elucidated by spectroscopic methods. The compounds were examined for their effects on 3T3-L1 adipocytes. In the MTT assay, it was found that the compounds had no cytotoxic effects up to 25?µM. Adipocyte differentiation analysis was carried out by Oil Red O staining method. In the presence of adipogenic cocktail (MDI), it was found that friedelin and lanosterol enhanced intracellular fat accumulation by 2.02 and 2.18-fold, respectively, compared with the vehicle-treated cells. Deoxyglucose uptake assay was used to examine the insulin sensitivity of adipocytes in the presence of the compounds. It was found that friedelin was able to stimulate glucose uptake up to 1.8-fold compared with insulin-treated cells. It was suggested that friedelin and lanosterol may be beneficial to mimic insulin action that would be useful in the treatment of diabetes type 2 patients. PMID:22988818

Susanti, Deny; Amiroudine, Mohamed Zaffar Ali Mohamed; Rezali, Mohamad Fazlin; Taher, Muhammad



[A study on alterations of gene expression in a flat revertant R1 from ras-oncogene transformed NIH/3T3 cells].  


The flat revertant cell line R1, isolated from human activated Ha-ras oncogene transformed NIH/3T3 cells (EJ-NIH/3T3) by mutagen treatment, expresses a variant form of the actin-regulatory protein gelsolin, designated p92-5.7. To clone the gene encoding p92-5.7, gelsolin cDNAs were isolated from a cDNA library of R1 cells. In vitro transcription-translation and nucleotide sequence analyses of the cloned cDNAs identified a point mutation in codon 321 at the cause for the expression of p92-5.7. Considering gelsolin's function as an actin binding protein, the expression of alpha-actin, which is downregulated in many transformed fibroblasts, was analyzed. In EJ-NIH/3T3 cells no alpha-actin transcript was detected, whereas in R1 cells alpha-actin mRNA expression was restored to a level similar to NIH/3T3 cells. Immunofluorescence staining of the cells with an alpha-actin specific monoclonal antibody did not detect any alpha-actin containing microfilaments in EJ-NIH/3T3 cells, but revealed an ordered microfilament pattern in R1 and NIH/3T3 cells. In order to identify other genetic alterations that may also contribute to the revertant phenotype, genes with an elevated expression in R1 cells compared with the parental EJ-NIH/3T3 cells were isolated by using a differential hybridization approach. The identified sequences represented mitochondrial (cytochrome b, cytochrome c oxidase subunit II, NADH dehydrogenase subunits 1 and 4) and alpha 2 (type I) collagen genes. In summary, these results suggest that a complex alteration of the expression of cytoskeletal, mitochondrial and extracellular matrix components is closely associated with the flat reversion of R1 cells. PMID:7693563

Müllauer, L



Layered patterning of hepatocytes in co-culture systems using microfabricated stencils  

PubMed Central

Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling microenvironments in which cell behavior can be observed. Here we present a novel approach to generate layered patterning of hepatocytes on micropatterned fibroblast feeder layers using microfabricated polydimethylsiloxane (PDMS) stencils. We fabricated PDMS stencils to pattern circular holes with diameters of 500 µm. Hepatocytes were co-cultured with 3T3-J2 fibroblasts in two types of patterns to evaluate and characterize the cellular interactions in the co-culture systems. Results of this study demonstrated uniform intracellular albumin staining and E-cadherin expression, increased liver-specific functions, and active glycogen synthesis in the hepatocytes when the heterotypic interface between hepatocytes and fibroblasts was increased by the layered patterning technique. This patterning technique can be a useful experimental tool for applications in basic science, drug screening, and tissue engineering, as well as in the design of bioartificial liver devices.

Cho, Cheul H.; Park, Jaesung; Tilles, Arno W.; Berthiaume, Francois; Toner, Mehmet; Yarmush, Martin L.



Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes.  

PubMed Central

Platelet-derived growth factor (PDGF) stimulates the incorporation of 32P from [gamma-32P]ATP into a Mr approximately 170,000 protein by an endogenous tyrosine-specific protein kinase in membrane preparations of Swiss mouse 3T3 cells. Epidermal growth factor (EGF), but not fibroblast growth factor (FGF) or insulin, stimulates limited incorporation of 32P into a protein of similar molecular weight. The ligand concentration required for half-maximal activity (S0.5) for PDGF stimulation of phosphorylation is 50 ng/ml; saturation is achieved at 300 ng/ml. The S0.5 for ATP is 15 microM. Mg2+ or Mn2+ is required for protein kinase activity. Stimulation of PDGF results in the preferential phosphorylation of tyrosine residues in this Mr approximately 170,000 membrane protein. The Mr approximately 170,000 protein can be resolved into Mr approximately 180,000 and 160,000 components in 4% NaDodSO4 gels. PDGF stimulates 32P incorporation preferentially into the Mr approximately 180,000 and less extensively into the Mr approximately 160,000 protein. EGF stimulates 32P incorporation predominantly into a protein of Mr approximately 160,000. The similarity of PDGF and EGF in stimulating phosphotyrosine-specific protein kinase activity and the stimulation of a similar activity by viral transformation (src) genes suggest that a common mechanism may exist for the phenotypic expression of increased DNA synthesis and cell growth stimulated by these separate factors. Images

Nishimura, J; Huang, J S; Deuel, T F



Amine oxidase substrates mimic several of the insulin effects on adipocyte differentiation in 3T3 F442A cells.  

PubMed Central

We have previously reported that substrates of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) exert short-term insulin-like effects in rat adipocytes, such as stimulation of glucose transport. In the present work, we studied whether these substrates could also mimic long-term actions of insulin. Adipose differentiation of 3T3 F442A cells, which is highly insulin-dependent, served as a model to test the effects of sustained administration of amine oxidase substrates. Daily treatment of confluent cells with 0.75 mM tyramine (a substrate of MAO and SSAO) or benzylamine (a substrate of SSAO) over 1 week caused the acquisition of typical adipocyte morphology. The stimulation of protein synthesis and triacylglycerol accumulation caused by tyramine or benzylamine reached one half of that promoted by insulin. This effect was insensitive to pargyline (an MAO inhibitor), but was inhibited by semicarbazide (an SSAO inhibitor) and by N-acetylcysteine (an antioxidant agent), suggesting the involvement of the H(2)O(2) generated during SSAO-dependent amine oxidation. Chronic administration of amine oxidase substrates also induced the emergence of adipose conversion markers, such as aP2, glycerol-3-phosphate dehydrogenase, the glucose transporter GLUT4, and SSAO itself. Moreover, cells treated with amines acquired the same insulin sensitivity regarding glucose transport as adipocytes classically differentiated with insulin. In all, most of the adipogenic effects of amines were additive to insulin. Our data reveal that amine oxidase substrates partially mimic the adipogenic effect of insulin in cultured preadipocytes. Furthermore, they suggest that SSAO not only represents a novel late marker of adipogenesis, but could also be directly involved in the triggering of terminal adipocyte differentiation.

Fontana, E; Boucher, J; Marti, L; Lizcano, J M; Testar, X; Zorzano, A; Carpene, C



Beta-Mecaptoethanol Suppresses Inflammation and Induces Adipogenic Differentiation in 3T3-F442A Murine Preadipocytes  

PubMed Central

Preadipocytes are present in adipose tissues throughout adult life that can proliferate and differentiate into mature adipocytes in response to environmental cues. Abnormal increase in adipocyte number or size leads to fat tissue expansion. However, it is now recognized that adipocyte hypertrophy is a greater risk factor for metabolic syndrome whereas fat tissue that continues to produce newer and smaller fat cells through preadipocyte differentiation is “metabolically healthy”. Because adipocyte hypertrophy is often associated with increased oxidant stress and low grade inflammation, both are linked to disturbed cellular redox, we tested how preadipocyte differentiation may be regulated by beta-mercaptoethanol (BME), a pharmacological redox regulator and radical scavenger, using murine 3T3-F442A preadipocytes as the cell model. Effects of BME on adipogenesis were measured by microphotography, real-time PCR, and Western analysis. Our data demonstrated that preadipocyte differentiation could be regulated by extracellular BME. At an optimal concentration, BME enhanced expression of adipogenic gene markers and lipid accumulation. This effect was associated with BME-mediated down-regulation of inflammatory cytokine expression during early differentiation. BME also attenuated TNFalpha-induced activation of NFkappaB in differentiating preadipocytes and partially restored TNFalpha-mediated suppression on adipogenesis. Using a non-adipogenic HEK293 cell line transfected with luciferase reporter genes, we demonstrated that BME reduced basal and TNFalpha-induced NFkappaB activity and increased basal and ciglitazone-induced PPARgamma activity; both may contribute to the pro-adipogenic effect of BME in differentiating F442A preadipocytes.

Guo, Wen; Li, Yahui; Liang, Wentao; Wong, Siu; Apovian, Caroline; Kirkland, James L.; Corkey, Barbara E.



Sensitivity of transformation to small differences in population density during serial passage of NIH 3T3 cells.  

PubMed Central

Early passages of the NIH 3T3 mouse cell line undergo spontaneous neoplastic transformation leading to the development of transformed foci if grown to confluence in 2% (vol/vol) calf serum (CS) and left there for more than a week. Transfer of the postconfluent cultures results in the appearance of large numbers of transformed foci; many of them are larger and denser than those in the original culture. If the cells are continually kept at low population densities by frequent passages in 10% CS, they lose the capacity to undergo spontaneous transformation. If however the low-density passages are made in 2% CS or in 10% (vol/vol) fetal bovine serum, both of which support lower growth rates and saturation densities than does 10% CS, they gain the capacities to grow to high saturation densities and produce more foci when grown to confluence in 2% CS. These increases are proportional to the population densities used in the frequent passages, although the densities are all kept well below confluence. We conclude that the combined constraints of submaximal serum plus those of the limited cell contacts of the low cell densities used here elicit an adaptive response that endows the entire population with increased growth capacity. The increased growth capacity of the heterogeneous population in turn increases the capacity of a fraction of the population to initiate distinctive transformed foci. Similar studies have indicated that the capacity of cells to produce tumors and metastases in mice and rats is enhanced by prior maintenance at high density in culture. We propose the concept of progressive state selection to account for the general increase in the growth capacity of cells that is elicited by moderate constraints on their growth and metabolism.

Yao, A; Rubin, H



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

SciTech Connect

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.

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.



Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells  

PubMed Central

Background Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting. Results We identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes. Conclusions We conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis.



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


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

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



A role for Rab14 in the endocytic trafficking of GLUT4 in 3T3-L1 adipocytes  

PubMed Central

Summary Insulin enhances the uptake of glucose into adipocytes and muscle cells by promoting the redistribution of the glucose transporter isoform 4 (GLUT4) from intracellular compartments to the cell surface. Rab GTPases regulate the trafficking itinerary of GLUT4 and several have been found on immunopurified GLUT4 vesicles. Specifically, Rab14 has previously been implicated in GLUT4 trafficking in muscle although its role, if any, in adipocytes is poorly understood. Analysis of 3T3-L1 adipocytes using confocal microscopy demonstrated that endogenous GLUT4 and endogenous Rab14 exhibited a partial colocalisation. However, when wild-type Rab14 or a constitutively-active Rab14Q70L mutant were overexpressed in these cells, the colocalisation with both GLUT4 and IRAP became extensive. Interestingly, this colocalisation was restricted to enlarged ‘ring-like’ vesicular structures (mean diameter 1.3?µm), which were observed in the presence of overexpressed wild-type Rab14 and Rab14Q70L, but not an inactive Rab14S25N mutant. These enlarged vesicles contained markers of early endosomes and were rapidly filled by GLUT4 and transferrin undergoing endocytosis from the plasma membrane. The Rab14Q70L mutant reduced basal and insulin-stimulated cell surface GLUT4 levels, probably by retaining GLUT4 in an insulin-insensitive early endosomal compartment. Furthermore, shRNA-mediated depletion of Rab14 inhibited the transit of GLUT4 through early endosomal compartments towards vesicles and tubules in the perinuclear region. Given the previously reported role of Rab14 in trafficking between endosomes and the Golgi complex, we propose that the primary role of Rab14 in GLUT4 trafficking is to control the transit of internalised GLUT4 from early endosomes into the Golgi complex, rather than direct GLUT4 translocation to the plasma membrane.

Reed, Sam E.; Hodgson, Lorna R.; Song, Shuang; May, Margaret T.; Kelly, Eoin E.; McCaffrey, Mary W.; Mastick, Cynthia C.; Verkade, Paul; Tavare, Jeremy M.



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


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

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



Protecting distribution feeders for simultaneous faults  

Microsoft Academic Search

Overhead distribution systems may experience faults involving more than one feeder. During simultaneous faults, the transformer low-voltage-side overcurrent relay measures a current greater than the current measured by faulted feeder relays. Therefore, the transformer relay may trip faster than faulted feeder relays. Transformer relay misoperation affects service availability in circuits not involved with the fault. In this paper, we describe

J. Betanzos Manuel; H. E. Lemus Zavala; E. Alcazar Ramirez; D. Sanchez Escobedo; H. J. Altuve



Economic feeder for recharging and “topping off”  

Microsoft Academic Search

Increasing the size of the melt charge significantly increases yield and reduces costs. Siemens Solar Industries is optimizing a method to charge additional material after meltdown (top-off) using an external feeder system. A prototype feeder system was fabricated consisting of a hopper and feed delivery system. The low-cost feeder is designed for simple operation and maintenance. The system is capable

Bryan Fickett; G Mihalik



Power flow analysis on simplified feeder modeling  

Microsoft Academic Search

Three novel models to simplify distribution network analysis are presented including equivalent load model (ELM), equivalent load density model (ELDM), and discrete equivalent load density model (DELDM). The voltages and the power on\\/through both ends of a feeder are used to describe the load and its distribution pattern within the feeder line. Only real time field data from feeder circuit

Jian Liu; Bi Pengxiang; Zhang Yanqing; Wu Xiaomeng



Feedback Control for Electromagnetic Vibration Feeder  

Microsoft Academic Search

An electromagnetic-type vibratory feeder of is a typical transportation device used in automatic weighers. As existing feeders are driven by feedforward control, the so-called ``firing angle control'', the driver cannot negate sudden disturbances. In this study, we consider applying a feedback control for such a feeder system. First, we give the two details of modelings for the vibration part and

Tomoharu Doi; Koji Yoshida; Yutaka Tamai; Katsuaki Kono; Kazufumi Naito; Toshiro Ono



Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells  

Microsoft Academic Search

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms\\u000a that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly,\\u000a feeder cells generally lose their capacity

Sergey V. Anisimov; Nicolaj S. Christophersen; Ana S. Correia; Vanessa J. Hall; Ingrid Sandelin; Jia-Yi Li; Patrik Brundin



Nr4a1 siRNA expression attenuates ?-MSH regulated gene expression in 3T3-L1 adipocytes.  


Several recent investigations have underscored the growing role of melanocortin signaling in the peripheral regulation of lipid, glucose, and energy homeostasis. In addition, the melanocortins play a critical role in the central control of satiety. These observations, and the latest reports highlighting the emerging role of the nuclear hormone receptor (NR) 4A subgroup in metabolism, have prompted us to investigate the cross talk between [Nle(4), d-Phe(7)] (NDP)-?-MSH and Nr4a signaling in adipose. We have shown that NDP-MSH strikingly and preferentially induces the expression of the NR4A subgroup (but not any other members of the NR superfamily) in differentiated 3T3-L1 adipocytes. Utilization of quantitative PCR on custom-designed metabolic TaqMan low-density arrays identified the concomitant and marked induction of the mRNAs encoding Il-6, Cox2, Pdk4, and Pck-1 after NDP-MSH treatment. Similar experiments demonstrated that the mRNA expression profile induced by cAMP and NDP-MSH treatment displayed unique but also overlapping properties and suggested that melanocortin-mediated induction of gene expression involves cAMP-dependent and -independent signaling. Nr4a1/Nur77 small interfering RNA (siRNA) expression suppressed NDP-MSH-mediated induction of Nr4a1/Nur77 and Nr4a3/Nor-1 (but not Nr4a2/Nurr1). Moreover, expression of the siRNA-attenuated NDP-MSH mediated induction of the mRNAs encoding Il-6, Cox2/Ptgs2, and Pck-1 expression. In addition, Nur77 siRNA expression attenuated NDP-MSH-mediated glucose uptake. In vivo, ip administration of NDP-MSH to C57 BL/6J (male) mice significantly induced the expression of the mRNA encoding Nur77 and increased IL-6, Cox2, Pck1, and Pdk4 mRNA expression in (inguinal) adipose tissue. We conclude that Nur77 expression is necessary for MSH-mediated induction of gene expression in differentiated adipocytes. Furthermore, this study demonstrates cross talk between MSH and Nr4a signaling in adipocytes. PMID:21239615

Wang, S-C Mary; Myers, Stephen A; Eriksson, Natalie A; Fitzsimmons, Rebecca L; Muscat, George E O



Enhanced liver functions of hepatocytes cocultured with NIH 3T3 in the alginate/galactosylated chitosan scaffold.  


Formation of primary hepatocyte spheroids in the hydrogel scaffold is a promising approach for enhancing liver-specific functions in liver tissue engineering as well as for developing bioartificial liver (BAL) devices. In the present study, a highly porous hydrogel scaffold composed of alginate (AL) and galactosylated chitosan (GC) as a synthetic extracellular matrix (ECM) for hepatocytes was fabricated with 150-200 microm pore size in diameter. Cell adhesion onto AL/GC and AL/chitosan film was 72.7 and 45% at 1 wt% of GC (or chitosan) to AL content whereas cell adhesion onto AL film was 28.5%. The optimal concentration of GC in AL/GC sponge was 1 wt% to AL content by the measurement of albumin secretion. Cell viabilities performed on AL and AL/GC sponges were 72.2+/-3.6 and 81.3+/-3.5% of control, respectively, after 10 days incubation. Hepatocytes were aggregated to form multicellular spheroids in AL/GC sponge with diameter enlarged up to about 100 microm, 36 h postseeding, whereas most of them in the AL sponge remained as single cells and only a few cells began to form aggregates. Intercellular molecules such as connexin32 and E-cadherin genes related with cell-cell contact were expressed in hepatocytes within AL/GC sponge at 36 h after incubation, but not in AL sponge. Treatment with a gap junctional intercellular communication (GJIC) inhibitor, 18beta-glycyrrhetinic acid, resulted in a 1.5-fold marked decrease in albumin secretion levels in AL/GC sponge. Specially, coculture of hepatocytes in AL and AL/GC sponges with NIH3T3 in a transwell insert resulted in enhanced increase of liver-specific functions, such as albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1, compared to those in hepatocyte monoculture. The results suggest that formation of hepatocyte spheroids in coculture system enhances liver-specific functions for the AL/GC sponge as a new synthetic ECM to design developed BAL devices. PMID:16188312

Seo, Seog-Jin; Kim, In-Yong; Choi, Yun-Jaie; Akaike, Toshihiro; Cho, Chong-Su



Rho family GTP binding proteins are involved in the regulatory volume decrease process in NIH3T3 mouse fibroblasts  

PubMed Central

The role of Rho GTPases in the regulatory volume decrease (RVD) process following osmotic cell swelling is controversial and has so far only been investigated for the swelling-activated Cl? efflux. We investigated the involvement of RhoA in the RVD process in NIH3T3 mouse fibroblasts, using wild-type cells and three clones expressing constitutively active RhoA (RhoAV14). RhoAV14 expression resulted in an up to fourfold increase in the rate of RVD, measured by large-angle light scattering. The increase in RVD rate correlated with RhoAV14 expression. RVD in wild-type cells was unaffected by the Rho kinase inhibitor Y-27632 and the phosphatidyl-inositol 3 kinase (PI3K) inhibitor wortmannin. The maximal rates of swelling-activated K+ (86Rb+ as tracer) and taurine ([3H]taurine as tracer) efflux after a 30 % reduction in extracellular osmolarity were increased about twofold in cells with maximal RhoAV14 expression compared to wild-type cells, but were unaffected by Y-27632. The volume set points for activation of release of both osmolytes appeared to be reduced by RhoAV14 expression. The maximal taurine efflux rate constant was potentiated by the tyrosine phosphatase inhibitor Na3VO4, and inhibited by the tyrosine kinase inhibitor genistein. The magnitude of the swelling-activated Cl? current (ICl,swell) was higher in RhoAV14 than in wild-type cells after a 7.5 % reduction in extracellular osmolarity, but, in contrast to 86Rb+ and [3H]taurine efflux, similar in both strains after a 30 % reduction in extracellular osmolarity. ICl,swell was inhibited by Y-27632 and strongly potentiated by the myosin light chain kinase inhibitors ML-7 and AV25. It is suggested that RhoA, although not the volume sensor per se, is an important upstream modulator shared by multiple swelling-activated channels on which RhoA exerts its effects via divergent signalling pathways.

Pedersen, Stine F; Beisner, Kristine H; Hougaard, Charlotte; Willumsen, Berthe M; Lambert, Ian H; Hoffmann, Else K



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

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

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



Effect of a Seaweed Extract on Fatty Acid Accumulation and Glycerol3Phosphate Dehydrogenase Activity in 3T3-L1 Adipocytes  

Microsoft Academic Search

This study was to determine the effect of a seaweed Ascophyllum nodosum extract (SE) containing 220 mg g?1 phlorotannins on differentiation and fatty acid accumulation in differentiating 3T3-L1 adipocytes. 3T3-L1 cells (2 × 104 mL?1) were seeded to 24-well plates and proliferated to reach confluence and then were treated with media containing 0, 12.5,\\u000a 25, 50, 75 and 100 ?g mL?1 SE for 8 days. Dexamethasone,

M. L. He; Y. Wang; J. S. You; P. S. Mir; T. A. McAllister



PDGF-induces the glutathione-dependent enzyme PGH2/PGE2 isomerase in NIH3T3 and pEJ transformed fibroblasts.  


Exposure of NIH3T3 and pEJ serum-starved cells to platelet derived growth factor results in a 16 fold increase in the glutathione-dependent enzyme prostaglandin H2/prostaglandin E2 isomerase activity (EC The response is rapid as a detectable increase in NIH3T3 cells occurs after only 7 minutes of exposure to the growth factor. Only a mild increase in another microsomal glutathione-dependent enzyme, microsomal glutathione transferase (EC, was detected after a 2 hour exposure to the growth factor. PMID:8292033

Kelner, M J; Uglik, S F



Dominant negative ?-subunit of farnesyl- and geranylgeranyl-transferase I inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes  

Microsoft Academic Search

OBJECTIVE: To investigate whether the expression of a dominant negative (DN) farnesyl- and geranygeranyl-transferase I (FTase\\/GGTase I) ?-subunit in 3T3-L1 pre-adipocytes can inhibit insulin's ability to induce differentiation.DESIGN: 3T3-L1 pre-adipocytes were stably transfected with vector alone or vector expressing a mutated DN FTase\\/GGTase I ?-subunit (S60A)(S62A) and incubated in serum-free medium in the absence and presence of insulin.MEASUREMENTS: Various assays

C S Solomon; J W Leitner; M L Goalstone



Conjugated Linoleic Acid Inhibits Proliferation but Stimulates Lipid Filling of Murine 3T3-L1 Preadipocytes1,2,3  

Microsoft Academic Search

This study documented the effects of conjugated linoleic acid (CLA) on the proliferation and differentiation of 3T3-L1 preadipocytes. During proliferation, preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM), 100 g\\/L fetal bovine serum (FBS), 0.584 g\\/L L-glutamine and 0 (control), 0.5, 1.0, 5.0 or 10.0 mg\\/L CLA. Proliferation of 3T3-L1 preadipocytes was measured directly by cell counting and indirectly

David L. Satory; Stephen B. Smith


Inhibition of Calcium-Independent Phospholipases A2beta or A2gamma Inhibit Hormone-Induced Differentiation of 3T3-L1 Preadipocytes.  

National Technical Information Service (NTIS)

A method for identifying an agonist exhibiting molecular or pharmacologic inhibition which is effective against the activity of at least one of iPLA(sub 2)beta and iPLA(sub 2)gamma which comprises culturing 3T3-L1 cells and transfecting them with negative...

R. W. Gross



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

PubMed Central

SUMMARY Survivin is an inhibitor of apoptosis (IAP) 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 3 (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 that this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.

Du, Jiamu; Kelly, Alexander E.; Funabiki, Hironori; Patel, Dinshaw J.



Injectable calcium phosphate-alginate-chitosan microencapsulated MC3T3-E1 cell paste for bone tissue engineering in vivo.  


Osteoblasts or stem cells have been delivered into injectable calcium phosphate cement (CPC) to improve its effectiveness and biological function. However, the osteogenic potential of the new construct in vivo has been rarely reported, and there are no reports on alginate-chitosan microencapsulated osteoblasts mixed with CPC. This study aimed to develop alginate-chitosan microencapsulated mouse osteoblast MC3T3-E1 cells (AC-cells), evaluate the osteogenic potential of a calcium phosphate cement complex with these AC-cells (CPC-AC-cell), and trace the implanted MC3T3-E1 cells in vivo. MC3T3-E1 cells were embedded in alginate microcapsules, cultured in osteogenic medium for 7days, and then covered with chitosan before mixing with a paste of ?-tricalcium phosphate/calcium phosphate cement (?-TCP/CPC). The construct was injected into the dorsal subcutaneous area of nude mice. Lamellar-bone-like mineralization, newly formed collagen and angiogenesis were observed at 4weeks. At 8weeks, areas of newly formed collagen expanded; further absorption of ?-TCP/CPC and osteoid-like structures could be seen. Cell tracing in vivo showed that implanted MC3T3-E1 cells were clearly visible at 2weeks. These in vivo results indicate that the novel injectable CPC-AC-cell construct is promising for bone tissue engineering applications. PMID:24094170

Qiao, Pengyan; Wang, Juan; Xie, Qiufei; Li, Fangfang; Dong, Limin; Xu, Tao



Structure-based hybridization of the bioactive natural products rhizonin A and ternatin leading to a selective fat-accumulation inhibitor against 3T3-L1 adipocytes  

Microsoft Academic Search

Based on the structural similarity between the naturally occurring cyclic heptapeptides rhizonin A and ternatin, two novel analogues were designed. The synthetic analogues were assessed with regard to their fat-accumulation inhibitory effect against 3T3-L1 adipocytes, and this led to the discovery of a potent and selective fat-accumulation inhibitor compared to the parent compound rhizonin A.

Kenichiro Shimokawa; Kaoru Yamada; Daisuke Uemura



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


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

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



NIH3T3 cells overexpressing CD98 heavy chain resist early G1 arrest and apoptosis induced by serum starvation.  


CD98 is a heterodimeric glycoprotein of 125-kDa, which consists of a 90-kDa heavy chain (hc) subunit and 35-kDa to 55-kDa light chain (lc) subunits. It is strongly expressed on the surface of proliferating normal cells and almost all tumor cells. To investigate the participation of CD98 in cellular proliferation and malignant transformation, we analyzed cell-cycle progression of NIH3T3 clones transfected with cDNA of human CD98hc. Although NIH3T3 and control transfectant cells grown to the subconfluent state were arrested in the G0/G1 phase by serum starvation, considerable portions of CD98hc-transfected cells resided at S and G2/M phases. Under serum-starved and confluent conditions, significant fractions (20-25%) of NIH3T3 and control transfectant cells contained less than 2n content DNA, indicating occurrence of apoptosis, whereas no apoptotic cells were detected in CD98hc-transfectant cells. Under serum-starved conditions, a marked increase in the levels of cyclin D1 and cyclin E and a decrease in p16 were observed in CD98hc-transfectant cells. The reverse was true for NIH3T3 and control transfectant cells. Our results suggest that resistance to G1 arrest and apoptosis by CD98 overexpression are associated with high G1-cyclins and low p16 levels. PMID:22497681

Hara, Kaori; Ueda, Shiho; Ohno, Yoshiya; Tanaka, Toshiyuki; Yagi, Hideki; Okazaki, Shogo; Kawahara, Rieko; Masayuki, Takechi; Enomoto, Takemi; Hashimoto, Yoshiyuki; Masuko, Kazue; Masuko, Takashi



Combined effects of 60?Hz electromagnetic field exposure with various stress factors on cellular transformation in NIH3T3 cells.  


Epidemiological studies have suggested that extremely low-frequency magnetic fields (ELF-MF) are associated with an increased incidence of cancer. Studies using in vitro systems have reported mixed results for the effects of ELF-MF alone, and the World Health Organization (WHO) Research Agenda published in 2007 suggested that high priority research should include an evaluation of the co-carcinogenic effects of ELF-MF exposure using in vitro models. Here, the carcinogenic potential of ELF-MF exposure alone and in combination with various stress factors was investigated in NIH3T3 mouse fibroblasts using an in vitro cellular transformation assay. NIH3T3 cells were exposed to a 60 Hz ELF-MF (1 mT) alone or in combination with ionizing radiation (IR), hydrogen peroxide (H?O?), or c-Myc overexpression, and the resulting number of anchorage-independent colonies was counted. A 4 h exposure of NIH3T3 cells to ELF-MF alone produced no cell transformation. Moreover, ELF exposure did not influence the transformation activity of IR, H?O?, or activated c-Myc in our in vitro assay system, suggesting that 1 mT ELF-MF did not affect any additive or synergistic transformation activities in combination with stress factors such as IR, H?O?, or activated c-Myc in NIH3T3 cells. PMID:21898471

Lee, Hae-June; Jin, Yeung Bae; Lee, Jae Seon; Choi, Jong-Il; Lee, Ju-Woon; Myung, Sung Ho; Lee, Yun-Sil



Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens  

SciTech Connect

BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B{sub 1} (AFT{sub 1}), and 4-nitroquinoline-N-oxide (4-NQO)). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUA{sup R}) was examined, the authors found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUA{sup R} locus in this system, while AFB{sub 1} was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (CIN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. CIN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.

Lubet, R.A. (National Cancer Institute, Frederick, MD (USA)); Kouri, R.E.; Curren, R.A.; Putman, D.L.; Schechtman, L.M. (Microbiological Associates, Bethesda, MD (USA))



Ultrasound-targeted microbubble destruction enhances gene transduction of adeno-associated virus in a less-permissive cell type, NIH/3T3  

PubMed Central

Adeno-associated virus (AAV) is a common vector utilized in gene therapy. The NIH/3T3 cell line, which is a potential induced pluripotent stem (iPS) cell type, was identified to be a less-permissive cell type to AAV due to its defective endosomal processing. Ultrasound-targeted microbubble destruction (UTMD) enhanced the gene transduction of AAV in permissive cells. However, there are no data concerning UTMD enhancement in less-permissive cells, and the exact mechanism of UTMD enhancement in cellular uptake is unclear. Greater knowledge concerning the rate-limiting steps in NIH/3T3 cells would aid in the elucidation of the mechanism of UTMD enhancement in the gene transduction of AAV. In the present study, UTMD enhanced the gene transduction of AAV in NIH/3T3 cells, suggesting that UTMD-enhanced AAV-mediated gene transduction may be beneficial for gene therapy in iPS cells. The dose dependence of UTMD enhancement indicated that mechanisms other than sonoporation were involved in the cellular uptake of AAV. However, UTMD did not greatly increase the gene transduction of AAV in NIH/3T3 cells. Additionally, the similar degree of enhancement in the two cell types resulted in no correlation between UTMD and endosomal processing. Future studies on UTMD-mediated AAV transduction in other non- or less-permissive cell types may aid in elucidating the exact mechanism of UTMD enhancement in cellular uptake.




Simvastatin suppresses leptin expression in 3T3-L1 adipocytes via activation of the cyclic AMP-PKA pathway induced by inhibition of protein prenylation.  


Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyses conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at 1 microM markedly inhibited adipocyte differentiation measured by Oil Red O staining in preadipocyte cells (3T3-L1), while expression of leptin, a marker of adipocyte differentiation, was suppressed by 1 muM simvastatin for up to 12 days of culture. Next, to elucidate mechanisms underlying the reduction of leptin expression induced by simvastatin, differentiated 3T3-L1 adipocytes were treated with various inhibitors with mevalonate or its metabolite in the presence or absence of simvastatin. Simvastatin time- and dose-dependently suppressed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by 10 muM simvastatin, while stability of the mRNA was not changed by treatment with simvastatin in transcription-arrested 3T3-L1 cells. Simvastatin inhibition of leptin gene transcription was not abrogated by pre-treatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate (GGPP), a mevalonate metabolite, abolished simvastatin-induced inhibition of leptin expression in 3T3-L1 cells. Suppression of expression was observed upon addition of GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor. Expression was suppressed by treatment with hydroxyfasudil, a protein prenylation inhibitor. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells. Simvastatin dose-dependently increased intra-cellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells, with maximal stimulation obtained at 10 muM. Addition of GGPP abolished simvastatin-induced stimulation of cAMP accumulation and protein kinase A (PKA) activity. H89, an inhibitor of PKA, completely abolished simvastatin-induced suppression of leptin expression. These results suggested that simvastatin reduced geranylgeranylprotein prenylation followed by deactivation of PI3K, leading to cAMP accumulation and subsequent activation of PKA in differentiated 3T3-L1 adipocytes. Finally, PKA inhibited leptin gene transcription without new protein synthesis. PMID:19254925

Maeda, Toyonobu; Horiuchi, Noboru



The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein beta.  


Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein beta (PI-TPbeta, SPIbeta cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPIbetaS262A cells overexpressing a mutant PI-TPbeta that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable with wtNIH3T3 cells. This suggests that the phosphorylation of Ser-262 in PI-TPbeta is involved in the regulation of apoptosis. Conditioned medium (CM) from wtNIH3T3 cells contains bioactive factors, presumably arachidonic acid metabolites [H. Bunte, et al., 2006; M. Schenning, et al., 2004] that are able to protect SPIbeta cells against UV-induced apoptosis. CM from SPIbeta cells lacks this protective activity. However, after heat denaturation CM from SPIbeta cells regains a protective activity comparable with that of wtNIH3T3 cells. This indicates that CM from SPIbeta cells contains an antagonistic factor interfering with the anti-apoptotic activity present. SPIbetaS262A cells do not produce the antagonist suggesting that phosphorylation of Ser-262 is required. Moreover, in line with the apparent lack of anti-apoptotic activity, CM from SPIbeta cells does not induce the expression of COX-2 or the activation of p42/p44 MAP kinase in SPIbeta cells. In contrast, CM from wtNIH3T3 and SPIbetaS262A cells or heat-treated CM from SPIbeta cells does induce these anti-apoptotic markers. Since we have previously shown that some of the arachidonic acid metabolites present in CM from wtNIH3T3 cells are prostaglandin (PG) E(2) and PGF(2alpha), we investigated the effect of these PGs on cell survival. Although PGE(2) and PGF(2alpha) were found to protect wtNIH3T3 and SPIbetaS262A cells against UV-induced apoptosis, these PGs failed to rescue SPIbeta cells. The fact that the concentrations of PGE(2) and PGF(2alpha) in the CM from SPIbeta cells and wtNIH3T3 cells were found to be comparable suggests that the failure of these PGs to protect SPIbeta cells could render these cells more apoptosis sensitive. Concomitantly, upon incubation with PGE(2) and PGF(2alpha), an increased expression of COX-2 and activation of p42/p44 MAP kinase were observed in wtNIH3T3 and SPIbetaS262A cells but not in SPIbeta cells. Hence, it appears that specific mechanisms of cell survival are impaired in SPIbeta cells. PMID:17683809

Schenning, Martijn; van Tiel, Claudia M; Wirtz, Karel W A; Snoek, Gerry T



Microscopic filter feeders near boundaries  

NASA Astrophysics Data System (ADS)

We show through calculations, simulations, and experiments that the eddies often observed near sessile filter feeders are due to the presence of nearby boundaries. We model the common filter feeder Vorticella, which is approx 50 ?m across and which feeds by removing bacteria from ocean or pond water that it draws towards itself. We use an analytic stokeslet model and a Brinkman flow approximation with the organism modeled as a cylinder with two different boundary conditions to predict the size of the eddy caused by two parallel no-slip boundaries that represent the slides between which experimental observations are often made. We also use three-dimensional finite-element simulations to fully solve for the flow around a model Vorticella. Additionally, we track particles around live feeding Vorticella in order to determine the experimental flow field. Our models are in good agreement both with each other and with the experiments. We also show through calculations that filter feeders such as Vorticella can greatly enhance their nutrient uptake by feeding at an angle rather than perpendicular to a substrate.

Pepper, Rachel; Roper, Marcus; Ryu, Sangjin; Matsudiara, Paul; Stone, Howard



Erythropoietin improves insulin resistance via the regulation of its receptor-mediated signaling pathways in 3T3L1 adipocytes.  


Recombinant human erythropoietin (rHuEPO) reduces serum insulin levels, increases insulin sensitivity, and reduces insulin resistance (IR). However, the mechanisms behind these effects are unclear. This study aimed to investigate the mechanism by which rHuEPO effects IR in 3T3L1 adipocytes. After treatment with different concentrations of rHuEPO, glucose consumption, and tumor necrosis factor (TNF-?), adiponectin, and leptin levels were assayed with a commercial enzyme-linked immunosorbent assays. Endogenous erythropoietin receptor (EPOR) expression was inhibited using small interfering RNA (siRNA). EPOR protein and mRNA expression was detected via immunofluorescence and real-time PCR analyses, respectively. The expression of pAKT/AKT and p-STAT5/STAT5 was determined via Western blot analysis. rHuEPO treatment improved glucose uptake, increased adiponectin levels, and reduced TNF-? and leptin levels in 3T3L1 adipocytes with dexamethasone-induced IR. Whereas EPOR protein and gene expression was absent in preadipocytes, it was observed in mature 3T3L1 adipocytes. However, the expression of EPOR in insulin resistant 3T3L1 adipocytes was significantly decreased (p<0.05). rHuEPO increased the expression of EPOR, and upregulated the expression of pAKT/AKT and pSTAT5/STAT5 in 3T3L1 adipocytes (p<0.05), which was blocked by siEPOR, the phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002, and a STAT5 inhibitor, respectively. In summary, rHuEPO reduced IR in adipocytes by increasing glucose uptake and improving the adipokine profile. rHuEPO-induced EPOR protein expression and subsequent induction of pAKT and pSTAT5 suggest that the EPO-EPOR system may play a role in glucose metabolism within adipocytes. PMID:23313788

Pan, Yu; Shu, Jin Lian; Gu, Hui Fang; Zhou, Dong Chi; Liu, Xiao Li; Qiao, Qing Yan; Fu, Shun Kun; Gao, Feng Hou; Jin, Hui Min



The 3T3 neutral red uptake phototoxicity test: practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop.  


This is the report from the "ECVAM-EFPIA workshop on 3T3 NRU Phototoxicity Test: Practical Experience and Implications for Phototoxicity Testing", jointly organized by ECVAM and EFPIA and held on the 25-27 October 2010 in Somma Lombardo, Italy. The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 within the European Commission Joint Research, based on a Communication from the European Commission (1991). The main objective of ECVAM is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine and replace the use of laboratory animals. The European Federation of Pharmaceuticals Industries and Association (EFPIA) represent the pharmaceutical industry operating in Europe. Through its direct membership of 31 national associations and 40 leading pharmaceutical companies, EFPIA is the voice on the EU scene of 2200 companies committed to researching, developing and bringing to patients new medicines that improve health and the quality of life around the world. The workshop, co-chaired by Joachim Kreysa (ECVAM) and Phil Wilcox (GSK, EFPIA) involved thirty-five experts from academia, regulatory authorities and industry, invited to contribute with their experiences in the field of phototoxicology. The main objectives of the workshop were: -to present 'in use' experience of the pharmaceutical industry with the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU-PT), -to discuss why it differs from the results in the original validation exercise, -to discuss technical issues and consider ways to improve the usability of the 3T3 NRU-PT for (non-topical) pharmaceuticals, e.g., by modifying the threshold of chemical light absorption to trigger photo-toxicological testing, and by modifying technical aspects of the assay, or adjusting the criteria used to classify a positive response. During the workshop, the assay methodology was reviewed by comparing the OECD Test Guideline (TG 432) with the protocols used in testing laboratories, data from EFPIA and JPMA 'surveys' were presented and possible reasons for the outcomes were discussed. Experts from cosmetics and pharmaceutical industries reported on their experience with the 3T3 NRU-PT and evidence was presented for phototoxic clinical symptoms that could be linked to certain relevant molecules. Brainstorming sessions discussed if the 3T3 NRU-PT needed to be improved and whether alternatives to the 3T3 NRU-PT exist. Finally, the viewpoint from EU and US regulators was presented. In the final session, the conclusions of the meeting were summarized, with action points. It was concluded that the 3T3 NRU-PT identifies phototoxicological hazards with a 100% sensitivity, and thus is accepted as the tier one test that correctly identifies the absence of phototoxic potential. Consequently, positive results in the 3T3 NRU-PT often do not translate into a clinical phototoxicity risk. Possible ways to improve the practical use of this assay include: (i) adaptation of changed UV/vis-absorption criteria as a means to reduce the number of materials tested, (ii) reduction of the highest concentration to be tested, and (iii) consideration of modifying the threshold criteria for the prediction of a positive call in the test. PMID:22687423

Ceridono, Mara; Tellner, Pär; Bauer, Daniel; Barroso, Joăo; Alépée, Nathalie; Corvi, Raffaella; De Smedt, Ann; Fellows, Mick D; Gibbs, Neil K; Heisler, Eckhard; Jacobs, Abigail; Jirova, Dagmar; Jones, David; Kandárová, Helena; Kasper, Peter; Akunda, Jacqueline Kinyamu; Krul, Cyrille; Learn, Douglas; Liebsch, Manfred; Lynch, Anthony M; Muster, Wolfgang; Nakamura, Kazuichi; Nash, J Frank; Pfannenbecker, Uwe; Phillips, Gareth; Robles, Catherine; Rogiers, Vera; Van De Water, Femke; Liminga, Ulla Wändel; Vohr, Hans-Werner; Wattrelos, Olivier; Woods, Julie; Zuang, Valérie; Kreysa, Joachim; Wilcox, Phil



Characterization of Extracellular Matrix (ECM) Produced by MC3T3 Cells Using Thickness Shear Mode (TSM) Resonators  

Microsoft Academic Search

Quartz thickness shear mode (TSM) resonators for monitoring the attachment and spreading of mammalian cells have been investigated in the past years. Recent studies have shown that the TSM resonator signal is not only contributed by cellular body closed to the resonator substrate, but also contributed by the extracellular matrix (ECM), which is a protein layer between the cellular body

Fang Li; Qing-Ming Wang; James H.-C. Wang



The dual-effects of LaCl? on the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells.  


A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, alkaline phosphatase (ALP) activity measurement, alizarin red S stain and measurement, quantitative real-time reverse transcriptase polymerase chain reaction, and Western blot analysis were employed to assess the effects of LaCl? on the proliferation, osteogenic differentiation, and mineralization of a murine preosteoblast cell line MC3T3-E1 at cell and molecular levels. The results indicated that LaCl? had dual effects on the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells. First, LaCl? promoted the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells at lower concentrations, then had no effects and further turned to inhibit the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells with increasing concentrations. The expression of runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (BMP2), ALP, bone sialoprotein (BSP), collagen I (Col I), and osteocalcin (OCN) genes was upregulated in the presence of 0.0001 and 0.1 ?M LaCl?, but these genes were downregulated in the MC3T3-E1 cells treated with 1,000 ?M LaCl?. In addition, the expression of BMP2, Runx2, and OCN proteins was promoted by LaCl? at the concentration of 0.0001 ?M, but these proteins were downregulated after 1,000 ?M LaCl? treatment. The results suggest that LaCl? likely up- or downregulates the expression of Runx2, which subsequently up- or downregulates osteoblasts marker genes Col I and BMP2 at early stages and ALP and OCN at later stages of differentiation, thus causes to promote or inhibit the proliferation, osteogenic differentiation and mineralization of MC3T3-E1 cells. The results will be helpful for understanding the mechanisms of bone metabolism and application of lanthanum-based compounds in the future. PMID:22886987

Liu, Dandan; Zhang, Jinchao; Wang, Guifang; Liu, Xiaolong; Wang, Shuxiang; Yang, MengSu



Titanium Immobilized with an Antimicrobial Peptide Derived from Histatin Accelerates the Differentiation of Osteoblastic Cell Line, MC3T3-E1  

PubMed Central

The objective of this study was to evaluate the effect of titanium immobilized with a cationic antimicrobial peptide (JH8194) derived from histatin on the biofilm formation of Porphyromonas gingivalis and differentiation of osteoblastic cells (MC3T3-E1). The titanium specimens (Ti) were immobilized with JH8194, according to the method previously described. The colonization of P. gingivalis on JH8194-Ti was significantly lower than that on control- and blocking-Ti. JH8194-Ti enhanced the mRNA expressions of Runx2 and OPN, and ALPase activity in the MC3T3-E1, as compared with those of control- and blocking-Ti. These results, taken together, suggested the possibility that JH8194-Ti may be a potential aid to shorten the period of acquiring osseointegration.

Makihira, Seicho; Shuto, Takahiro; Nikawa, Hiroki; Okamoto, Keishi; Mine, Yuichi; Takamoto, Yuko; Ohara, Masaru; Tsuji, Koichiro



Unraveling the mode of action of an obesogen: mechanistic analysis of the model obesogen tributyltin in the 3T3-L1 cell line.  


Obesogenic compounds are chemicals that have an influence on obesity development. This study was designed to unravel the molecular mechanisms of the model obesogen TBT, using microarray analysis in the 3T3-L1 in vitro system, and to evaluate the use of toxicogenomics for obesogen screening. The microarray results revealed enrichment of Gene Ontology terms involved in energy and fat metabolism after 10 days of TBT exposure. Pathway analysis unveiled PPAR signalling pathway as the sole pathway significantly enriched after 1 day and the most significantly enriched pathway after 10 days of exposure. To our knowledge, this is the first study delivering an in depth mechanistic outline of the mode of action of TBT as an obesogen, combining effects on both cell physiological and gene expression level. Furthermore, our results show that combining transcriptomics with 3T3-L1 cells is a promising tool for screening of potential obesogenic compounds. PMID:23428407

Pereira-Fernandes, Anna; Vanparys, Caroline; Hectors, Tine L M; Vergauwen, Lucia; Knapen, Dries; Jorens, Philippe G; Blust, Ronny



Identification of benzophenone C-glucosides from mango tree leaves and their inhibitory effect on triglyceride accumulation in 3T3-L1 adipocytes.  


A 70% ethanol-water extract from the leaves of Mangifera indica L. (Anacardiaceae) inhibited triglyceride (TG) accumulation in 3T3-L1 cells. From the active fraction, seven new benzophenone C-glycosides, foliamangiferosides A (1), A(1) (2), A(2) (3), B (4), C(1) (5), C(2) (6), and C(3) (7), together with five known compounds were isolated and the structures were elucidated on the basis of chemical and physicochemical evidence. The effects of these compounds on TG and the free fatty acid level in 3T3-L1 cells were determined, and the structure-activity relationship was discussed. On the basis of the AMPK signaling pathway, several compounds were found to increase the AMPK enzyme expression and down-regulate lipogenic enzyme gene expression such as SREBP1c, FAS, and HSL. PMID:21923172

Zhang, Yi; Qian, Qian; Ge, Dandan; Li, Yuhong; Wang, Xinrui; Chen, Qiu; Gao, Xiumei; Wang, Tao



31 P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na + and K + and mitogenic stimulation  

Microsoft Academic Search

Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional31P and19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular

Mortimer M. Civan; Stephen R. Williams; David G. Gadian; Enrique Rozengurt



Identification of genes potentially involved in supporting hematopoietic stem cell activity of stromal cell line MC3T3-G2\\/PA6  

Microsoft Academic Search

Although coculture of hematopoietic stem cells (HSCs) with stromal cells is a useful system to study hematopoiesis in the\\u000a niche, little is known regarding the precise cellular and molecular mechanisms of maintaining HSCs through cell–cell interactions.\\u000a The murine preadipose stromal cell line MC3T3-G2\\/PA6 (PA6) has been demonstrated to support HSCs in vitro. In this study,\\u000a microarray analysis was performed on

Natsumi Shimizu; Shinichi Noda; Kazufumi Katayama; Hitoshi Ichikawa; Hiroaki Kodama; Hiroyuki Miyoshi



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


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. PMID:22705593

St?pnik, Maciej; Arkusz, Joanna; Smok-Pieni??ek, Anna; Bratek-Skicki, Anna; Salvati, Anna; Lynch, Iseult; Dawson, Kenneth A; Gromadzi?ska, Jolanta; De Jong, Wim H; Rydzy?ski, Konrad



Effect of Pinus radiata bark extracts with different molecular weight distributions on cell growth of NIH\\/3T3 fibroblasts and dendrite retraction of B16 melanoma cells  

Microsoft Academic Search

Hot water extract (HWE) of Pinus radiata bark was separated into monomeric polyphenol (MPP), oligomeric proanthocyanidin (OPA), and polymeric proanthocyanidin (PPA)\\u000a fractions by monitored chromatography using a Sephadex LH 20 column and an UV detector at 250 nm. The effects of these fractions\\u000a on NIH\\/3T3 fibroblasts and B16 melanoma cells were examined by evaluating cell viability, melanogenesis (melanin content),\\u000a morphological

Chang Sub Ku; Sung Phil Mun


Effects of Ca 2+Ionophore A23187 and Calmodulin Antagonists on Regulatory Mechanisms of Glycolysis and Cell Viability of NIH-3T3 Fibroblasts  

Microsoft Academic Search

We studied here, in NIH-3T3 fibroblasts, the effect of the Ca2+-ionophore A23187 (which is known to increase intracellular-free Ca2+) on the control of glycolysis and cell viability and the action of calmodulin antagonists. Time-response studies with Ca2+-ionophore A23187 have revealed dual effects on the distribution of phosphofructokinase (PFK) (EC, the rate-limiting enzyme of glycolysis, between the cytoskeletal and cytosolic

Michal Ashkenazy-Shahar; Rivka Beitner



Effect of trans 8, cis 10+ cis 9, trans 11 Conjugated Linoleic Acid Mixture on Lipid Metabolism in 3T3-L1 Cells  

Microsoft Academic Search

Evidence suggests that minor isomers of conjugated linoleic acid (CLA), such as trans8, cis10 CLA, can elicit unique biological effects of their own. In order to determine the effect of a mixture of t8, c10+c9, t11 CLA isomers on selected aspects of lipid metabolism, 3T3-L1 preadipocytes were differentiated for 8 days in the presence\\u000a of 100 ?M linoleic acid (LA); t8, c10+c9,

Shama V. Joseph; Jessica R. Miller; Roger S. McLeod; Hélčne Jacques



Elucidation of a Signaling Pathway Induced by FGF2 Leading to uPA Gene Expression in NIH 3T3 Fibroblasts  

Microsoft Academic Search

Fibroblast growth factors (FGFs) play a role in biological processes such as cell growth and development, angiogenesis, and wound healing. Several genes have been shown to be induced by FGFs, but the underlying mechanisms have not been elucidated. We investigated the effect of FGF-2 (basic FGF) on the urokinase-type plasminogen activator (uPA) gene in NIH 3T3 fibroblasts. We found that

Daniel Besser; Marco Presta; Yoshikuni Nagamine



Topoisomerase I(-mediated DNA Breaks and Cytotoxicity in Relation to Cell Proliferation and the Cell Cycle in NIH 3T3 Fibroblasts and L1210 Leukemia Cells  

Microsoft Academic Search

The DNA intercalato!-, 4'-{9-acridinylamino)methanesuIfon-\\/n-anisi- dide (m-AMSA) and the nonintercalator, etoposide (VP-16) produce topoisomerase Il-mediated protein-linked DNA strand breaks. This func tion of topoisomerase II was investigated in relation to cell proliferation and cell cycle. Mouse fibroblasts NIH 3T3 and mouse leukemia LI 210 cells stop proliferation when they reach a certain density. Nuclei were isolated from proliferative or quiescent cells

Judith Markovits; Yves Pommier; Donna Kerrigan; Joseph M. Covey; Eugene J. Tilchen; Kurt W. Kohn



Ras Transformation Results in an Elevated Level of Cyclin D1 and Acceleration of G1Progression in NIH 3T3 cells  

Microsoft Academic Search

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower




Reversion of v-H- ras-Trasformed NIH 3T3 Cells by Apigenin Through Inhibiting Mitogen-Activated Protein Kinase and Its Downstream Oncogenes  

Microsoft Academic Search

Apigenin, a plant flavonoid, induced the reversion of transformed phenotypes of v-H-ras-transformed NIH 3T3 cells at a quite low concentration of 12.5 ?M. In the present study, we have examined the components of this Ras-mediated signaling transduction to determine whether they were involved in the apigenin-induced reversion process. Interestingly, the consitutively activated mitogen activated protein kinase (MAPK) in the ras

M. L. Kuo; N. C. Yang



Invasiveness and Metastasis of NIH 3T3 Cells Induced by Met-Hepatocyte Growth Factor\\/Scatter Factor Autocrine Stimulation  

Microsoft Academic Search

The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor\\/scatter factor (HGF\\/SF). NIH 3T3 cells express HGF\\/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF\\/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH

Sing Rong; Shraga Segal; Miriam Anver; James H. Resau; George F. Vande Woude



External Cl--Dependent Formation of Watery Vacuoles by Long-Term Hypotonic Shock in 3T3-L1 Cells  

Microsoft Academic Search

Osmotic shock transiently induces a volume change in the cells, followed by a restoration of the cell volume due to intracellular water regulation. Effect of long-term osmotic shock on the water regulation is not completely understood. Vacuole formation by long-term osmotic shock was investigated to clarify the water exclusion mechanism from cytoplasm into intracellular vacuoles in 3T3-L1 cells. Incubation of

Yoshiko Iwasa; Chikara Hirono; Makoto Sugita; Kazuhisa Takemoto; Yoshiki Shiba



Direct Comparison of the Spread Area, Contractility, and Migration of balb\\/c 3T3 Fibroblasts Adhered to Fibronectin and RGD-Modified Substrata  

Microsoft Academic Search

Native proteins are often substituted by short peptide sequences. These peptides can recapitulate key, but not all biofunctional properties of the native proteins. Here, we quantify the similarities and differences in spread area, contractile activity, and migration speed for balb\\/c 3T3 fibroblasts adhered to fibronectin- (FN) and Arg-Gly-Asp (RGD)-modified substrata of varying surface density. In both cases spread area has

Padmavathy Rajagopalan; William A. Marganski; Xin Q. Brown; Joyce Y. Wong



RNA interference of PPAR? using fiber-modified adenovirus vector efficiently suppresses preadipocyte-to-adipocyte differentiation in 3T3-L1 cells  

Microsoft Academic Search

The peroxisome proliferator-activated receptor (PPAR) ? is regarded as a “master regulator” of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPAR? in adipose, RNA interference (RNAi) of PPAR? should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult

Tetsuji Hosono; Hiroyuki Mizuguchi; Kazufumi Katayama; Naoya Koizumi; Kenji Kawabata; Teruhide Yamaguchi; Shinsaku Nakagawa; Yoshiteru Watanabe; Tadanori Mayumi; Takao Hayakawa



Differential Tumorigenicity of 3T3 Cells Transformed in Vitro with Polyoma Virus and i\\/i VivoSelection for High Tumorigenicity1  

Microsoft Academic Search

BALB\\/c 3T3 cells transformed in vitro with a temperature-sensitive mutant of polyoma virus were cloned. Forty-eight clones examined dem onstrated heterogeneity with respect to doubling-time in vitro and tumor- igenicity in syngeneic mice in vivo. Observation periods that lasted in certain cases as long as 2 years showed that some clones exhibited a relatively high tumorigenicity, i.e., they yielded a

Eliezer Halachmi; Isaac P. Witz



Requirement of Phosphatidylinositol 3-Kinase-Dependent Pathway and Src for Gas6-Axl Mitogenic and Survival Activities in NIH 3T3 Fibroblasts  

Microsoft Academic Search

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires




Adipocyte differentiation of 3T3-L1 preadipocytes is dependent on lipoxygenase activity during the initial stages of the differentiation process.  


Adipocytes play a central role in whole-body energy homoeostasis. Complex regulatory transcriptional networks control adipogensis, with ligand-dependent activation of PPARgamma (peroxisome proliferator-activated receptor gamma) being a decisive factor. Yet the identity of endogenous ligands promoting adipocyte differentiation has not been established. Here we present a critical evaluation of the role of LOXs (lipoxygenases) during adipocyte differentiation of 3T3-L1 cells. We show that adipocyte differentiation of 3T3-L1 preadipocytes is inhibited by the general LOX inhibitor NDGA (nordihydroguaiaretic acid) and the 12/15-LOX selective inhibitor baicalein. Baicalein-mediated inhibition of adipocyte differentiation was rescued by administration of rosiglitazone. Treatment with baicalein during the first 4 days of the differentiation process prevented adipocyte differentiation; supplementation with rosiglitazone during the same period was sufficient to rescue adipogenesis. Accordingly, we demonstrate that adipogenic conversion of 3T3-L1 cells requires PPARgamma ligands only during the first 4 days of the differentiation process. We show that the baicalein-sensitive synthesis of endogenous PPARgamma ligand(s) increases rapidly upon induction of differentiation and reaches a maximum on days 3-4 of the adipocyte differentiation programme. The conventional platelet- and leucocyte-type 12(S)-LOXs and the novel eLOX-3 (epidermis-type LOX-3) are expressed in white and brown adipose tissue, whereas only eLOX-3 is clearly expressed in 3T3-L1 cells. We suggest that endogenous PPARgamma ligand(s) promoting adipocyte differentiation are generated via a baicalein-sensitive pathway involving the novel eLOX-3. PMID:18320708

Madsen, Lise; Petersen, Rasmus K; Sřrensen, Morten B; Jřrgensen, Claus; Hallenborg, Philip; Pridal, Lone; Fleckner, Jan; Amri, Ez-Zoubir; Krieg, Peter; Furstenberger, Gerhard; Berge, Rolf K; Kristiansen, Karsten



Tiamulin inhibits human CYP3A4 activity in an NIH\\/3T3 cell line stably expressing CYP3A4 cDNA  

Microsoft Academic Search

Tiamulin is an antibiotic frequently used in veterinary medicine. The drug has been shown to produce clinically important interactions with other compounds that are administered simultaneously. An NIH\\/3T3 cell line, stably expressing human cytochrome P450 (EC cDNA (CYP3A4), was used to study the effect of tiamulin on CYP3A4 activity. The 6?-hydroxylation activity of testosterone, which is increased in CYP3A4-expressing

Els M. De Groene; Sandra M. Nijmeijer; G. J. M. Jean Horbach; Renger F. Witkamp



Bone morphogenetic protein-2 (BMP2) and transforming growth factor-?1 (TGF-?1) alter connexin 43 phosphorylation in MC3T3-E1 Cells  

Microsoft Academic Search

BACKGROUND: Bone morphogenetic proteins (BMPs) and transforming growth factor-?s (TGF-?s) are important regulators of bone repair and regeneration. BMP-2 and TGF-?1 have been shown to inhibit gap junctional intercellular communication (GJIC) in MC3T3-E1 cells. Connexin 43 (Cx43) has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We

Lance E Wyatt; Chi Y Chung; Brian Carlsen; Akiko Iida-Klein; George H Rudkin; Kenji Ishida; Dean T Yamaguchi; Timothy A Miller



Contrasting Signaling Pathways of  1A- and  1B-Adrenergic Receptor Subtype Activation of Phosphatidylinositol 3Kinase and Ras in Transfected NIH3T3 Cells  

Microsoft Academic Search

Activation of protein kinases is an important inter- mediate step in signaling pathways of many G pro- tein-coupled receptors including a1-adrenergic re- ceptors. The present study was designed to investigate the capacity of the three cloned sub- types of human a1-receptors, namely, a1A, a1B and a1D, to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21ras in transfected NIH3T3 cells. Norepinephrine activated

Zhuo-Wei Hu; Xiao-You Shi; Richard Z. Lin; Brian B. Hoffman



Transformation of NIH3T3 fibroblasts by the c-Kit receptor tyrosine kinase: effect of receptor density and ligand-requirement  

Microsoft Academic Search

Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in

Georgina Caruana; Antony C Cambareri; Thomas J Gonda; Leonie K Ashman



Connexin43 Associated with an N-cadherin-containing Multiprotein Complex Is Required for Gap Junction Formation in NIH3T3 Cells  

Microsoft Academic Search

Previous studies have indicated an intimate linkage between gap junction and adherens junction formation. It was suggested this could reflect the close membrane- membrane apposition required for junction formation. In NIH3T3 cells, we observed the colocalization of con- nexin43 (Cx431) gap junction protein with N-cadherin, p120, and other N-cadherin-associated proteins at re- gions of cell-cell contact. We also found that

Chih-Jen Wei; Richard Francis; Xin Xu; Cecilia W. Lo



Inhibition of Anchorage-independent Growth of Transformed NIH3T3 Cells by Epithelial Protein Lost in Neoplasm (EPLIN) Requires Localization of EPLIN to Actin Cytoskeleton  

Microsoft Academic Search

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein characterized by the presence of a single centrally located lin-11, isl-1, and mec-3 (LIM) domain. We have reported previously that EPLIN is down-regulated in transformed cells. In this study, we have investigated whether ectopic expression of EPLIN affects transformation. In untransformed NIH3T3 cells, retroviral-mediated transduction of EPLIN did not alter

Yuhong Song; Raymond S. Maul; C. Sachi Gerbin; David D. Chang



Effect of quinupristin\\/dalfopristin on 3T3 and Eahy926 cells in vitro in comparison to other antimicrobial agents with the potential to induce infusion phlebitis  

Microsoft Academic Search

Infusion phlebitis is a common clinical problem that is observed with some antimicrobial agents, when being administered intravenously.\\u000a In this study, cultured murine fibroblasts and immortalised human endothelial cells were exposed to three antibiotics at clinically\\u000a relevant concentrations to assess their toxic potential in two established cytotoxicity assays. BALB\\/c 3T3 fibroblasts and\\u000a Eahy926 endothelial cells were exposed to quinupristin\\/dalfopristin (QD),

Matthias Kruse; Bülent Kilic; Burkhard Flick; Ralf Stahlmann



Phototoxicity of bituminous tars—correspondence between results of 3T3 NRU PT, 3D skin model and experimental human data  

Microsoft Academic Search

Bituminous tars (Ichthammol and Ichthyol Pale) are widely used in pharmaceutical, veterinary and cosmetic industries for their anti-microbial, anti-inflammatory and anti-pruritic effects. In contrast to coal tar, no phototoxicity of bituminous tars has been reported in man, although both Ichthammol and Ichthyol Pale exhibit UV absorption which is higher and broader for the former. The validated 3T3 NRU phototoxicity test

D. J??rová; K. Kejlová; H. Bendová; D. Ditrichová; M. Mezulán??ková



Reversine stimulates adipocyte differentiation and downregulates Akt and p70 s6k signaling pathways in 3T3-L1 cells  

Microsoft Academic Search

In this study, we investigate the ability of reversine to stimulate adipocyte differentiation and its effect on cellular signaling pathways associated with adipocyte differentiation. Our data show that reversine treatment of 3T3-L1 cells under differentiation conditions synergistically enhances adipocyte differentiation and the expression of adipogenic marker genes such as aP2, PPAR-?, resistin, C\\/EBP?, and adiponectin. In parallel, reversine treatment leads

Yong Kee Kim; Hye-Young Choi; Nam Hyun Kim; Woojung Lee; Dong-Wan Seo; Dong-Won Kang; Hoi Young Lee; Jeung-Whan Han; Sahng Wook Park; Su-Nam Kim



Nitric Oxide Donors Selectively Potentiate Thrombin-Stimulated p70 S6k Activity and Morphological Changes in Swiss 3T3 Cells  

Microsoft Academic Search

Thrombin stimulates both DNA synthesis and cell morphological changes in Swiss 3T3 cells, although the mechanism of signal coordination leading to these responses is unknown. We report here that nitric oxide (NO) donors selectively enhance thrombin-stimulated p70S6k activity by 40–60%, an effect that was sustained for 24 h. Potentiation of p70S6k also was observed with cGMP analogues indicating that this

Leise A. Berven; Ian J. Frew; Michael F. Crouch



Ameliorating Effects of Fermented Rice Bran Extract on Oxidative Stress Induced by High Glucose and Hydrogen Peroxide in 3T3-L1 Adipocytes  

Microsoft Academic Search

In this study, we investigated whether fermented rice bran (FRB) can ameliorate the oxidative stress induced by high glucose\\u000a and hydrogen peroxide (H2O2) in 3T3-L1 adipocytes by analyzing reactive oxygen species (ROS), oil red O staining, as well as the expression of mRNAs\\u000a related to glucose homeostasis and adipogenesis. It was first confirmed that rice bran fermented by Issatchenkia orientalis

Dongyeop Kim; Gi Dong Han


Effect of extracts from safflower seeds on osteoblast differentiation and intracellular calcium ion concentration in MC3T3-E1 cells  

Microsoft Academic Search

Although safflower seeds have long been used in Korea as herbal medicines, very little research has been published on the effects of safflower seed on bone formation or bone density. The study reported here therefore examined bone nodule formation, calcium uptake, alkaline phosphatase activity, and intracellular concentration of calcium ion [Ca]i in murine osteoblastic cells of the MC3T3-E1 line that

Hye-Ock Jang; Young-Sik Park; Jong-Hwa Lee; Jun-Bong Seo; Kyo-Il Koo; Soo-Cheol Jeong; Seong-Deok Jin; Young-Ho Lee; Hyun-Sup Eom; IL Yun



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

SciTech Connect

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.

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



Effect of Beta-Alanyl-L-Histidinato Zinc on Osteoblastic MC3T3-E1 Cells: Increases in Alkaline Phosphatase and Proliferation  

Microsoft Academic Search

The effect of ?-alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37° C in a CO2 incubator in plastic dishes containing ?-modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1 % bovine serum albumin plus various concentrations

Masayoshi Yamaguchi; Junko Ohtaki



Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase  

Microsoft Academic Search

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in a-minimal essential medium containing either vehicle, genistein (10–7–10–5 M) or daidzein (10–7–10–5 M). The 5,500 g supernatant of cell homogenate was used for assay

Masayoshi Yamaguchi; Emi Sugimoto



Requirement of Atypical Protein Kinase Cl for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes  

Microsoft Academic Search

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimu- lation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCz and PKCl) have been impli- cated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCl in 3T3-L1 adipocytes




Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells  

SciTech Connect

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phase of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.

Horiuchi, Shinji [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan) and Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Jyounanku Fukuoka 814-0180 (Japan); Kato, Kiyoko [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan)]. E-mail:; Suga, Shin [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Takahashi, Akira [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Ueoka, Yousuke [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Arima, Takahiro [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Nishida, Jun-ichi [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Hachisuga, Toru; Kawarabayashi, Tatsuhiko [Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Jyounanku Fukuoka 814-0180 (Japan); Wake, Norio [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan)



Wnt5b partially inhibits canonical Wnt/beta-catenin signaling pathway and promotes adipogenesis in 3T3-L1 preadipocytes.  


To elucidate the functional roles of Wnt5b in adipogenesis, we characterized gene expression profiles in Wnt5b overexpressing 3T3-L1 cells using microarray analysis. Of the approximately 20,000 genes screened, we found that 85 genes were up-regulated and 211 genes were down-regulated in 3T3-L1 cells overexpressing Wnt5b. Among the genes regulated by Wnt5b, the expressions of insulin like growth factor-1 (IGF-1), vascular endothelial growth factor-C (VEGF-C), and WNT1 inducible signaling pathway protein 1 (WISP-1), which were known to be up-regulated by Wnt1/beta-catenin signaling, were decreased in the Wnt5b overexpressing cells. This result was subsequently confirmed by real-time quantitative RT-PCR (IGF-1; 0.74+/-0.08 and 0.56+/-0.08, WISP-1; 0.71+/-0.03 and 0.56+/-0.08, and VEGF-C; 0.67+/-0.01 and 0.80+/-0.07, mean+/-SEM, compared with the control at zero and two days after induction of differentiation, respectively). We also found that Wnt5b overexpression in 3T3-L1 preadipocytes was able to partially prevent the inhibitory effect of Wnt3a on adipogenesis. Furthermore, the overexpression of Wnt5b was able to inhibit Wnt3a-induced activation of the canonical Wnt/beta-catenin pathway as evidenced by the reduced translocation of beta-catenin into the nucleus. These findings indicate that Wnt5b may promote adipogenesis in 3T3-L1 cells, at least in part, by antagonizing the canonical Wnt/beta-catenin pathway. PMID:15796911

Kanazawa, Akio; Tsukada, Shuichi; Kamiyama, Masumi; Yanagimoto, Toru; Nakajima, Masatoshi; Maeda, Shiro



Regulation of CCAAT\\/Enhancer Binding Protein? (C\\/EBP?) Gene Expression by Thiazolidinediones in 3T3-L1 Adipocytes  

Microsoft Academic Search

Thiazolidinediones are a class of antidiabetic drugs that induce preadipocyte differentiation by binding and activating peroxisome proliferator-activated receptor ?2. Although thiazolidinediones are commonly thought of as insulin-sensitizing agents, these drugs have opposing and antagonistic effects to that of insulin on CCAAT\\/enhancer binding protein ? (C\\/EBP?) gene expression in fully differentiated 3T3-L1 adipocytes. Thiazolidinediones induce expression of C\\/EBP? mRNA and protein,

Nahid Hemati; Robin L. Erickson; Sarah E. Ross; Raymond Liu; Ormond A. MacDougald



Growth hormone activates mitogen-activated protein kinase and S6 kinase and promotes intracellular tyrosine phosphorylation in 3T3-F442A preadipocytes.  

PubMed Central

Physiological concentrations of growth hormone induced a rapid and transient activation of mitogen-activated protein kinase (MAP kinase) and S6 kinase in 3T3-F442A preadipocytes. These effects were abrogated by staurosporine and in cells chronically pretreated with phorbol esters, suggesting that protein kinase C is involved in the mechanism of activation. In addition, three cytosolic proteins exhibited a growth-hormone-dependent increase in tyrosine phosphorylation. Images Fig. 2.

Anderson, N G



Bis(alpha-furancarboxylato)oxovanadium(IV) prevents and improves dexamethasone-induced insulin resistance in 3T3-L1 adipocytes.  


Previous studies showed that bis(alpha-furancarboxylato)oxovanadium(IV) (BFOV), an orally active anti-diabetic organic vanadium complex, could improve insulin resistance in animals with type 2 diabetes. The present study has been carried out to evaluate the effects of BFOV on insulin-resistant glucose metabolism using dexamethasone-treated 3T3-L1 adipocytes as an in-vitro model of insulin resistance. The results showed that BFOV, similar to vanadyl sulfate and rosiglitazone, caused a concentration-dependent increase in glucose consumption by insulin-resistant adipocytes. Moreover, BFOV enhanced the action of insulin and completely prevented the development of insulin resistance induced by dexamethasone, leading to glucose consumption equal to that by normal cells. In addition, dexamethasone reduced the mRNA expression of insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT4) in 3T3-L1 adipocytes, while BFOV normalized the expression of IRS-1 and GLUT4. These findings suggest that BFOV prevents and improves dexamethasone-induced insulin resistance in 3T3-L1 adipocytes by enhancing expression of IRS-1 and GLUT4 mRNA. PMID:18812026

Zuo, Yi-Qing; Liu, Wei-Ping; Niu, Yan-Fen; Tian, Chang-Fu; Xie, Ming-Jin; Chen, Xi-Zhu; Li, Ling



Plumbagin activates ERK1/2 and Akt via superoxide, Src and PI3-kinase in 3T3-L1 cells.  


Plumbagin, derived from the plant Plumbago zeylanica, has been shown to chronically activate ERK1/2 and inhibit Akt activity in cancer cells. However, the acute effects of plumbagin on ERK1/2 and Akt activities remain unknown. In this study, we examined the effects of plumbagin on ERK1/2 and Akt activities in 3T3-L1 cells. Exposure of 3T3-L1 cells to plumbagin generated superoxide and activated both ERK1/2 and Akt. The plumbagin-stimulated ERK1/2 and Akt activities were sensitive to an antioxidant NAC, superoxide dismutase mimetic MnTBAP, superoxide scavenger Tiron and NAD(P)H oxidase inhibitor DPI. Plumbagin-stimulated ERK1/2 activity was attenuated by the MEK1/2 inhibitor PD98059 and Ras inhibitor manumycin A, whereas plumbagin-stimulated Akt activity was blocked by the PI3K inhibitor LY294002. Both plumbagin-stimulated ERK1/2 and Akt activities were attenuated by PP2, a Src inhibitor. Interestingly, inhibition of phosphatidylinositol 3-kinase (PI3-kinase), but not Akt, activity leaded to attenuation of plumbagin-stimulated ERK1/2 activity. These results suggest that plumbagin activates NAD(P)H oxidase, Src, and PI3K, and that the activated PI3K or PDK1 subsequently stimulate Akt and Ras-Raf-MEK1/2-ERK1/2 in 3T3-L1 cells. PMID:20420821

Yang, Su-Jung; Chang, Shi-Chuan; Wen, Hui-Chin; Chen, Ching-Yu; Liao, Jyh-Fei; Chang, Chung-Ho



Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.  

PubMed Central

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

Goruppi, S; Ruaro, E; Varnum, B; Schneider, C



A novel role for fatty acid transport protein 1 in the regulation of tricarboxylic acid cycle and mitochondrial function in 3T3-L1 adipocytes  

PubMed Central

Fatty acid transport proteins (FATPs) are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. Whereas some FATP1 translocates to the plasma membrane in response to insulin, the majority of FATP1 remains within intracellular structures and bioinformatic and immunofluorescence analysis of FATP1 suggests the protein primarily resides in the mitochondrion. To evaluate potential roles for FATP1 in mitochondrial metabolism, we used a proteomic approach following immunoprecipitation of endogenous FATP1 from 3T3-L1 adipocytes and identified mitochondrial 2-oxoglutarate dehydrogenase. To assess the functional consequence of the interaction, purified FATP1 was reconstituted into phospholipid-containing vesicles and its effect on 2-oxoglutarate dehydrogenase activity evaluated. FATP1 enhanced the activity of 2-oxoglutarate dehydrogenase independently of its acyl-CoA synthetase activity whereas silencing of FATP1 in 3T3-L1 adipocytes resulted in decreased activity of 2-oxoglutarate dehydrogenase. FATP1 silenced 3T3-L1 adipocytes exhibited decreased tricarboxylic acid cycle activity, increased cellular NAD+/NADH, increased fatty acid oxidation, and increased lactate production indicative of altered mitochondrial energy metabolism. These results reveal a novel role for FATP1 as a regulator of tricarboxylic acid cycle activity and mitochondrial function.

Wiczer, Brian M.; Bernlohr, David A.



Two Compartments for Insulin-Stimulated Exocytosis in 3t3-L1 Adipocytes Defined by Endogenous Acrp30 and Glut4  

PubMed Central

Insulin stimulates adipose cells both to secrete proteins and to translocate the GLUT4 glucose transporter from an intracellular compartment to the plasma membrane. We demonstrate that whereas insulin stimulation of 3T3-L1 adipocytes has no effect on secretion of the ?3 chain of type VI collagen, secretion of the protein hormone adipocyte complement related protein of 30 kD (ACRP30) is markedly enhanced. Like GLUT4, regulated exocytosis of ACRP30 appears to require phosphatidylinositol-3-kinase activity, since insulin-stimulated ACRP30 secretion is blocked by pharmacologic inhibitors of this enzyme. Thus, 3T3-L1 adipocytes possess a regulated secretory compartment containing ACRP30. Whether GLUT4 recycles to such a compartment has been controversial. We present deconvolution immunofluorescence microscopy data demonstrating that the subcellular distributions of ACRP30 and GLUT4 are distinct and nonoverlapping; in contrast, those of GLUT4 and the transferrin receptor overlap. Together with supporting evidence that GLUT4 does not recycle to a secretory compartment via the trans-Golgi network, we conclude that there are at least two compartments that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes: one for ACRP30 secretion and one for GLUT4 translocation.

Bogan, Jonathan S.; Lodish, Harvey F.



Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPARgamma activation.  


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

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



3T3-L1 adipocytes possess anandamide- and epinephrine-responsive machinery for MDM2 distribution to the plasma membrane.  


The effects of biomolecules on peripheral tissues and their responsive machinery are not well understood. We examined MDM2 level in the plasma membrane (PM) and total MDM2 level of 3T3-L1 adipocytes treated with biomolecular anandamide, epinephrine, and other agents for 15 min. We also examined biomolecular responses in cells treated with mithramycin A, a binding inhibitor, or cells exposed to cooling and cell viability. Immunoblotting revealed that PM MDM2 level increased and total MDM2 level was not altered following treatment with anandamide, epinephrine, capsaicin, CL316243, and aluminum fluoride. PM MDM2 distribution caused by a biomolecular concentration was maintained by treatment with mithramycin A and exposure of cells to 28°C or 32°C but not to 18°C, and PM MDM2 levels after treatment with high concentrations of biomolecules were altered upon exposure to the inhibitor and mild hypothermia. These conditions did not decrease cell viability. Our findings indicate that 3T3-L1 adipocytes possess molecular machinery that responds differentially to anandamide and epinephrine under the inhibitor treatment and cool temperature conditions and that is sensitive to other agents (which mimic biomolecular responses); these machineries can induce subcellular alterations in molecular interactions. We provide information helpful for clarifying biomolecular responsive machinery present in 3T3-L1 adipocytes. PMID:23682023

Ohsaka, Yasuhito; Nishino, Hoyoku



The roots of Atractylodes japonica Koidzumi promote adipogenic differentiation via activation of the insulin signaling pathway in 3T3-L1 cells  

PubMed Central

Background Type 2 diabetes (T2D) is a complex metabolic disorder characterized by insulin resistance and hyperglycemia. Peroxisome proliferator-activated receptor gamma (PPAR?) is a key transcription factor and plays an important role in the regulation of genes involved in adipogenic differentiation, glucose metabolism and insulin signal transduction. Methods In this study, the effects of the root extract of Atractylodes japonica Koidzumi (Atractylodis Rhizoma Alba, ARA) on the differentiation of 3T3-L1 preadipocytes and the possible mechanism of glucose transport were investigated. 3T3-L1 cells were cultured with insulin and ARA extract. Results In 3T3-L1 cells, ARA extract significantly enhanced adipogenic differentiation and upregulated the expression of PPAR? genes and protein in a dose-dependent manner. ARA also promoted glucose transport by increasing the glucose transporter 4 (GLUT-4), phosphatidylinositol 3-kinase (PI3K) and insulin receptor substrates-1 (IRS-1) levels. Conclusion Our results suggest that ARA extract may be an attractive therapeutic agent for managing T2D via promoting the differentiation of adipocytes with the upregulation of PPAR? levels and the activation of the insulin signaling pathway.



Differentiation to adipocytes in accompanied by an increase in the amounts of Gi- and Go-proteins in 3T3-L1 cells  

SciTech Connect

Treatment of cultures of 3T3-L1 cells with methylisobutyl-xanthine and dexamethasone has been shown to result in accumulation of lipid and conversion to the morphology of adipocytes in more than 90% of the cells. The status of the stimulatory (Gs), inhibitory (Gi) and Go-proteins during the course of 3T3-L1 differentiation was examined. The amount of alpha subunit of Gs (..cap alpha..Gs), assayed by radiolabeling in the presence of cholera toxin and (/sup 32/P)NAD/sup +/, increased upon differentiation as previously described by others. The amounts of ..cap alpha..Gi and ..cap alpha..Go assayed by radiolabeling in the presence of pertussis toxin and (/sup 32/P)NAD/sup +/ increased 3-fold upon differentiation. Immunoblots of cell membranes subjected to gel electrophoresis in sodium dodecyl sulfate were probed with two rabbit antisera raised against bovine brain ..cap alpha..Go and with one raised against the..beta..-subunit of the bovine rod-outer-segment G-protein, referred to as transducin. The immunoblotting data confirm the increase upon differentiation of ..cap alpha..Go and also demonstrate an increase in the amount of the ..beta..-subunit. Thus differentiation of 3T3-L1 cells is accompanied by dramatic changes in the complexion of G-proteins in the membranes.

Watkins, D.C.; Northup, J.K.; Malbon, C.C.



The requirements for viral entry differ from those for virally induced syncytium formation in NIH 3T3/DTras cells exposed to Moloney murine leukemia virus.  


Moloney murine leukemia virus (Mo-MuLV) has the unique ability to infect different cells via either a low-pH-dependent or a pH-independent entry pathway. Only the pH-independent mechanism of Mo-MuLV entry has been associated with Mo-MuLV-induced syncytium formation. We have now identified a transformed cell line (NIH 3T3/DTras) which efficiently forms syncytia when exposed to Mo-MuLV, yet is low pH dependent for Mo-MuLV entry. Treatment of NIH 3T3/DTras cells with chloroquine, an agent which raises endosomal pH, blocks Mo-MuLV entry, but not Mo-MuLV-induced syncytium formation. This demonstrates that fusion which accompanies viral entry and fusion which is respons