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1

Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan  

PubMed Central

A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

Bisson, Francis; Rochefort, Eloise; Lavoie, Amelie; Larouche, Danielle; Zaniolo, Karine; Simard-Bisson, Carolyne; Damour, Odile; Auger, Francois A.; Guerin, Sylvain L.; Germain, Lucie

2013-01-01

2

The use of peripheral blood feeder layers as a source of GM-CSF for human bone marrow cultures  

Microsoft Academic Search

The use of peripheral blood feeder layers as a source of stimulus for colony formation by human granulocytic progenitor cells in semi-solid agar cell cultures was examined. Comparison with various conditioned media demonstrated that cultures stimulated by peripheral blood feeder layers produced the greatest number and largest colonies. The cell concentration in the feeder layers was more important than the

R. D. Brown; K. A. Rickard; E. Yuen; H. Kronenberg

1983-01-01

3

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

PubMed

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

2012-10-01

4

Mesenchymal Stem Cells as a Feeder Layer Can Prevent Apoptosis of Expanded Hematopoietic Stem Cells Derived from Cord Blood  

PubMed Central

Umbilical cord blood (UCB) has been used for transplantation in the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). Because of insufficient number of cord blood CD34+ cells, the expansion of these cells seems to be important for clinical application. Mesenchymal stromal cells (MSCs), playing an important role in HSCs maintenance, were used as feeder layer. Apoptosis and cell cycle distribution of expanded cells were analyzed in MSCs co-culture and cytokine conditions and results were compared. Three culture conditions of cord blood HSCs were prepared ex-vivo for 14 days: cytokines (SCF, TPO and Flt3L) with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs without cytokines. Expansion was followed by measuring the total nucleated cells (TNCs), CD34+? cells and colony-forming unit (CFU) output. Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells. Maximum cord blood CD34+ cells expansion was observed in day 10. The mean fold change of TNCs and CD34+ cells at day 10 in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). The highest apoptosis rate and the least number of cells in Go/G1 phase were observed in cytokine culture without feeder layer (p=0.041). The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.

Mehrasa, Roya; Vaziri, Hamidreza; Oodi, Arezoo; Khorshidfar, Mona; Nikogoftar, Mahin; Golpour, Monireh; Amirizadeh, Naser

2014-01-01

5

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.

2010-01-01

6

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.

2013-01-01

7

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

PubMed Central

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

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

2013-01-01

8

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

PubMed

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. PMID:21156717

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

2011-03-01

9

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.

2011-01-01

10

Use of BRL-conditioned medium in combination with feeder layers to isolate a diploid embryonal stem cell line  

Microsoft Academic Search

The derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129\\/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and

Alan H. Handyside; Gerard T. O'Neill; Mary Jones; Martin L. Hooper

1989-01-01

11

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

SciTech Connect

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

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

2010-02-19

12

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.

1979-01-01

13

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro.  

PubMed

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, ?6-integrin, ?1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells. PMID:22289219

Nasiri, Z; Hosseini, S M; Hajian, M; Abedi, P; Bahadorani, M; Baharvand, H; Nasr-Esfahani, M H

2012-05-01

14

Regulated renin release from 3T3-L1 adipocytes.  

PubMed

Whereas adipose tissue possesses a local renin-angiotensin system, the synthesis and regulated release of renin has not been addressed. To that end, we utilized differentiating 3T3-L1 cells and analyzed renin expression and secretion. Renin mRNA expression and protein enzymatic activity were not detectable in preadipocytes. However, upon differentiation, renin mRNA and both intracellular and extracellular renin activity were upregulated. In differentiated adipocytes, forskolin treatment resulted in a 28-fold increase in renin mRNA, whereas TNFalpha treatment decreased renin mRNA fourfold. IL-6, insulin, and angiotensin (Ang) II were without effect. In contrast, forskolin and TNFalpha each increased renin protein secretion 12- and sevenfold, respectively. Although both forskolin and TNFalpha induce lipolysis in adipocytes, fatty acids, prostaglandin E(2), and lipopolysaccharide had no effect on renin mRNA or secretion. To evaluate the mechanism(s) by which forskolin and/or TNFalpha are able to regulate renin secretion, a general lipase inhibitor (E600) and PKA inhibitor (H89) were used. Both inhibitors attenuated forskolin-induced renin release, whereas they had no effect on TNFalpha-regulated secretion. In contrast, E600 potentiated forskolin-stimulated renin mRNA levels, whereas H89 had no effect. Neither inhibitor had any influence on TNFalpha regulation of renin mRNA. Relative to lean controls, renin expression was reduced 78% in the epididymal adipose tissue of obese male C57Bl/6J mice, consistent with TNFalpha-mediated downregulation of renin mRNA in the culture system. In conclusion, the expression and secretion of renin are regulated under a complex series of hormonal and metabolic determinants in mature 3T3-L1 adipocytes. PMID:19293336

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

2009-06-01

15

Alteration of glycolipids in ras-transfected NIH 3T3 cells  

SciTech Connect

Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. The authors analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, they found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by /sup 125/I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1 as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, they found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. These results indicated that ras oncogenes derived form human tumors are capable of inducing alterations in glycolipid composition.

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

1987-09-01

16

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

1989-01-01

17

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

PubMed

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. PMID:2764539

Kaminsky, R; Zweygarth, E

1989-06-01

18

Feeder Ninja  

NSDL National Science Digital Library

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

19

Kinetic analysis of deviation from the undifferentiated state in colonies of human induced pluripotent stem cells on feeder layers.  

PubMed

Understanding of the fundamental mechanisms that govern unintentional differentiation of human induced pluripotent stem cells (hiPSCs) provides key strategies to maintain their undifferentiated state during cell expansion. This study focused on deviation from the undifferentiated state in hiPSC colonies during culture with feeder cells. Deviated cells from the undifferentiated state of hiPSCs in cultures with SNL and MEF feeder cells were observed at the center and periphery of the colonies, respectively, accompanied by dramatic changes in the cell morphology from small to large flattened shapes. It was found that the deviation of undifferentiated hiPSCs in culture with SNL feeder cells caused deviated cells in the center of the colony through spontaneous occurrence in a colony size-dependent manner, whereas the deviation of undifferentiated hiPSCs in culture with MEF feeder cells caused deviated cells in the periphery of the colonies through accidental events during migration in a colony size-independent manner. Based on a kinetic analysis of time-lapse images of single hiPSC colonies, the specific growth rate for replication of deviated cells from the undifferentiated state in culture with SNL feeder cells was 1.83 and 3.57 times higher than those of undifferentiated cells and transformation, respectively, meaning that the deviation of undifferentiated hiPSCs dramatically expanded through replication of deviated cells from the undifferentiated state and transformation once deviation from the undifferentiated state had occurred. In the case of MEF feeder cells, the specific growth rates for replication of deviated cells from the undifferentiated state was 3.12 times higher than that of undifferentiated cells, whereas the rate by transformation exhibited a negligible level compared with the rates of replication for undifferentiated cells and deviated cells from undifferentiated state, meaning that deviation of undifferentiated hiPSCs dramatically expanded only through replication of deviated cells from the undifferentiated state. These results suggest that once deviation has occurred in a colony, the deviated cells from undifferentiated state undertake dramatic invasion to occupy the colony. Maintenance of the undifferentiated state in subcultures inevitably requires vigilant care to remove any colonies that include deviated cells from the undifferentiated state. Biotechnol. Bioeng. 2014;111: 1128-1138. © 2014 Wiley Periodicals, Inc. PMID:24420640

Kim, Mee-Hae; Masuda, Eri; Kino-Oka, Masahiro

2014-06-01

20

Impact of stress hormone on adipogenesis in the 3T3-L1 adipocytes.  

PubMed

Stress hormone is known to play a vital role in lipolysis and adipogenesis in fat cells. The present study was carried out to evaluate the effect of epinephrine on adipogenesis in the 3T3-L1 cells. The investigation on adipogenesis was done in both mono and co-cultured 3T3-L1 cells. 3T3-L1 preadipocytes and C2C12 cells were grown independently on transwell plates and transferred to differentiation medium. Following differentiation, C2C12 cells transferred to 3T3-L1 plate and treated with medium containing 10 ?g/ml of epinephrine. Adipogenic markers such as fatty acid binding protein 4, peroxisome proliferator activating receptor, CCAAT/enhancer-binding protein, adiponectin, lipoprotein lipase and fatty acid synthase mRNA expressions were evaluated in the 3T3-L1 cells. Epinephrine treatment reduced adipogenesis, evidenced by reducing adipogenic marker mRNA expression in the 3T3-L1 cells. In addition, glycerol accumulation and oil red-O staining supported the reduced rate of adipogenesis. Taking all together, it is concluded that the stress hormone, epinephrine reduces the rate of adipogenesis in the mono and co-cultured 3T3-L1 cells. In addition, the rate of adipogenesis is much reduced in the co-cultured 3T3-L1 cells compared monocultured 3T3-L1 cells. PMID:23943061

Pandurangan, Muthuraman; Ravikumar, Sambandam

2014-08-01

21

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

PubMed

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

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

2000-10-24

22

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.

1989-04-01

23

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

2010-01-01

24

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

SciTech Connect

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

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

2013-01-18

25

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

SciTech Connect

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

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

2012-02-15

26

Loss of Differentiation Control in Transformed 3T3 T Proadipocytes1  

Microsoft Academic Search

Nontransformed 3T3 T mesenchymal\\/proadipocyte stem cells can be readily induced to differentiate, yet previous work has shown that 3T3 T cells that are spontaneously or virally transformed not only lose their normal growth control mechanisms but also lose the ability to differenti ate. Loss of growth control can be due to autocrine mechanisms in some transformed cells, but the mechanisms

Rodney L. Sparks; Blake J. Allen; Andrea I. Zygmunt; Ethan E. Strauss

27

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

1996-01-01

28

Esculetin Induces Apoptosis and Inhibits Adipogenesis in 3T3-L1 Cells  

Microsoft Academic Search

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3-L1 adipocyte apoptosis and adipogenesis.Research Methods and Procedures: 3T3-L1 pre-confluent preadipocytes and lipid-filled adipocytes were incubated with esculetin (0 to 800 ?M) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis

Jeong-Yeh Yang; Mary Anne Della-Fera; Diane L. Hartzell; Cass Nelson-Dooley; Dorothy B. Hausman; Clifton A. Baile; A. Baile

2006-01-01

29

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

1998-01-01

30

mdm-2 gene amplification in 3T3-L1 preadipocytes  

Microsoft Academic Search

In this study the regulation of the murine double minute-2 (mdm-2) gene was examined in NIH 3T3-L1 preadipocytes. The 3T3-L1 cell line, under proper conditions, has the capacity to differentiate from fibroblasts into adipocytes [15]. A recent report demonstrated that mdm-2 overexpression could block myogenesis [12]. While examining the regulation of the mdm-2 gene during adipogenesis, it was discovered that

Steven J. Berberich; Vaughn Litteral; Lindsey D. Mayo; Dara Tabesh; David Morris

1999-01-01

31

Anti-obesity effect of grape skin extract in 3T3-L1 adipocytes  

Microsoft Academic Search

The aim of the present study was to determine the effects of grape skin which was well known as natural antioxidant using\\u000a 3T3-L1 adipocyte, representative cells with morphological and biochemical characteristics of adipocytes. Grape skin extract\\u000a (GSE) suppressed the differentiation of 3T3-L1 adipocytes, and decreased the triglycerides content with lipid accumulation.\\u000a The adipogenesis inhibitory effect of GSE was confirmed in

Yoo Seok Jeong; Hee Kyoung Jung; Kyung-Hyun Cho; Kwang-Sup Youn; Joo-Heon Hong

2011-01-01

32

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

PubMed Central

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

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

2014-01-01

33

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

PubMed

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

2011-07-01

34

Illudins C2 and C3 Stimulate Lipolysis in 3T3-L1 Adipocytes and Suppress Adipogenesis in 3T3-L1 Preadipocytes.  

PubMed

The secondary metabolites illudins C2 (1) and C3 (2), obtained from the culture broth of Coprinus atramentarius, have been shown to possess antimicrobial activity. In the present study, we discovered novel biological activities of 1 and 2 in lipolysis of differentiated 3T3-L1 adipocytes and adipogenesis of 3T3-L1 preadipocytes. Compounds 1 and 2 exhibit a dose-dependent increase in glycerol release and thereby reduce intracellular lipid accumulation. The stimulatory effects of 1 and 2 on lipolysis are prevented by cAMP-dependent protein kinase (PKA) and extracellular signal-regulated kinase (ERK) inhibitors. Compounds 1 and 2 down-regulated perilipin and also affected the mRNA and protein levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). However, 1 and 2 treatment leads to a significant increase in PKA-mediated phosphorylation of HSL at S563 and S660. In addition, 1 and 2 treatment in 3T3-L1 preadipocytes induces down-regulation of the critical transcription factors, CCAAT/enhancer binding protein ? and ? (C/EBP? and C/EBP?), and peroxisome proliferator activated receptor ? (PPAR?), which are required for adipogenesis, and accordingly inhibits adipogenesis. These results suggest that 1 and 2 might be useful for treating obesity due to their modulatory effects on fat by affecting adipocyte differentiation and fat mobilization. PMID:24597820

Kim, Sun-Ok; Sakchaisri, Krisada; Asami, Yukihiro; Ryoo, In-Ja; Choo, Soo-Jin; Yoo, Ick-Dong; Soung, Nak-Kyun; Kim, Young Sang; Jang, Jae-Hyuk; Kim, Bo Yeon; Ahn, Jong Seog

2014-04-25

35

Lentiviral vectors efficiently transduce quiescent mature 3T3-L1 adipocytes.  

PubMed

Obesity is associated with many serious afflictions such as cardiovascular disease, cancer, and diabetes. One of the main cellular systems used to study the underlying physiological and biological processes is the 3T3-L1 preadipocyte differentiation model. However, studies on 3T3-L1 adipocytes are hampered by the fact that genetic modification of mature adipocytes is notoriously difficult. In this report, we evaluated the use of lentivirus-mediated gene transfer into 3T3-L1 mature adipocytes. We demonstrate that quiescent, fully differentiated 3T3-L1 adipocytes as well as 3T3-L1 preadipocytes can be efficiently transduced with HIV-1-derived lentiviral vectors. Upon transduction using LV-PGK-GFP lentiviral vector at 100 ng p24 per 10(5) cells, more than 95% of the 3T3-L1 adipocytes in the culture expressed the GFP reporter gene. There were no overt signs of toxicity or cytopathogenicity in the cultures. Furthermore, modification of undifferentiated preadipocytes did not affect their capacity to differentiate. In addition, insulin-induced glucose uptake was not affected by the procedure. In contrast, adenoviral-mediated gene transfer into 3T3-L1 adipocytes is associated with marked cytopathogenicity. From these data, we conclude that lentiviral vectors are the gene-transfer system of choice for genetic modification of mature adipocytes. The availability of an efficient vector system may stimulate the use of adipose tissue as a target for gene therapy in obesity and other disorders. PMID:14759805

Carlotti, Françoise; Bazuine, Merlijn; Kekarainen, Tuija; Seppen, Jurgen; Pognonec, Philippe; Maassen, J Antonie; Hoeben, Rob C

2004-02-01

36

[Multipotent mesenchymal stem cells of desquamated endometrium: isolation, characterization and use as feeder layer for maintenance of human embryonic stem cell lines].  

PubMed

In this study, we characterize new multipotent human mesenchymal stem cell (MSC) lines derived from desquamated (shedding) endometrium in menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSC of any origin. The eMSCs have positive expression of CD73, CD90, CD105, CD13, CD29, CD44 markers and the absence of expression of the hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130 and HLA-DR (class II). Multipotency of the established eMSC is confirmed by their ability to differentiate into other mesodermal cell types such as osteocytes and adipocytes. Besides, the isolated eMSC lines partially (over 50%) express the pluripotency marker SSEA-4, but do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and beta-III-tubulin. This suggests a neural predisposition of the established eMSC. These cells are characterized by high rate of cell proliferation (doubling time 22-23 h) and high cloning efficiency (about 60%). In vitro the eMSCs undergo more than 45 population doublings revealing normal karyotype without karyotipic abnormalilies. We demonstrate, that the mititotically inactivated eMSCs are perfect feeder cells for human embryonic stem cell lines (hESC) C612 and C910. The eMSC being a feeder culture maintain the pluripotent status of the hESC, which is revealed by the expression of Oct-4, alkaline phosphatase and SSEA-4. When co-culturing, hESC retain their morphology, proliferative rate for more than 40 passages and capability for spontaneous differentiation into embryoid bodies comprising the three embryonic germ layers. Thus, an easy and non-invasive extraction of the eMSC in menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESC to clinical setting. PMID:22359950

Zemel'ko, V I; Grinchuk, T M; Domnina, A P; Artsybasheva, I V; Zenin, V V; Kirsanov, A A; Bichevaia, N K; Korsak, V S; Nikol'ski?, N N

2011-01-01

37

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

PubMed

Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBP? and PPAR? was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBP?, PPAR?, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis. PMID:23237809

Lai, Peng-Yeh; Tsai, Chong-Bin; Tseng, Min-Jen

2013-01-18

38

TRPM7 channels regulate proliferation and adipogenesis in 3T3-L1 preadipocytes.  

PubMed

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. PMID:23765921

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

2014-01-01

39

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

PubMed Central

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

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

2009-01-01

40

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

PubMed

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

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

2010-01-01

41

Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells  

SciTech Connect

The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with ({sup 14}C)thymidine and ({sup 3}H)leucine. The evaluation of the DNA turnover and additional ({sup 3}H)thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.

Papadopoulos, T.; Pfeifer, U. (Univ. of Wuerzburg (West Germany))

1987-07-01

42

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.

1985-04-01

43

Growth changes of 3T3 cells in the presence of mineral fibers  

SciTech Connect

The relationship between exposure to asbestos fibers and the development of mesothelioma or bronchial carcinoma prompted many countries to ban its use from commercial products. The biological mechanism by which asbestos induces or promotes mesothelioma or carcinoma is still unknown. In order to study the influence of fibers on the cell surface, 3T3 fibroblasts were cultured in the presence of various mineral fibers. The acute cytotoxicity produced by mineral fibers was evaluated by the trypan blue dye exclusion method; growth of 3T3 cells was measured as well as the maximum cell density at saturation. It was found that growth of 3T3 cells was slower in the presence of chrysotile while light microscopy revealed an increased cellular chromogenicity and a modification of the cell-cell arrangement in the presence of this fiber. An assay is described in which chrysotile causes an increase in the maximum cell density at saturation.

Dumas, L.; Page, M.

1986-01-01

44

Cell cycle dependency of murine cytomegalovirus replication in synchronized 3T3 cells.  

PubMed Central

Synchronized murine 3T3 cells have been used to investigate the possible dependency of murine cytomegalovirus replication upon the cell cycle. The normal latent period of 12 h characteristic of asynchronous 3T3 cells was protracted to more than 24 h after an early G1 infection in synchronous cells. In this case viral progeny were not detected until after the initiation of the host S-phase. Cells maintained in the G1 phase did not replicate virus. This failure could not be explained by a decrease in virus penetration but was apparently due to a requirement for an event associated with the host S-phase. Thymidine-induced inhibition of cell cycle traverse also blocked virus replication. Viral DNA synthesis did not initiate until after the initiation of host DNA. In contrast, herpes simplex virus type 1 replicated in 3T3 cells independently of the cell cycle.

Muller, M T; Hudson, J B

1977-01-01

45

Polyomavirus transforms rat F111 and mouse NIH 3T3 cells by different mechanisms.  

PubMed Central

Polyomavirus middle tumor antigen (mT) was expressed in a line of mouse NIH 3T3 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promotor. Contrary to rat F111 cells which were rendered anchorage independent by mT expression alone (L. Raptis, H. Lamfrom, and T.L. Benjamin, Mol. Cell. Biol. 5:2476-2487, 1985), mT-producing NIH 3T3 cells were unable to grow in agar even after full mT induction. The mT:pp60c-src-associated phosphatidylinositol kinase was activated in these cells to a degree similar to that in fully transformed cells expressing the small and large T antigens, in addition to mT. We therefore propose that the stimulation of this phosphatidylinositol kinase, although apparently necessary, is not sufficient for transformation of NIH 3T3 cells by polyomavirus. Images

Raptis, L; Bolen, J B

1989-01-01

46

Inhibitory activity of Phellodendri cortex extracts on differentiation of 3T3-L1 preadipocytes  

Microsoft Academic Search

The purpose of the present study was to investigate the inhibitory effect of beberine-rich fraction of Phellodendri cortex extract (PC) on adipogenesis in 3T3-L1 cells. PC effectively prevented TG accumulation in differentiation of 3T3-L1 preadipocytes\\u000a in a dose-dependent manner. Compared to controls, PC at a concentration of 75 ?g\\/mL significantly decreased lipid droplets\\u000a by 79.5%. Beberine exhibited similar inhibitory effect

Duk Kwon Choi; Tae Seok Oh; Jong Won Yun

2011-01-01

47

Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells.  

PubMed

Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca(2+) chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca(2+) release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction. PMID:24164256

Suh, K S; Choi, E M; Rhee, S Y; Kim, Y S

2014-02-01

48

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

2013-01-01

49

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

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

50

Inhibition of Adipogenesis by Tempol in 3T3-L1 Cells  

PubMed Central

Obesity is highly associated with an increased risk of serious health conditions including hypertension, cardiovascular disease, diabetes, and cancer. Changes in redox status with increased oxidative stress have been linked with obesity. Previous studies have shown that administration of the antioxidant Tempol in the food of mice prevents obesity causing significant weight loss without toxicity. To gain a better understanding of the molecular mechanism(s) underlying this effect, the influence of Tempol on the differentiation of mouse 3T3-L1 pre-adipocytes was studied. Tempol inhibited differentiation of 3T3-L1 cells resulting in a reduction in cellular lipid storage, down-regulation of protein levels of key adipogenesis transcription factors (PPAR-? and PPAR-?), down regulation of prolyl hydroxylase, and up-regulation of HIF-1?. Mice on a Tempol diet demonstrated reduced systemic levels of IGF-1, in qualitative agreement with that observed in vitro in 3T3-L1 cells, which also show lower IGF-1 levels as a result of Tempol treatment. These results show that treatment of 3T3-L1 cells with Tempol inhibits the expression of key adipogenesis factors, adipose differentiation, and lipid storage and may underlie, at least in part, some of the in vivo effects of Tempol on body weight.

Samuni, Yuval; Cook, John A.; Choudhuri, Rajani; DeGraff, William; Sowers, Anastasia L.; Krishna, Murali C.; Mitchell, James B.

2010-01-01

51

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

52

Silicon tetrachloride spray feeder  

NASA Technical Reports Server (NTRS)

Silicon tetrachloride spray feeder mechanism is incorporated into high-temperature reactor for production of highly pure silicon intended for solar cells. Feeder supplies silicon tetrachloride as liquid droplets that rapidly vaporize in high temperature (2,000 to 2,200 K) reactor zone.

Meyer, T. N.; Wolf, C. B.

1979-01-01

53

Pleiotrophin Transforms NIH 3T3 Cells and Induces Tumors in Nude Mice  

NASA Astrophysics Data System (ADS)

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

Chauhan, Anil K.; Li, Yue-Sheng; Deuel, Thomas F.

1993-01-01

54

Expression of Nanog gene promotes NIH3T3 cell proliferation  

SciTech Connect

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

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

2005-12-16

55

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

PubMed Central

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 rearrangements. 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. Images

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

1986-01-01

56

Ultrasound associated uptake of chitosan nanoparticles in MC3T3-E1 cells  

NASA Astrophysics Data System (ADS)

Chitosan is a natural linear polysaccharide that has been well known for its applications in drug delivery system due to its unique physicochemical and biological properties. However, challenges still remain for it to become a fully realized therapeutic agent. In this study, we investigated the uptake of chitosan nanoparticles (CNP) under the ultrasound stimulation, using a model cell culture system (MC3T3-E1 mouse pre-osteoblasts). The CNP were fabricated by an ionic gelation method and were lyophilized prior to characterization and delivery to cells. Particle size and zeta potential were measured using Dynamic Light Scattering (DLS); the efficiency of chitosan complexation was measured using the ninhydrin assay. Cytotoxicity was examined by neutral red assay within 48 hours after delivery. The effect of ultrasound (US) on the efficiency of nanoparticle delivery to the MC3T3-E1 cells was examined at 1MHz and at either 1 or 2 W/cm2. Fluorescein isothiocyanate (FITC)-conjugated-CNP were used to visualize the internalized particles within the cytosol. The uptake of FITC-CNP exhibits a dose and time dependent effect, a strong FITC fluorescence was detected at the concentration of 500microg/mL under fluorescence microscope. Ultrasound assisted uptake of FITC-CNP performed a significant positive effect at 2W/cm2 with 60s of ultrasound exposure time. CNP displayed a slightly decrease in cell viability from 25microg/mL to 100microg/mL, while higher concentration of CNP facilitates the proliferation of MC3T3-E1 cells. Less than 10% of reduction in cell viability was observed for US at 1W/cm2 and 2W/cm2 with 30s and 60s of exposure time, which suggest a mild effect of US to MC3T3-E1 cell line.

Wu, Junyi

57

Tissue transglutaminase expression affects hypusine metabolism in BALB\\/c 3T3 cells  

Microsoft Academic Search

Post-translational formation of hypusine in eukaryotic initiation factor 5A (eIF-5A) is essential for cell viability. Recently, we showed that hypusine protein is an in vitro substrate for transglutaminases (TGases). We report the effect of tissue TGase expression on the in vivo hypusine metabolic pathway. The stable expression of tTGase in BALB\\/c 3T3 cells induced a 100-fold reduction of hypusine levels

S Beninati; V Gentile; M Caraglia; A Lentini; P Tagliaferri; A Abbruzzese

1998-01-01

58

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

Microsoft Academic Search

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

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

2004-01-01

59

Isoproterenol, TNF?, and insulin downregulate adipose triglyceride lipase in 3T3-L1 adipocytes  

Microsoft Academic Search

Recently, adipose triglyceride lipase (ATGL, also called desnutrin and calcium-independent phospholipase A2 [iPLA2] ?) was isolated as a novel adipose-expressed triglyceride lipase which is downregulated in obesity and may contribute to obesity-associated metabolic disorders such as hyperlipidemia and insulin resistance. To clarify expression and regulation of this fat-derived lipase, ATGL mRNA was measured in 3T3-L1 adipocytes by quantitative real-time reverse

Susan Kralisch; Johannes Klein; Ulrike Lossner; Matthias Bluher; Ralf Paschke; Michael Stumvoll; Mathias Fasshauer

2005-01-01

60

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

2000-01-01

61

Simvastatin Promotes Osteoblast Differentiation and Mineralization in MC3T3-E1 Cells  

Microsoft Academic Search

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the bone effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity

Toyonobu Maeda; Ayako Matsunuma; Tetsuya Kawane; Noboru Horiuchi

2001-01-01

62

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

PubMed

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

2012-05-01

63

Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts  

Microsoft Academic Search

Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of\\u000a cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in\\u000a proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized\\u000a by Nile red staining, positivity to adipophilin

Giacomo Diaz; Barbara Batetta; Francesca Sanna; Sabrina Uda; Camilla Reali; Fabrizio Angius; Marta Melis; Angela Maria Falchi

2008-01-01

64

Berberine inhibits 3T3-L1 adipocyte differentiation through the PPAR? pathway  

Microsoft Academic Search

Berberine (BBR), a compound purified from Cortidis rhizoma, reduces serum cholesterol, triglycerides, and LDL-cholesterol of hypercholesterolemic patients and high fat diet fed animals, and increases hepatic LDLR mRNA and protein levels through a post-transcriptional mechanism. BBR also enhances the hypoglycemic action of insulin in diabetic animal models. Here, we show that BBR inhibits the differentiation of 3T3-L1 preadipocytes induced by

Cheng Huang; Yuebo Zhang; Zhenwei Gong; Xiaoyan Sheng; Zongmeng Li; Wei Zhang; Ying Qin

2006-01-01

65

The Effects of AICAR on Adipocyte Differentiation of 3T3-L1 Cells  

Microsoft Academic Search

The AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has been found to inhibit the differentiation of 3T3-L1 adipocytes, if added at an early phase of differentiation. AICAR blocks the expression of the late adipogenic markers, fatty acid synthase and acetyl-CoA carboxylase, and of the transcription factors, C\\/EBP? and PPAR?. It also inhibits early clonal expansion of pre-adipocytes, prevents the

Susan A. Habinowski; Lee A. Witters

2001-01-01

66

l Carnitine protects against apoptosis of murine MC3T3-E1 osteoblastic cells  

Microsoft Academic Search

Summary.  \\u000a l-Carnitine (LC), an amino acid with a major role in cellular energy metabolism, has positive effects on bone metabolism. However,\\u000a the effect of LC on apoptosis of osteoblast in vitro has not been reported. The aim of this study was to investigate the action\\u000a of LC on apoptosis of mouse osteoblastic cell line MC3T3-E1. Cell apoptosis was measured by

H. Xie; S.-Y. Tang; H. Li; X.-H. Luo; L.-Q. Yuan; D. Wang; E.-Y. Liao

2008-01-01

67

Chronic exposure to stress hormones promotes transformation and tumorigenicity of 3T3 mouse fibroblasts  

PubMed Central

Epinephrine and norepinephrine are produced during psychological stress and can directly bind to cells to induce DNA damage. These effects may have more long-lasting consequences such as DNA mutations resulting in an increased potential for cellular transformation and/or tumor progression. This study examined the molecular effects of a chronic (24 h) in vitro exposure to these stress hormones on murine 3T3 cells. Long exposures (24 h) in dose–response experiments with norepinephrine or epinephrine induced significant increases in DNA damage in treated cells compared to that of untreated controls as measured by the alkaline comet assay. Pre-treatment with a blocking agent (the ?-adrenergic receptor antagonist propranolol) eliminated this increase in damage. In addition, both norepinephrine and epinephrine increased cellular transformation, as assessed by growth in soft agar, and 3T3 cells pre-treated with either norepinephrine or epinephrine induced a more rapid onset of tumors and more aggressive tumor growth in nude mice. In summary, incubation of 3T3 cells with catecholamines results in long-term DNA damage as measured by increased transformed phenotypes and tumor progression, indicating that they are important mediators of stress effects on genomic instability and vulnerability to tumor formation.

FLINT, MELANIE S.; BAUM, ANDREW; EPISCOPO, BRITTENY; KNICKELBEIN, KELLY Z.; LIEGEY DOUGALL, ANGELA J.; CHAMBERS, WILLIAM H.; JENKINS, FRANK J.

2013-01-01

68

Rubi Fructus (Rubus coreanus) Inhibits Differentiation to Adipocytes in 3T3-L1 Cells  

PubMed Central

Rubi Fructus (RF) is known to exert several pharmacological effects including antitumor, antioxidant, and anti-inflammatory activities. However, its antiobesity effect has not been reported yet. This study was focused on the antidifferentiation effect of RF extract on 3T3-L1 preadipocytes. When 3T3-L1 preadipocytes were differentiating into adipocytes, 10–100??g/mL of RF was added. Next, the lipid contents were quantified by Oil Red O staining. RF significantly reduced lipid accumulation and downregulated the expression of peroxisome proliferator-activated receptor ? (PPAR?), CCAAT0-enhancer-binding proteins ? (C/EBP?), adipocyte fatty acid-binding protein 2 (aP2), resistin, and adiponectin in ways that were concentration dependent. Moreover, RF markedly upregulated liver kinase B1 and AMP-activated protein kinase (AMPK). Interestingly, pretreatment with AMPK? siRNA and RF downregulated the expression of PPAR? and C/EBP? protein as well as the adipocyte differentiation. Our study shows that RF is capable of inhibiting the differentiation of 3T3-L1 adipocytes through the modulation of PPAR?, C/EBP?, and AMPK, suggesting that it has a potential for therapeutic application in the treatment or prevention of obesity.

Jeong, Mi-Young; Kim, Hye-Lin; An, Hyo-Jin; Kim, Sung-Hoon; Kim, Su-Jin; So, Hong-Seob; Park, Raekil; Um, Jae-Young; Hong, Seung-Heon

2013-01-01

69

Protective effect of liquiritigenin against methylglyoxal cytotoxicity in osteoblastic MC3T3-E1 cells.  

PubMed

Methylglyoxal (MG), a reactive dicarbonyl compound, is a metabolic byproduct of glycolysis and elevated MG levels contribute to diabetic complications. Glycation reactions of MG with amino acids can induce oxidative stress, leading to subsequent cytotoxicity. In the present study, the effect of liquiritigenin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells. Pretreatment of MC3T3-E1 cells with liquiritigenin prevented the MG-induced cell death and production of protein adduct, intracellular reactive oxygen species, mitochondrial superoxide, cardiolipin peroxidation, and TNF-? in osteoblastic MC3T3-E1 cells. In addition, liquiritigenin increased the activity of glyoxalase I inhibited by MG. These findings suggest that liquiritigenin provides a protective action against MG-induced cell damage by reducing oxidative stress and by increasing MG detoxification. Pretreatment with liquiritigenin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss. Additionally, the nitric oxide and PGC-1? levels were significantly increased by liquiritigenin, suggesting that liquiritigenin may induce mitochondrial biogenesis. Our findings indicate that liquiritigenin might exert its therapeutic effects via enhancement of glyoxalase I activity and mitochondrial function, and anti-oxidant and anti-inflammatory activities. Taken together, liquiritigenin has potential as a preventive agent against the development of diabetic osteopathy related to MG-induced oxidative stress in diabetes. PMID:24789098

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

2014-06-25

70

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

PubMed

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

2013-06-01

71

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

2013-01-01

72

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

PubMed

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

1995-01-01

73

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

SciTech Connect

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

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

2013-02-01

74

Effects of 6-Hydroxyflavone on Osteoblast Differentiation in MC3T3-E1 Cells  

PubMed Central

Osteoblast differentiation plays an essential role in bone integrity. Isoflavones and some flavonoids are reported to have osteogenic activity and potentially possess the ability to treat osteoporosis. However, limited information concerning the osteogenic characteristics of hydroxyflavones is available. This study investigates the effects of various hydroxyflavones on osteoblast differentiation in MC3T3-E1 cells. The results showed that 6-hydroxyflavone (6-OH-F) and 7-hydroxyflavone (7-OH-F) stimulated ALP activity. However, baicalein and luteolin inhibited ALP activity and flavone showed no effect. Up to 50??M of each compound was used for cytotoxic effects study; flavone, 6-OH-F, and 7-OH-F had no cytotoxicity on MC3T3-E1 cells. Moreover, 6-OH-F activated AKT and serine/threonine kinases (also known as protein kinase B or PKB), extracellular signal-regulated kinases (ERK 1/2), and the c-Jun N-terminal kinase (JNK) signaling pathways. On the other hand, 7-OH-F promoted osteoblast differentiation mainly by activating ERK 1/ 2 signaling pathways. Finally, after 5 weeks of 6-OH-F induction, MC3T3-E1 cells showed a significant increase in the calcein staining intensity relative to merely visible mineralization observed in cells cultured in the osteogenic medium only. These results suggested that 6-OH-F could activate AKT, ERK 1/2, and JNK signaling pathways to effectively promote osteoblastic differentiation.

Wu, Yu-Wei; Yeh, Shauh-Der; Lin, Yu-Hsaing; Tsai, Yu-Hui

2014-01-01

75

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

PubMed

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

2010-11-01

76

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation.  

PubMed

While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation. PMID:22906707

Mikami, Yoshikazu; Suzuki, Shinnosuke; Ishii, Yumiko; Watanabe, Nobukazu; Takahashi, Tomihisa; Isokawa, Keitaro; Honda, Masaki J

2012-12-01

77

Effects of 6-Hydroxyflavone on Osteoblast Differentiation in MC3T3-E1 Cells.  

PubMed

Osteoblast differentiation plays an essential role in bone integrity. Isoflavones and some flavonoids are reported to have osteogenic activity and potentially possess the ability to treat osteoporosis. However, limited information concerning the osteogenic characteristics of hydroxyflavones is available. This study investigates the effects of various hydroxyflavones on osteoblast differentiation in MC3T3-E1 cells. The results showed that 6-hydroxyflavone (6-OH-F) and 7-hydroxyflavone (7-OH-F) stimulated ALP activity. However, baicalein and luteolin inhibited ALP activity and flavone showed no effect. Up to 50? ? M of each compound was used for cytotoxic effects study; flavone, 6-OH-F, and 7-OH-F had no cytotoxicity on MC3T3-E1 cells. Moreover, 6-OH-F activated AKT and serine/threonine kinases (also known as protein kinase B or PKB), extracellular signal-regulated kinases (ERK 1/2), and the c-Jun N-terminal kinase (JNK) signaling pathways. On the other hand, 7-OH-F promoted osteoblast differentiation mainly by activating ERK 1/ 2 signaling pathways. Finally, after 5 weeks of 6-OH-F induction, MC3T3-E1 cells showed a significant increase in the calcein staining intensity relative to merely visible mineralization observed in cells cultured in the osteogenic medium only. These results suggested that 6-OH-F could activate AKT, ERK 1/2, and JNK signaling pathways to effectively promote osteoblastic differentiation. PMID:24795772

Lai, Chien-Hung; Wu, Yu-Wei; Yeh, Shauh-Der; Lin, Yu-Hsaing; Tsai, Yu-Hui

2014-01-01

78

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

PubMed Central

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

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

2013-01-01

79

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

PubMed

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

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

2013-01-01

80

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

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

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

81

EPAS1 promotes adipose differentiation in 3T3-L1 cells.  

PubMed

Adipose differentiation is regulated by several transcription factors, such as the CAAT/enhancer-binding protein family and peroxisome proliferator activator (PPAR) gamma2. Several recent studies have shown that the basic helix-loop-helix-PAS superfamily is also involved in the regulation of adipose differentiation. In this study, we investigated the roles played by EPAS1 (endothelial PAS domain protein 1) in adipogenesis. EPAS1, also referred to as hypoxia-inducible factor 2alpha, is a transcription factor known to play essential roles in catecholamine homeostasis, vascular remodeling, and the maintenance of reactive oxygen species, and so forth. During adipose differentiation in 3T3-L1 cells, the level of EPAS1 mRNA began to increase 6 days after the induction, and EPAS1 was highly expressed in differentiated cells. To examine whether EPAS1 is involved in adipogenesis, we first isolated stable clones from 3T3-L1 cells in which we could induce the expression of an EPAS1 C-terminal deletion mutant (designated EPAS1-(1-485)) with the insect hormone. The induction of EPAS1-(1-485) allowed the cells to accumulate only minimum amounts of intracellular lipid droplets. Consistent with the morphological observations, a minimum amount of aP2 and PPARgamma2 mRNA was induced in the EPAS1-(1-485) cells. We then examined whether or not EPAS1 was able to promote adipogenesis in NIH 3T3 cells, a relatively nonadipogenic cell line. Overexpression of EPAS1 in NIH 3T3 cells induced a significant amount of lipid accumulation compared with that of the control cells in the presence of the PPARgamma ligand. The results were also confirmed by measuring the expression of adipocyte-related genes. Adenovirus-mediated EPAS1-(1-485) expression resulted in the reduction of basal and insulin-dependent glucose transport in 3T3-L1 adipocytes. The mechanism involved the transcriptional regulation of GLUT1, GLUT4, and IRS3 expression by EPAS1. Taken together, these results suggest that EPAS1 plays several supporting roles in maintaining specific aspects of adipogenesis and adipocyte function including regulation of glucose uptake followed by lipid synthesis. PMID:15258146

Shimba, Shigeki; Wada, Taira; Hara, Shuntaro; Tezuka, Masakatsu

2004-09-24

82

[Effects of human mesenchymal stem cells and fibroblastoid cell line as feeder layers on expansion of umbilical cord blood CD34(+) cells in vitro].  

PubMed

To investigate the effects of human mesenchymal stem cells (MSC) and human fibroblastoid cell line (HFCL) as feeder layer on expansion of umbilical cord blood CD34(+) cells in vitro, (60)Co gamma-ray irradiated MSC and HFCL were used as feeder layer to expand cord blood CD34(+) cells in culture. The efficiencies of MSC and HFCL on expansion of CD34(+) cells in culture with or without cytokines were compared. The results showed that no matter whether cytokines (rhFL, rhSCF, rhTPO) were added, the proliferation of nucleated cells after expansion for 12 days in HFCL group was statistically higher than that in MSC group, i.e. with cytokines (9797 +/- 361)% vs (7061 +/- 418)%; without cytokines (5305 +/- 354)% vs (1992 +/- 247)%, when the cell numbers at day 0 was accounted as 100%), P < 0.01. The proliferation of propagated CD34(+) cells between MSC group and HFCL without addition of cytokines was not statistically different (820 +/- 191)% vs (825 +/- 305)%, P > 0.05. However, in the presence of cytokines, the propagating rate of MSC group was lower than that of HFCL group (939 +/- 212)% vs (1617 +/- 222)%, P < 0.01. MSC was better than HFCL in maintaining the LTC-IC of UCB CD34(+) cells, i.e. the number of CFU-GM colonies in the fifth week was (129.95 +/- 8.73) /10(5) seeded cells vs (89.81 +/- 10.29) colonies/10(5) cells, P < 0.05; with addition of cytokines, the effect was more obvious, i.e. the number of CFU-GM colonies in the fifth week (192.93 +/- 4.95)/10(5) seeded cells vs (90.47 +/- 14.28) colonies/10(5) seeded cells, P < 0.01. MSC mixed with a certain proportion of HFCL facilitated maintaining the LTC-IC of UCB CD34(+) cells. When the proportion was 4:1, the number of CFU-GM colonies was the highest (186.89 +/- 11.11)/10(5) seeded cells, which was higher than that of both 3:2 group [(138.92 +/- 14.84) colonies/10(5) seeded cells] and MSC only group, i.e. (64.63 +/- 6.11) colonies/10(5) seeded cells, both P < 0.01. It is concluded that HFCL is better than MSC in maintaining the expansion of CD34(+) cells and cytokines can enhance this effect, while MSC are stronger than HFCL in maintaining the LTC-IC of UCB CD34(+) cells in vitro. MSC with addition of a certain proportion of HFCL can significantly enhance the efficiency of CD34(+) cell expansion. PMID:17096895

Ma, Li-Jun; Gao, Lei; Zhou, Hong; Qiu, Hui-Ying; Hu, Xiao-Xia; Xie, Lin-Na; Wang, Jian-Min

2006-10-01

83

The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts  

Microsoft Academic Search

Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts

Marthe Rousseau; Lucilia Pereira-Mouričs; Maria-José Almeida; Christian Milet; Evelyne Lopez

2003-01-01

84

Survey of Screw Feeders.  

National Technical Information Service (NTIS)

This report presents the results of a survey to determine the availability of screw feeders for use in areas related to coal feeding in the field of coal conversion. 15 references. (ERA citation 09:037045)

G. Sine

1983-01-01

85

Human umbilical cord blood-derived stromal cell, a new resource of feeder layer to expand human umbilical cord blood CD34+ cells in vitro.  

PubMed

Allogeneic transplantation with human umbilical cord blood (hUCB) in adult recipients is mainly limited by a low CD34+ cell dose. To break the limit, hUCB as a novel source of hUCB-derived stromal cells was incorporated in an attempt to expand CD34+ cells from hUCB in vitro. Cord blood CD34 cells were separated by MACS system. HUCB-derived stromal cells were cultured by the Dexter system and characterized by morphologic, immunophenotypical, and functional analysis. We studied the effects of hUCB-derived stromal cells, cytokines, and hUCB-derived stromal cells combined with cytokines on expansion of hUCB CD34 cells. The CD34+ cells were assessed for the degree of expansion and the number of colony-forming units in semisolid culture. Our research found that hUCB-derived stromal cells were mainly composed of three kinds of cell components, with CD106, CD29, CD44, CD45, CD50, CD68, CD31, Fn, Lm, and collagen IV positive, but CD34 negative immunophenotype. Functionally, it was discovered by cell cycle and growth curve analyses that the capability of colony and parietal layer formation of hUCB-derived stromal cells was poorer than that of BM stromal cells, and the doubling time of hUCB-derived stromal cells was longer than that of BM stromal cells. It was indicated by ELISA and RT-PCR that hUCB-derived stromal cells express higher level of TPO and less GM-CSF and SCF than BM stromal cell. Adherent layer of hUCB-derived stromal cells alone or combining with cytokines, increased CD34+ cell expansion. In vitro formation of CFUs by expanded CCD34 cells was significantly higher than that of unexpanded CD34+ cells (P < 0.05). When cocultured with hUCB-derived stromal cells in the presence of cytokines, cell growth was significantly enhanced: CD34 cells by 8.02 +/- 0.96-fold, CFU-GM by 217.60 +/- 6.72-fold, CFU-E by 1940.80 +/- 52.78-fold, and CFU-Mg by 142.60 +/- 4.39-fold. HUCB-derived stromal cells have significant superiority on the expansion of CFU-Mg (P < 0.05). The results indicate that human umbilical cord blood-derived stromal cells may be a suitable feeder layer for expansion of hematopoietic progenitors from hUCB in vitro. PMID:16500123

Gao, Lei; Chen, Xinghua; Zhang, Xi; Liu, Yao; Kong, Peiyan; Peng, Xiangui; Liu, Lin; Liu, Hong; Zeng, Dongfeng

2006-01-01

86

Effects of crude drugs on lipolysis in differentiated 3T3-L1 adipocytes.  

PubMed

In the present study, aqueous fractions extracted from Radix Ginseng, Radix Rehmanniae, Radix Puerariae, Radix Asparagi, Cortex Phellodendri and Radix Scutellariae were investigated for their effects on lipolysis measured the glycerol release in cultured 3T3-L1 differentiated adipocytes cells. Following treatment of cells with various concentrations of water-soluble extracts ranging from 0.1, 1 to 10 mg/ml for 60 mim, the basal glycerol release from 3T3-L1 cells was changed from 71 nmole/mg protein of control to 48, 46 and 31 nmole/mg protein in Radix Ginseng-treated cells. Amount of glycerol was reduced to 60, 26 and 20 by Radix Rehmanniae. In the presence of Radix Puerariae, glycerol release was decreased to 35, 34 and 30, respectively. After exposure to Radix Asparagi, amount of glycerol became 108, 73 and 70 nmole/mg protein, respectively. In the case of Cortex Phellodendri, amount of glycerol was increased from 126, 112 to 90, respectively. In the presence of Radix Scutellariae, the glycerol was changed to 118, 77 and 29, respectively. In isoproterenol-stimulated cells, the glycerol release from cells by Radix Ginseng was changed from 169 of control to 76, 73 and 72 nmole/mg protein, respectively. After incubation with Radix Rehmanniae, amount of glycerol decreased to 52, 35 and 11, respectively. In the presence of Radix Puerariae, the glycerol was changed to 26, 25 to 20, respectively. In the presence of Radix Asparagi, the glycerol became 160, 96 and 64, respectively. In the case of Cortex Phellodendri, the glycerol was increased to 160, 92 to 88, respectively. In the presence of Radix Scutellariae, the glycerol was changed to 149, 83 and 50, respectively. These results indicated that the water-soluble substances from Radix Ginseng, Radix Rehmanniae and Radix Puerariae decreased the lipolysis in basal and isoproterenol-stimulated 3T3-L1 adipocytes. PMID:12164008

Hong, Show-Jen; Fong, Jim C; Hwang, Jia-Huae

2002-04-01

87

R-Ras induces malignant, but not morphologic, transformation of NIH3T3 cells.  

PubMed

Although previous studies have not identified transforming properties of the Ras-related protein R-Ras, two recent observations have prompted our further evaluation of R-Ras function. First, we observed that mutant forms of the closely related R-Ras2/TC21 protein (approximately 70% identity) exhibited the same potent transforming activity as oncogenic Ras proteins. Second, R-Ras association with Bcl-2 suggested a possible role for R-Ras in apoptotic growth control. Therefore, we have performed a detailed analysis of R-Ras transforming potential in NIH3T3 cells. Whereas expression of a mutant R-Ras protein (38V; analogous to the 12V activating Ras mutation) did not induce morphologic transformation of NIH3T3 cells, R-Ras(38V)-expressing cells proliferated in low serum, formed colonies in soft agar, and formed progressive tumors in nude mice. Like Ras-transformed cells, R-Ras(38V)-transformed cells exhibited constitutively activated mitogen activated protein kinases. Furthermore, R-Ras(38V) stimulated transcriptional activation of Ras-responsive promoter elements, and this activity (and transformation) was blocked by Raf dominant negative proteins. Finally, whereas co-expression of Bcl-2 did not cause significant alteration in wild type or mutant R-Ras transforming activity, coexpression of v-Myc and R-Ras(38V) induced a striking morphologic transformation of NIH3T3 cells. Taken together, these observations suggest that aberrant R-Ras function may stimulate malignant transformation, in the absence of morphologic transformation, via up-regulation of part of the Ras signal transduction pathway. PMID:7936652

Cox, A D; Brtva, T R; Lowe, D G; Der, C J

1994-11-01

88

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

PubMed

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

2013-06-01

89

Potent stimulation of vascular endothelial cell growth by differentiated 3T3 adipocytes.  

PubMed Central

3T3 cells that have undergone adipose differentiation in vitro secrete into the culture medium a potent growth stimulatory activity for bovine aortic endothelial cells. When medium containing 2% fetal calf serum, which does not support significant endothelial cell growth, is conditioned by 3T3-F442A adipocytes, the endothelial cells grow rapidly (doubling time, 24 hr) at a rate equal to the growth rate in 20% fetal calf serum. The potency of the conditioned medium is further shown by the fact that it can be diluted 1:5 with little apparent loss of activity and shows a half-maximal stimulation at 10 microliter/ml. Serum is not required for either the secretion of this mitogen by the adipocytes or its action on the endothelial cells, as shown by the fact that the latter are stimulated to divide in serum-free medium conditioned by the adipocytes. The growth stimulatory activity appears to be specific for vascular endothelial cells in that no other cell type examined, including vascular smooth muscle cells and pericytes, are significantly stimulated by medium conditioned by 3T3-F442A cells. Similarly, medium conditioned by no other cell type examined has more than 10% of the activity of medium conditioned by the adipocytes. The specificity and potency of the adipocyte-derived factor suggest that it may play a role in the vascularization of this tissue during development. Preliminary biochemical analysis indicates that the adipocyte factor is nondialyzable and is not inactivated by heat or proteases. The protease insensitivity distinguishes the adipocyte growth stimulatory activity from the low levels of activity secreted by fibroblasts and preadipocytes, suggesting that the adipocyte mitogen is a product specifically related to the differentiation process.

Castellot, J J; Karnovsky, M J; Spiegelman, B M

1980-01-01

90

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.

2006-07-01

91

Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes  

SciTech Connect

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

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

2013-08-16

92

Anti-adipogenic activity of Cordyceps militaris in 3T3-L1 cells.  

PubMed

Inhibition of adipocytes differentiation is suggested to be an important strategy for prevention and/or treatment of obesity. In our present study, Cordyceps militaris showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 preadipocytes as assessed by measuring fat accumulation using Oil Red O staining. Activity-guided fractionation led to the isolation of cordycepin (1), guanosine (2) and tryptophan (3) as active compounds. All the three compounds were more effective in the prevention of early stage of adipogenesis than in lipolysis. In addition, combinational treatment of three compounds significantly increased anti-adipogenic activity. PMID:22312720

Liu, Qing; Hong, In Pyo; Ahn, Mi-Jeong; Yoo, Hwan-Soo; Han, Sang-Bae; Hwang, Bang Yeon; Lee, Mi Kyeong

2011-12-01

93

The accumulation and metabolism of zidovudine in 3T3-F442A pre-adipocytes  

PubMed Central

Background and purpose: Cultured pre-adipocytes accumulate and metabolize zidovudine (ZDV), but its mode of accumulation into these cells is unclear. We investigated the mode of accumulation of [3H]-ZDV, and the impact of changes in external pH and modulators of drug transporters on its accumulation and metabolism. Experimental approach: The initial rate and steady-state accumulation of [3H]-ZDV were measured in 3T3-F442A cells. P-glycoprotein (P-gp) expression was detected by Western blotting. External pH was varied, and modulators of intracellular pH and drug transporters were used to study the mode of accumulation of ZDV. Phosphorylated ZDV metabolites were detected by high-performance liquid chromatography. Key results: Intracellular accumulation of ZDV was rapid, reaching equilibrium within 20 min; nigericin increased accumulation by 1.9-fold, but this did not alter the generation of ZDV mono-, di- and triphosphate. The accumulation and metabolism were pH dependent, being maximal at pH 7.4 and least at pH 5.1. Monensin, carbonyl cyanide p-trifluoromethoxy) phenyl hydrazone, brefeldin A, bafilomycin A1 and concanamycin A increased accumulation; 2-deoxyglucose, dipyridamole, thymidine and tetraphenylphosphonium inhibited accumulation. The accumulation was saturable; the derived Kd and capacity of binding were 250 nmol per 106 cells and 265 nM respectively. 3T3-F442A cells express P-gp; inhibitors of P-gp (XR9576 and verapamil), P-gp/BCRP (GF120918), multidrug resistance protein (MRP) (MK571) and MRP/OATP (probenecid) increased the accumulation of ZDV. Saquinavir, ritonavir, amprenavir and lopinavir increased accumulation. Conclusions and implications: The accumulation of ZDV in 3T3-F442A cells was rapid, energy dependent, saturable and pH sensitive. Western blot analysis showed that 3T3-F442A cells express P-gp, and direct inhibition assays suggest that ZDV is a substrate of P-gp and MRP.

Janneh, Omar; Owen, Andrew; Bray, Patrick G; Back, David J; Pirmohamed, Munir

2010-01-01

94

Effect of pycnogenol on glucose transport in mature 3T3-L1 adipocytes.  

PubMed

Pycnogenol, a procyanidins-enriched extract of Pinus maritima bark, possesses antidiabetic properties, which improves the altered parameters of glucose metabolism that are associated with type 2 diabetes mellitus (T2DM). Since the insulin-stimulated antidiabetic activities of natural bioactive compounds are mediated by GLUT4 via the phosphatidylinositol-3-kinase (PI3K) and/or p38 mitogen activated protein kinase (p38-MAPK) pathway, the effects of pycnogenol were examined on the molecular mechanism of glucose uptake by the glucose transport system. 3T3-L1 adipocytes were treated with various concentrations of pycnogenol, and glucose uptake was examined using a non-radioisotope enzymatic assay and by molecular events associated with the glucose transport system using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results show that pycnogenol increased glucose uptake in fully differentiated 3T3-L1 adipocytes and increased the relative abundance of both GLUT4 and Akt mRNAs through the PI3K pathway in a dose dependent manner. Furthermore, pycnogenol restored the PI3K antagonist-induced inhibition of glucose uptake in the presence of wartmannin, an inhibitor of the PI3K. Overall, these results indicate that pycnogenol may stimulate glucose uptake via the PI3K dependent tyrosine kinase pathways involving Akt. Further the results suggest that pycnogenol might be useful in maintaining blood glucose control. PMID:20658573

Lee, Hee-Hyun; Kim, Kui-Jin; Lee, Ok-Hwan; Lee, Boo-Yong

2010-08-01

95

Effect of Escherichia coli STb toxin on NIH-3T3 cells.  

PubMed

Previous studies have shown that STb causes microscopic histological alterations in animal intestinal models. Disrupted intestinal epithelium at the villous tips could be the result of an altered physiological cell state induced by the toxin. As a cellular model we used NIH-3T3 cells, a mouse fibroblast cell line, previously shown to be capable of internalizing the STb toxin. Using various probes specific for the cellular physiological state or cell organelles, we investigated STb activity using flow cytometry and confocal microscopy. In NIH-3T3 cells, labelled with propidium iodide and carboxyfluorescein diacetate, STb permeabilized the plasma membrane but the cellular esterases remained active. Confocal microscopy showed that fluorescein isothiocyanate (FITC)-labelled STb toxin molecules were internalized and were found scattered in the cytoplasm. Moreover, important clusters of FITC-STb were observed inside the cells after 6 h and these clusters matched with mitochondria labelling. After cell treatment with STb, using a fluorescent mitochondrial potential sensor, we observed mitochondria hyperpolarization, as an early event of intoxication. This phenomenon increased linearly with the dose of STb. The cell population treated with STb showed histological alterations such as membrane budding, granular cytoplasm and enlarged nucleus. Altogether, these results provide new information, at the cellular level, on the effect of the STb toxin. PMID:19222570

Gonçalves, Carina; Dubreuil, J Daniel

2009-04-01

96

Hormonal regulation of the novel adipocytokine visfatin in 3T3-L1 adipocytes.  

PubMed

Recently, visfatin was characterized as a novel adipo-cytokine that is upregulated in obesity and exerts insulin-mimetic effects in various tissues. To clarify expression and regulation of this adipocytokine, visfatin mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction in 3T3-L1 adipocytes during adipogenesis and after treatment with various hormones known to alter insulin sensitivity. Visfatin expression was about 6-fold higher in 3T3-L1 adipocytes in vitro as compared with epididymal fat in vivo and increased during adipogenic conversion more than 3-fold. Interestingly, 100 nM dexamethasone significantly increased visfatin mRNA by almost 1.5-fold. In contrast, 500 ng/ml growth hormone (GH), 10 ng/ml tumor necrosis factor (TNF) alpha, and 10 microM isoproterenol downregulated visfatin expression by 45%, 36%, and 43% respectively. Insulin did not influence synthesis of this adipocytokine. The effects of dexamethasone, GH, TNFalpha and isoproterenol were time- and dose-dependent. Furthermore, activation of G(s)-protein-coupled pathways by forskolin and cholera toxin was sufficient to significantly downregulate visfatin mRNA. Taken together, our results show a differential regulation of visfatin mRNA by insulin resistance-inducing hormones, supporting the view that this adipo-cytokine might be an interesting novel candidate linking core components of the metabolic syndrome such as obesity and insulin resistance. PMID:15930160

Kralisch, Susan; Klein, Johannes; Lossner, Ulrike; Bluher, Matthias; Paschke, Ralf; Stumvoll, Michael; Fasshauer, Mathias

2005-06-01

97

Zinc-chelated Vitamin C Stimulates Adipogenesis of 3T3-L1 Cells  

PubMed Central

Adipose tissue development and function play a critical role in the regulation of energy balance, lipid metabolism, and the pathophysiology of metabolic syndromes. Although the effect of zinc ascorbate supplementation in diabetes or glycemic control is known in humans, the underlying mechanism is not well described. Here, we investigated the effect of a zinc-chelated vitamin C (ZnC) compound on the adipogenic differentiation of 3T3-L1 preadipocytes. Treatment with ZnC for 8 d significantly promoted adipogenesis, which was characterized by increased glycerol-3-phosphate dehydrogenase activity and intracellular lipid accumulation in 3T3-L1 cells. Meanwhile, ZnC induced a pronounced up-regulation of the expression of glucose transporter type 4 (GLUT4) and the adipocyte-specific gene adipocyte protein 2 (aP2). Analysis of mRNA and protein levels further showed that ZnC increased the sequential expression of peroxisome proliferator-activated receptor gamma (PPAR?) and CCAAT/enhancer-binding protein alpha (C/EBP?), the key transcription factors of adipogenesis. These results indicate that ZnC could promote adipogenesis through PPAR? and C/EBP?, which act synergistically for the expression of aP2 and GLUT4, leading to the generation of insulin-responsive adipocytes and can thereby be useful as a novel therapeutic agent for the management of diabetes and related metabolic disorders.

Ghosh, Chiranjit; Yang, Seung Hak; Kim, Jong Geun; Jeon, Tae-Il; Yoon, Byung Hyun; Lee, Jai Young; Lee, Eun Young; Choi, Seok Geun; Hwang, Seong Gu

2013-01-01

98

Paprika Pigments Attenuate Obesity-Induced Inflammation in 3T3-L1 Adipocytes  

PubMed Central

Obesity is related to various diseases, such as diabetes, hyperlipidemia, and hypertension. Adipocytokine, which is released from adipocyte cells, affects insulin resistance and blood lipid level disorders. Further, adipocytokine is related to chronic inflammation in obesity condition adipocyte cells. Paprika pigments (PPs) contain large amounts of capsanthin and capsorubin. These carotenoids affect the liver and improve lipid disorders of the blood. However, how these carotenoids affect adipocyte cells remains unknown. Present study examined the effects of PP on adipocytokine secretion, which is related to improvement of metabolic syndrome. In addition, suppressive effects of PP on chronic inflammation in adipocyte cells were analyzed using 3T3-L1 adipocyte cells and macrophage cell coculture experiments. PP promoted 3T3-L1 adipocyte cells differentiation upregulated adiponectin mRNA expression and secretion. Further, coculture of adipocyte and macrophage cells treated with PP showed suppressed interleukin-6 (IL-6), tumor necrosis factor-? (TNF-?), monocyte chemotactic protein-1 (MCP-1), and resistin mRNA expression, similarly to treatment with troglitazone, which is a PPAR? ligand medicine. Conclusion. These results suggest that PP ameliorates chronic inflammation in adipocytes caused by obesity. PP adjusts adipocytokine secretion and might, therefore, affect antimetabolic syndrome diseases.

Maeda, Hayato; Saito, Shuuichi; Nakamura, Nozomi; Maoka, Takashi

2013-01-01

99

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 ..mu..m 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.

1986-03-05

100

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

PubMed

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

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

2014-07-01

101

Incorporation of xenobiotic carboxylic acids into lipids by cultured 3T3-L1 adipocytes.  

PubMed

The study was established to assess the potential for a variety of xenobiotic aromatic carboxylic acids to be incorporated into glycerolipids. The 14C-labelled xenobiotic acids were included in incubations of cultured 3T3-L1 adipocytes under defined conditions. Lipids were extracted and identified by TLC and radioscanning. Ibuprofen, 4-(2,4-dichlorophenoxy)-butanoic acid (2,4-DB), 4-(2-methyl-4-chlorophenoxy)-butanoic acid (MCPB) and 2-(2-methyl-4-chlorophenoxy)-propanoic acid (MCPP) (all 0.5 mM) were incorporated into lipid extracts at rates of 220, 227, 199 and 21 pmol microg(-1) phospholipid/h, respectively. 2,4-Dichlorophenoxyacetic acid (2,4-D), indomethacin, naproxen and fluroxypyr were incorporated at rates lower than MCPP or not at all. The incorporation of acids was first order to at least 1 mM acid (except MCPB: 300 microM). Triacylglycerol analogues were the major products with incorporation into diacylglycerol and phosphatidylcholine also observed. After digestion with pancreatic lipase, ibuprofen-containing triacylglycerol was unusual in that the main product was the monoacylglycerol analogue, suggesting that esterification had been at the sn-2 position. Incubation with cultured 3T3-L1 adipocytes is a useful and easy method to assess whether xenobiotic compounds can be incorporated into glycerolipids; of eight acids assessed, four (of which three have not previously been reported) were shown to form xenobiotic triacylglycerols. PMID:15801546

Vickery, S; Dodds, P F

2004-01-01

102

Adipogenetic effects of retrofractamide A derivatives in 3T3-L1 cells.  

PubMed

In a previous study, retrofractamide A from the fruit of Piper chaba was shown to promote adipogenesis in 3T3-L1 cells. In the present study, retrofractamide A and its derivatives were synthesized, and their adipogenetic effects in 3T3-L1 cells were examined. Among the tested compounds, an amide composed of 9-(3',4'-methylenedioxyphenyl)-nona-2E,4E,8E-trienoic acid and an n-butyl or n-pentyl amine showed strongest activity. Moreover, the amide with the n-pentyl amine moiety significantly increased the uptake of 2-deoxyglucose into the cells, and also increased the mRNA levels of adiponectin, peroxisome proliferator-activated receptor ?2 (PPAR?2), glucose transporter 4 (GLUT4), fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein (C/EBP) ? and ? in a similar manner as the PPAR? agonist troglitazone, although it had less agonistic activity against PPAR?. PMID:23910984

Mourad, Ahmed Aboul-Fotouh; Nakamura, Seikou; Ueno, Tsubasa; Minami, Takahiro; Yagi, Takanari; Yasue, Haruka; Komatsu, Ryoko; Yoshikawa, Masayuki; Taye, Ashraf Mohamed; El-Moselhy, Mohamed Ahmed; Khalifa, Mohamed Montaser; Matsuda, Hisashi

2013-09-01

103

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.

2009-01-01

104

Effects of crude drugs on glucose uptake in 3T3-L1 adipocytes.  

PubMed

In this study, various water-extracted crude drugs from Radix Asparagi, Radix Ginseng, Radix Scutellariae, Cortex Lycii Radicis, Cortex Phellodendri and Radix Ophiopogonis were investigated in their effects on [3H]-2-deoxyglucose uptake in 3T3-L1 adipocytes. Following treatment of cells with various crude drugs for 60 mim, the basal [3H]-2-deoxyglucose uptake in cultured 3T3-L1 cells was changed by Radix Asparagi from 140 pmole/min/mg protein of control to 513 (0.1 mg/ml), 201 (1 mg/ml) and 97 (10 mg/ml). Glucose uptake was changed to 324 (0.1 mg/ml), 146 (1 mg/ml) and 46 (10 mg/ml) with Radix Ginseng. In the presence of Radix Scutellariae, glucose uptake was changed to 215 (0.1 mg/ml), 213 (1 mg/ml) and 34 (10 mg/ml). In the presence of Cortex Lycii Radicis, glucose uptake was 230 (0.1 mg/ml), 188 (1 mg/ml) and 38 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, uptake was changed to 142 (0.1 mg/ml), 132 (1 mg/ml), 24 (10 mg/ml) and 489 (0.1 mg/ml), 374 (1 mg/ml), 344 (10 mg/ml), respectively. In insulin-stimulated cells, the [3H]-2-deoxyglucose uptake was changed by Radix Asparagi from 570 pmole/min/mg protein of the control to 816 (0.1 mg/ml), 674 (1 mg/ml) and 532 (10 mg/ml). After incubation with Radix Ginseng, the glucose uptake was changed to 254 (0.1 mg/mi), 123 (1 mg/mi) to 76 (10 mg/mi). In the presence of Radix Scutellariae, the glucose uptake was changed to 315 (0.1 mg/ml), 265 (1 mg/ml) and 33 (10 mg/ml). After incubation of Cortex Lycii Radicis, the uptake activity was changed to 281 (0.1 mg/ml), 248 (1 mg/ml) and 37 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, the activity of glucose uptake was measured as 747 (0.1 mg/ml), 523 (1 mg/ml), 33 (10 mg/ml) and 753 (0.1 mg/ml), 740 (1 mg/ml), and 421 (10 mg/ml), respectively. These results indicate that the water-extracted materials of Radix Asparagi and Radix Ophiopogonis increase the glucose uptake in basal and insulin-stimulated 3T3-L1 adipocytes. PMID:11271729

Hong, S J; Fong, J C; Hwang, J H

2000-09-01

105

Regulation of pyruvate carboxylase in 3T3-L1 cells.  

PubMed Central

When 3T3-L1 fibroblasts differentiate to adipocytes, the specific activity of pyruvate carboxylase (PC) increases about 25-fold in parallel with its intracellular protein concentration. The increase in PC protein concentration is accompanied by a 9-10-fold increase in the relative abundance of 4.2 kb PC mRNA measured by Northern-blot analysis using a cDNA probe encoding a segment of the PC gene of 3T3-L1 adipocytes. The effects of cyclic AMP (cAMP) alone and together with insulin on levels of cellular protein, PC activity, PC protein and on the relative abundance of PC mRNA were examined in mature 3T3-L1 adipocytes. Adipocytes exposed to cAMP for 24 h exhibited a 25% decrease in cellular protein and marked decreases in enzyme activity (88%) and PC mRNA abundance (98%) compared with untreated adipocyte controls. After 48 h of exposure to cAMP, PC activity and PC mRNA diminished to levels approaching their detection limits. When exposed to medium containing cAMP plus insulin, adipocyte enzyme activity and PC mRNA declined more slowly during the first 24 h exposure (about 20% decrease) but after 48 h fell to values comparable with those of adipocytes exposed to cAMP alone. Despite these decreases in enzyme activity, the PC protein content of adipocytes treated with cAMP alone or cAMP plus insulin are nearly identical with that of control adipocytes. The inactivation of PC in cAMP-treated adipocytes does not involve loss of the prosthetic group from the holoenzyme. Cross-linking experiments suggest that the spatial arrangement of protomers in inactive PC may differ from that in the active tetrameric enzyme. Data presented suggest that, in addition to inducing inactivation, cAMP may also regulate adipocyte PC by decreasing transcription of the PC gene and/or enhancing the rate of degradation of PC mRNA. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Zhang, J; Xia, W L; Ahmad, F

1995-01-01

106

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

PubMed Central

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, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18- (3), and the cytoplasmic compartment was stained with fluoresceinyl- dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close- contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close- contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.

1985-01-01

107

Ursolic Acid Inhibits Adipogenesis in 3T3-L1 Adipocytes through LKB1/AMPK Pathway  

PubMed Central

Background Ursolic acid (UA) is a triterpenoid compound with multiple biological functions. This compound has recently been reported to possess an anti-obesity effect; however, the mechanisms are less understood. Objective As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes. Methods and Results The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days. The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at concentrations ranging from 2.5 µM to 10 µM dose-dependently attenuated adipogenesis, accompanied by reduced protein expression of CCAAT element binding protein ? (C/EBP?), peroxisome proliferator-activated receptor ? (PPAR?), CCAAT element binding protein ? (C/EBP?) and sterol regulatory element binding protein 1c (SREBP-1c), respectively. Ursolic acid increased the phosphorylation of acetyl-CoA carboxylase (ACC) and protein expression of carnitine palmitoyltransferase 1 (CPT1), but decreased protein expression of fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Ursolic acid increased the phosphorylation of AMP-activated protein kinase (AMPK) and protein expression of (silent mating type information regulation 2, homolog) 1 (Sirt1). Further studies demonstrated that the anti-adipogenic effect of UA was reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1), the upstream kinase of AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK activation was diminished. Conclusions Ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK pathway. There is potential to develop UA into a therapeutic agent for the prevention or treatment of obesity.

He, Yonghan; Li, Ying; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

2013-01-01

108

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

PubMed

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

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

2012-01-01

109

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

SciTech Connect

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

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

1988-09-01

110

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

2013-01-01

111

Inhibitory effect of andrographolide in 3T3-L1 adipocytes differentiation through the PPAR? pathway.  

PubMed

Andrographolide (AG), an active compound found in Andrographis paniculate Nees, has been shown to exert anti-inflammatory, anticancer and anti-hyperglycemic effects. However, its biological activities against obesity have not been reported. The purpose of this study was to investigate the effect of AG on the differentiation of 3T3-L1 preadipocytes. We found AG significantly inhibited not only on adipocyte differentiation induced by standard adipogenic agents and MDI, but also on the adipogenesis-related transcription factor, peroxisome proliferator-activated receptor ? (PPAR?), as well as the expressions of the PPAR? targeted genes, such as CD36, LPL, FAS and other adiocyte markers. Taken together, our data showed AG inhibited the early stage of adipogenic differentiation, in part via the inhibition of PPAR?-dependent mechanisms. PMID:22449851

Jin, Lina; Fang, Wenqiang; Li, Bo; Shi, Guojun; Li, Xiaoying; Yang, Ying; Yang, Jian; Zhang, Zhiguo; Ning, Guang

2012-07-01

112

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: soh@inha.ac.kr

2007-12-14

113

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

PubMed

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

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

2014-04-15

114

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

PubMed

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

2012-05-01

115

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

2011-01-01

116

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

Microsoft Academic Search

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

Michelle N. Yarmo; Anne Landry; André S. D. Molgat; AnneMarie Gagnon; Alexander Sorisky

2009-01-01

117

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

PubMed

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

2008-05-01

118

Anti-obesity effect of Blumea balsamifera extract in 3T3-L1 preadipocytes and adipocytes.  

PubMed

Obesity, the leading metabolic disease in the world, is a serious health problem in industrialized countries. We investigated the anti-obesity effect of Blumea balsamifera extract on adipocyte differentiation of 3T3-L1 preadipocytes and anti-obesity effect of 3T3-L1 adipocytes. We found that treatment with an extract of Blumea balsamifera suppressed lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity without affecting cell viability in 3T3-L1 preadipocytes and adipocytes. Furthermore, Blumea balsamifera extract brought significant attenuation of expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor (PPAR)gamma, CCAAT element binding protein (C/EBPs) and leptin, however, induced up-regulation of adiponectin at the protein level in 3T3-L1 preadipocytes and adipocytes. These results suggest that Blumea balsamifera extract may block adipogenesis, at least in part, by decreasing key adipogenic transcription factors in 3T3-L1 preadipocytes and may have antiatherogenic, anti-inflammatory, and antidiabetic effects through up-regulation of adiponectin in 3T3-L1 adipocytes. PMID:19885945

Kubota, Hiroaki; Kojima-Yuasa, Akiko; Morii, Risako; Huang, Xuedan; Norikura, Toshio; Rho, Sook-Nyung; Matsui-Yuasa, Isao

2009-01-01

119

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 (10..mu..M) 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.

1986-03-05

120

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells  

SciTech Connect

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

Jang, Min Kyung; Kim, Cho Hee [School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735 (Korea, Republic of)] [School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735 (Korea, Republic of); Seong, Je Kyung [Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul 151-742 (Korea, Republic of)] [Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul 151-742 (Korea, Republic of); Jung, Myeong Ho, E-mail: jung0603@pusan.ac.kr [School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735 (Korea, Republic of)

2012-04-27

121

Measurement of lipolysis products secreted by 3T3-L1 adipocytes using microfluidics.  

PubMed

Glass microfluidic devices have been fabricated to monitor the secretion of glycerol or fatty acids from cultured murine 3T3-L1 adipocytes. In the current studies, adipocytes are perfused in a reversibly sealed cell chamber, and secreted products are analyzed by enzyme assay on either a single- or dual-chip device. The analysis of glycerol employed the use of a dual-chip system, which used separate chips for cell perfusion and sample analysis. An improved single-chip device integrated the cell perfusion chamber and analysis component on one platform. The performance of this device was demonstrated by the analysis of fatty acids but could also be applied to analysis of glycerol or other chemicals. The single-chip system required fewer cells and lower flow rates and provided improved temporal response. In both systems, cells were perfused with buffer to monitor basal response followed by lipolysis stimulation with the ?-adrenergic agonist isoproterenol. Measured basal glycerol concentration from 50,000 cells was 28 ?M, and when stimulated, a spike threefold higher than basal concentration was detected followed by a continuous release 40% above basal levels. Fatty acid basal concentration was 24 ?M, measured from 6200 cells, and isoproterenol stimulation resulted in a constant elevated concentration sevenfold higher than basal levels. PMID:24529440

Dugan, Colleen E; Kennedy, Robert T

2014-01-01

122

Proteomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3-L1 adipocyte differentiation.  

PubMed

In the present study, we performed a differential proteomic analysis using 2-DE combined with MS to clarify the molecular mechanism for the suppressive effect of chitosan oligosaccharides (CO) during differentiation of adipocyte 3T3-L1. Cell differentiation was significantly inhibited by CO at the concentration of 4 mg/mL. Protein mapping of adipocyte homogenates by 2-DE revealed that numerous protein spots were differentially altered in response to CO treatment. Out of 50 identified proteins showing significant alterations, six were up-regulated and 44 were down-regulated by CO treatment in comparison to control mature adipocytes. Among them, most of the proteins are associated with lipid metabolism, cytoskeleton, and redox regulation, in which the levels of farnesyl diphosphate synthetase (FDS), dedicator of cytokinesis 9 (DOCK9), and chloride intracellular channel 1 (CLIC1) were significantly reduced (>two-fold) with CO treatment. These results have not previously been examined in the context of adipogenesis, and thus can be used as novel biomarkers. Taken together with immunoblot analysis, it was concluded that the inhibitory effect of CO on adipocyte differentiation was mediated by C/EBPalpha and PPARgamma pathway through significant downregulations of important adipogenic molecules such as fatty acid binding protein and glucose transporter 4. PMID:18175373

Rahman, Atiar; Kumar, Suresh G; Kim, Sang Woo; Hwang, Hye Jin; Baek, Yu Mi; Lee, Sung Hak; Hwang, Hee Sun; Shon, Yun Hee; Nam, Kyung Soo; Yun, Jong Won

2008-02-01

123

Isoproterenol stimulates monocyte chemoattractant protein-1 expression and secretion in 3T3-L1 adipocytes.  

PubMed

Recently, monocyte chemoattractant protein (MCP)-1 has been characterized as a novel adipocytokine upregulated in obesity and insulin resistance which impairs insulin signaling in muscle and fat in vitro. Growing evidence, on the other hand, suggests that increased activity of the sympathetic nervous system is an integral part in the development of insulin resistance. In the current study, the impact of the beta-adrenergic agonist isoproterenol on MCP-1 mRNA synthesis and secretion was determined in 3T3-L1 adipocytes. Interestingly, isoproterenol increased MCP-1 secretion 3-fold. Furthermore, 10 microM isoproterenol acutely induced MCP-1 mRNA by up to 5.3-fold in a time-dependent fashion with significant stimulation seen at concentrations as low as 0.3 microM effector. Studies using pharmacological inhibitors suggested that basal and isoproterenol-induced MCP-1 expressions are mediated via beta-adrenergic receptors and protein kinase A. Moreover, acute activation of adenylyl cyclase by forskolin was sufficient to mimic the effects of isoproterenol. Taken together, our results demonstrate that isoproterenol induces MCP-1 expression and secretion via a classical GS-protein-coupled pathway and support the notion that MCP-1 might be an interesting novel candidate linking obesity and insulin resistance. PMID:16644035

Kralisch, Susan; Klein, Johannes; Lossner, Ulrike; Bluher, Matthias; Paschke, Ralf; Stumvoll, Michael; Fasshauer, Mathias

2006-07-15

124

Neuropeptide Y potentiates beta-adrenergic stimulation of lipolysis in 3T3-L1 adipocytes.  

PubMed

Recently, we have shown that neuropeptide Y (NPY) is produced and upregulated in visceral adipose tissue of an early-life programmed rat model of central obesity. Moreover, we have demonstrated that NPY promotes proliferation of adipocyte precursor cells and contributes to the pathogenesis of obesity. However, the role of NPY in regulating adipocyte metabolism is poorly understood. The present study was designed to examine the effects of NPY on adipocyte metabolic function using 3T3-L1 adipocytes as an in vitro cell model system. We found that although it did not affect basal lipolysis, NPY potentiated isoproterenol (a ?-adrenergic receptor agonist) stimulated lipolysis. Furthermore, this potentiation occurred upstream of adenylyl cyclase, since NPY did not enhance forskolin (an activator of adenylyl cyclase) stimulated lipolysis. In addition, NPY also augmented isoproterenol-stimulated phosphorylation of hormone sensitive lipase. In contrast, NPY did not alter the expression of several key lipolytic and lipogenic enzymes/proteins. Taken together, our results revealed a novel cross talk between the NPY and ?-adrenergic signaling pathways in regulating lipolysis. Thus, the present findings add a new dimension to the dynamic role NPY plays in regulating energy balance. PMID:22750277

Li, Raymond; Guan, Haiyan; Yang, Kaiping

2012-10-10

125

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

SciTech Connect

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

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

1987-03-01

126

Withaferin A induces apoptosis and inhibits adipogenesis in 3T3-L1 adipocytes.  

PubMed

Withaferin A (WA), a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera (also called ashwagandha) has been reported to have anti-tumor, anti-angiogenesis, and pro-apoptotic activity. We investigated the effects of WA on viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. Pre- and post-confluent preadipocytes and mature adipocytes were treated with WA (1-25 microM) up to 24 hrs. Viability and apoptosis were measured by CellTiter-Blue Cell Viability Assay and single strand DNA ELISA Assay, respectively. WA decreased viability and induced apoptosis in all stages of cells. Induction of apoptosis by WA in mature adipocytes was mediated by increased ERK1/2 phosphorylation and altered Bax and Bcl2 protein expression. The effect of WA on adipogenesis was examined by AdipoRed Assay after treating with WA (0.1-1 microM) during the differentiation period. WA decreased lipid accumulation in a dose-dependent manner and decreased the expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha and adipocyte fatty acid binding protein. The effects on apoptosis and lipid accumulation were also confirmed with Hoechst staining and Oil Red O staining, respectively. These results show that WA acts on adipocytes to reduce cell viability and adipogenesis and also induce apoptosis. PMID:19346589

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

2008-01-01

127

Echinacea purpurea root extract enhances the adipocyte differentiation of 3T3-L1 cells.  

PubMed

Echinacea purpurea has been shown to have anti-diabetic activities; for example, it activates peroxisome proliferator-activated receptor ? (PPAR?) and increases insulin-stimulated glucose uptake. Adipogenesis has been used to study the insulin signaling pathway and to screen anti-diabetic compounds. The present study was conducted to investigate the effects of an ethanol extract of E. purpurea (EEEP) and its constituents on the insulin-induced adipocyte differentiation of 3T3-L1 preadipocytes. When adipocyte differentiation was induced with insulin plus 3-isobutyl-1-methylxanthine and dexamethasone, the accumulation of lipid droplets and the cellular triglyceride content were significantly increased by EEEP. The expressions of PPAR? and C/EBP? in adipocytes treated with EEEP were gradually increased as compared with control cells. Fat accumulation and triglyceride content of adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly increased as compared with control cells. The expressions of PPAR? and C/EBP? in adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly higher than in control cells. These results suggest EEEP promotes the adipogenesis that is partially induced by insulin and that dodeca-2(E),4(E)-dienoic acid isobutylamide appears to be responsible for EEEP-enhanced adipocyte differentiation. PMID:24085629

Shin, Dong-Mi; Choi, Kyeong-Mi; Lee, Youn-Sun; Kim, Wonkyun; Shin, Kyong-Oh; Oh, Seikwan; Jung, Jae-Chul; Lee, Mi Kyeong; Lee, Yong-Moon; Hong, Jin Tae; Yun, Yeo-Pyo; Yoo, Hwan-Soo

2014-06-01

128

Evidence that downregulation of hexose transport limits intracellular glucose in 3T3-L1 fibroblasts  

SciTech Connect

Measurements of initial glucose entry rate and intracellular glucose concentration in cultured cells are difficult because of rapid transport relative to intracellular volume and a substantial extracellular space from which glucose cannot be completely removed by quick exchanges of medium. In 3T3-L1 cells, we obtained good estimates of initial entry of ({sup 14}C)methylglucose and D-({sup 14}C)glucose with (1) L-({sup 3}H)glucose as an extracellular marker together with the ({sup 14}C)glucose or ({sup 14}C)methylglucose in the substrate mixture, (2) sampling times as short as 2 s, (3) ice-cold phloretin-containing medium to stop uptake and rinse away the extracellular label, and (4) nonlinear regression of time courses. Methylglucose equilibrated in two phases--the first with a half-time of 1.7 s and the second with a half-time of 23 s; it eventually equilibrated in an intracellular space of 8 microliters/mg protein. Entry of glucose remained almost linear for 10 s, making its transport kinetics easier to study (Km = 5.7 mM, Vmax = 590 nmol.s-1.ml-1 cell water). Steady-state intracellular glucose concentration was 75-90% of extracellular glucose concentration. Cells grown in a high-glucose medium (24 mM) exhibited a 67% reduction of glucose-transport activity and a 50% reduction of steady-state ratio of intracellular glucose to extracellular glucose.

Whitesell, R.R.; Regen, D.M.; Pelletier, D.; Abumrad, N.A. (Vanderbilt Univ. School of Medicine, Nashville, TN (USA))

1990-10-01

129

Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts.  

PubMed

Vanadium compounds are shown to have a mitogenic effect on fibroblast cells. The effects of vanadate, vanadyl and pervanadate on the proliferation and morphological changes of Swiss 3T3 cells in culture are compared. Vanadium derivatives induced cell proliferation in a biphasic manner, with a toxic-like effect at doses over 50 microM, after 24 h of incubation. Vanadyl and vanadate were equally potent at 2.5-10 microM. At 50 microM vanadate inhibited cell proliferation, whereas slight inhibition was observed at 100 microM of vanadyl. At 10 microM pervanadate was as potent as vanadate and vanadyl in stimulating fibroblast proliferation, but no effect was observed at lower concentrations. A pronounced cytotoxic-like effect was induced by pervanadate at 50 microM. All of these effects were accompanied by morphological changes: transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation; and loss of lamellar processes. The magnitude of these transformations correlates with the potency of vanadium derivatives to induce a cytotoxic-like effect: pervanadate > vanadate > vanadyl. These data suggest that the oxidation state and coordination geometry of vanadium determine the degree of the cytotoxicity. PMID:9210295

Cortizo, A M; Sálice, V C; Vescina, C M; Etcheverry, S B

1997-04-01

130

Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis.  

PubMed

Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis. PMID:24740755

Krautbauer, Sabrina; Eisinger, Kristina; Hader, Yvonne; Neumeier, Markus; Buechler, Christa

2014-08-01

131

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

PubMed Central

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

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

2014-01-01

132

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

NASA Technical Reports Server (NTRS)

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.

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

1998-01-01

133

A proteomic approach to investigate AuNPs effects in Balb/3T3 cells.  

PubMed

Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell-nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study the effects of AuNPs. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. Of interest was the difference observed in the protein pattern expression of cells exposed to AuNPs. It was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, and inflammatory response. Moreover, SPR analysis showed an increase of ECM proteins biosynthesis in cells exposed to AuNPs. We observed by TEM analysis that NPs are internalized and confined mainly in autophagosomes. Endoplasmic reticulum stressed and modification at mitochondrial level occurred. This study aims to improve existing knowledge necessary for a correct assessment of the balance between AuNPs potential adverse and beneficial effects and might have important implications for biomedical applications (e.g. nanomedicine). To conclude proteomics link to system biology analysis is a valuable tool to understand and predict nanoparticles' toxicity, furthermore it has the potential to reveal pathways that may not be immediately evident with classical toxicological assays. PMID:24780912

Gioria, Sabrina; Chassaigne, Hubert; Carpi, Donatella; Parracino, Antonietta; Meschini, Stefania; Barboro, Paola; Rossi, François

2014-07-15

134

Isoproterenol, TNFalpha, and insulin downregulate adipose triglyceride lipase in 3T3-L1 adipocytes.  

PubMed

Recently, adipose triglyceride lipase (ATGL, also called desnutrin and calcium-independent phospholipase A2 [iPLA(2)] zeta) was isolated as a novel adipose-expressed triglyceride lipase which is downregulated in obesity and may contribute to obesity-associated metabolic disorders such as hyperlipidemia and insulin resistance. To clarify expression and regulation of this fat-derived lipase, ATGL mRNA was measured in 3T3-L1 adipocytes by quantitative real-time reverse transcription-polymerase chain reaction after treatment with isoproterenol, tumor necrosis factor (TNF) alpha, insulin, and growth hormone (GH) which have been shown to influence lipolysis and insulin sensitivity profoundly. Interestingly, treatment of adipocytes with 100 nM isoproterenol, 30 ng/ml TNF alpha, and 100 nM insulin for 16 h significantly decreased ATGL mRNA to 74%, 17%, and 49% of control levels, respectively. GH did not influence ATGL synthesis. The effect of isoproterenol, TNFalpha, and insulin on ATGL expression was time- and dose-dependent. Similarly, HSL mRNA was downregulated by the three hormones. Furthermore, signaling studies suggested that activation of Gs-protein-coupled pathways by forskolin and cholera toxin is sufficient to significantly downregulate ATGL mRNA. Moreover, p44/42 mitogen-activated protein kinase appears to partly mediate the negative effect of insulin but not TNFalpha on ATGL. Taken together, downregulation of ATGL by isoproterenol, TNFalpha, and insulin might contribute to dysregulated expression and function of this lipase in obesity, hyperlipidemia, and insulin resistance. PMID:16009485

Kralisch, Susan; Klein, Johannes; Lossner, Ulrike; Bluher, Matthias; Paschke, Ralf; Stumvoll, Michael; Fasshauer, Mathias

2005-08-30

135

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.

1987-05-01

136

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

PubMed Central

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

2014-01-01

137

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

PubMed

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

2012-08-01

138

Characterization of a cellular RNA that activates PKR in 3T3-F442A cells.  

PubMed

The double-stranded RNA dependent eIF-2 alpha kinase (PKR) has been implicated in the regulation of a number of cellular processes including cell growth and differentiation. Previous studies using embryonic mouse 3T3-F442A cells have indicated that PKR undergoes phosphorylation and activation in vivo. This activation of PKR has been attributed to a subset of poly(A)(+)-rich cellular RNA (R-RNA) having sufficient secondary structure to interact with the kinase. To characterize the R-RNA activity, a cDNA was prepared which, when transcribed in vitro, gave rise to an RNA transcript that retained its property to activate PKR. The ability of the transcript to activate PKR was sensitive to ribonuclease V1 and was abolished by the addition of high concentrations of poly(I).poly(C). The cloned cDNA was utilized for liquid RNA/DNA hybridization experiments, which disrupted the secondary structure of the R-RNA and for Northern blot analysis. The results from these studies indicated that the measurable RRNA activity was represented in a specific cellular RNA which was responsible for the activation of PKR. Furthermore it was found that the R-RNA was specifically associated with PKR and that this complex could be detected directly in cell extracts. The nucleotide sequence of the cDNA was determined. We propose that this novel cellular RNA may play a critical role in regulating the activation of PKR and thus be an important component in the control of cell growth and differentiation. PMID:21528262

Li, J; Yu, Z; Slomiany, D; Petryshyn, R

1997-10-01

139

CREB Activation Induces Adipogenesis in 3T3-L1 Cells  

PubMed Central

Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several “adipocyte-specific” transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues.

Reusch, Jane E. B.; Colton, Lilliester A.; Klemm, Dwight J.

2000-01-01

140

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells.  

PubMed

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

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

2012-04-27

141

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

PubMed

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

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

2010-07-01

142

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

2012-01-01

143

Effect of Black Soybean Koji Extract on Glucose Utilization and Adipocyte Differentiation in 3T3-L1 Cells  

PubMed Central

Adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and progression of diseases such as insulin resistance and obesity. Black soybean koji (BSK) is produced by the fermentation of black soybean with Aspergilllus awamori. Previous study indicated that BSK extract has antioxidative and multifunctional bioactivities, however, the role of BSK in the regulation of energy metabolism is still unclear. We aimed to investigate the effect of glucose utilization on insulin-resistant 3T3-L1 preadipocytes and adipogenesis-related protein expression in differentiated adipocytes with BSK treatment. Cytoxicity assay revealed that BSK did not adversely affect cell viability at levels up to 200 ?g/mL. The potential for glucose utilization was increased by increased glucose transporter 1 (GLUT1), GLUT4 and protein kinase B (AKT) protein expression in insulin-resistant 3T3-L1 cells in response to BSK treatment. Simultaneously, BSK inhibited lipid droplet accumulation in differentiated 3T3-L1 cells. The inhibitory effect of adipogenesis was associated with downregulated peroxisome proliferator-activated receptor ? (PPAR?) level and upregulated Acrp30 protein expression. Our results suggest that BSK extract could improve glucose uptake by modulating GLUT1 and GLUT4 expression in a 3T3-L1 insulin-resistance cell model. In addition, BSK suppressed differentiation and lipid accumulation in mature 3T3-L1 adipocytes, which may suggest its potential for food supplementation to prevent obesity and related metabolic abnormalities.

Huang, Chi-Chang; Huang, Wen-Ching; Hou, Chien-Wen; Chi, Yu-Wei; Huang, Hui-Yu

2014-01-01

144

Mechanisms of resveratrol-induced inhibition of clonal expansion and terminal adipogenic differentiation in 3T3-L1 preadipocytes.  

PubMed

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

2013-11-01

145

Effect of black soybean koji extract on glucose utilization and adipocyte differentiation in 3T3-L1 cells.  

PubMed

Adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and progression of diseases such as insulin resistance and obesity. Black soybean koji (BSK) is produced by the fermentation of black soybean with Aspergilllus awamori. Previous study indicated that BSK extract has antioxidative and multifunctional bioactivities, however, the role of BSK in the regulation of energy metabolism is still unclear. We aimed to investigate the effect of glucose utilization on insulin-resistant 3T3-L1 preadipocytes and adipogenesis-related protein expression in differentiated adipocytes with BSK treatment. Cytoxicity assay revealed that BSK did not adversely affect cell viability at levels up to 200 µg/mL. The potential for glucose utilization was increased by increased glucose transporter 1 (GLUT1), GLUT4 and protein kinase B (AKT) protein expression in insulin-resistant 3T3-L1 cells in response to BSK treatment. Simultaneously, BSK inhibited lipid droplet accumulation in differentiated 3T3-L1 cells. The inhibitory effect of adipogenesis was associated with downregulated peroxisome proliferator-activated receptor g (PPAR?) level and upregulated Acrp30 protein expression. Our results suggest that BSK extract could improve glucose uptake by modulating GLUT1 and GLUT4 expression in a 3T3-L1 insulin-resistance cell model. In addition, BSK suppressed differentiation and lipid accumulation in mature 3T3-L1 adipocytes, which may suggest its potential for food supplementation to prevent obesity and related metabolic abnormalities. PMID:24821545

Huang, Chi-Chang; Huang, Wen-Ching; Hou, Chien-Wen; Chi, Yu-Wei; Huang, Hui-Yu

2014-01-01

146

Red yeast rice stimulates osteoblast proliferation and increases alkaline phosphatase activity in MC3T3-E1 cells.  

PubMed

Red yeast (Monascus purpureus) is used as a traditional hypocholesterolemic dietary food component in Asia due to its bioactive component, lovastatin. Recently, new evidence suggesting that the statins in red yeast enhance bone formation has been reported, but more research is still needed in order to support these claims of osteogenic effects. Therefore, in this study, we hypothesized that red yeast rice (in which red yeast is fermented) can improve osteogenic function through osteoblast cell proliferation and differentiation. We studied the effect of methanol extract of red yeast rice powder (RYRP) on osteoblast proliferation and differentiation by measuring mitochondrial enzyme activity and bone marker alkaline phosphatase (ALP) activity, respectively. Osteoblast-like MC3T3-E1 cells were cultured in various concentrations of RYRP methanol extract (0.001-1 mg/mL) during the osteoblast differentiation period (1, 5, 10, and 15 days). As measured by 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay, RYRP extracts stimulated cell proliferation during a 24-hour period, compared to cooked white rice powder extract. The most pronounced effect was observed at the concentration range between 0.075 and 0.1 mg/mL. This RYRP stimulatory effect for cell proliferation was observed during the whole osteogenic period. Cellular (synthesized) ALP activity was increased at a RYRP extract concentration of 0.075 mg/mL during 15 days of culture, but the medium (secreted) ALP activity did not show any significant change. This cellular ALP activity stimulation by RYRP extract was confirmed by the staining of ALP activity on cell matrix layers for matrix calcification. The results imply that RYRP extract may increase osteogenic effect by stimulating cell proliferation and ALP activity in osteoblastic cells. PMID:20797483

Cho, Young-Eun; Alcantara, Ethel; Kumaran, Santhy; Son, Kun-Ho; Sohn, Ho-Yong; Lee, Jong-Hwa; Choi, Chung-Sig; Ha, Tae-Youl; Kwun, In-Sook

2010-07-01

147

Inhibitory effects of garcinol and pterostilbene on cell proliferation and adipogenesis in 3T3-L1 cells.  

PubMed

The aim of this work was to study the effects of garcinol and pterostilbene on cell proliferation and adipogenesis in 3T3-L1 cells. The results showed that garcinol and pterostilbene decreased the cell population growth and caused cell cycle arrest at the G2/M phase in 3T3-L1 preadipocytes. During adipocyte differentiation, both garcinol and pterostilbene had inhibitory effects on fat droplet formation and triacylglycerol accumulation. The data indicated that garcinol and pterostilbene could inhibit the glycerol-3-phosphate dehydrogenase (GPDH) activity by 97.8 and 61.5%, respectively, as compared to the control. Both garcinol and pterostilbene significantly attenuated the protein expressions of PPAR? and C/EBP? during 3T3-L1 adipocyte differentiation. Moreover, garcinol and pterostilbene caused an inhibition of lipid accumulation in the 3T3-L1 adipocyte differentiation phase. Garcinol and pterostilbene also significantly up-regulated the gene expression of adiponectin as well as down-regulated the gene expressions of leptin, resistin, and fatty acid synthase (FAS) in 3T3-L1 adipocyte differentiation. In 3T3-L1 adipocytes, garcinol significantly down-regulated the protein expressions of PPAR? and FAS as well as up-regulated the protein expressions of adipose triglyceride lipase (ATGL) and adiponectin. Garcinol also significantly up-regulated the gene expression of adiponectin as well as down-regulated the gene expressions of leptin and FAS. These results suggest that garcinol and pterostilbene have anti-adipogenic effects on preadipocytes and adipocytes. PMID:22094440

Hsu, Chin-Lin; Lin, Yu-Jyun; Ho, Chi-Tang; Yen, Gow-Chin

2012-01-01

148

Overexpression of PGC1? improves insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes  

Microsoft Academic Search

The co-transcription factor peroxisome proliferator-activated receptor ? coactivator-1? (PGC-1?) was first identified in 2002.\\u000a Although the function of PGC-1? in white adipose tissue (WAT) is largely unknown, it has been studied extensively in the liver,\\u000a cardiac muscle, and skeletal muscle. Herein, we investigated PGC-1? overexpression in 3T3-L1 adipocytes. The main findings\\u000a were as follows: (i) 3T3-L1 adipocytes overexpressing PGC-1? showed

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

2011-01-01

149

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.

1992-01-01

150

Human lung-derived mature mast cells cultured alone or with mouse 3T3 fibroblasts maintain an ultrastructural phenotype different from that of human mast cells that develop from human cord blood cells cultured with 3T3 fibroblasts.  

PubMed Central

Culture systems designed to maintain or develop human mast cells have proved difficult, yet these systems would provide valuable resources for future investigations of human mast cell biology. Cocultures of either isolated mature human lung mast cells (Levi-Schaffer et al., J Immunol 1987, 139:494-500) or human cord blood mononuclear cells (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043) with 3T3 embryonic mouse skin fibroblasts have implicated fibroblasts as an important factor in the successful maintenance and development of human mast cells in vitro. The authors cultured isolated, mature human lung mast cells either with or without 3T3 cells for 1 month and examined their ultrastructural phenotype. Mast cell viability in each circumstance was equivalent, but mast cell yield was improved in the presence of 3T3 cells. The ultrastructural phenotype was identical in both culture systems. Mast cells were shown to maintain the phenotype of their in vivo lung counterparts (ie, scroll granules predominanted, and numerous lipid bodies were present). This ultrastructural phenotype differs from that of mast cells that develop in cocultures of human cord blood cells and 3T3 cells, where developing mast cells with crystalline granules and few lipid bodies prevail, a phenotype much like that of human skin mast cells in vivo (Furitsu, Proc Natl Acad Sci USA 1989, 86:10039-10043). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Dvorak, A. M.; Furitsu, T.; Estrella, P.; Ishizaka, T.

1991-01-01

151

Robot agua-feeder  

US Patent & Trademark Office Database

This invention generally relates to a fully automatic operation system for feeder and more specifically to an unmanned apparatus which is fully automated so that the self-guiding, charging, and scheduled feed-spraying by the apparatus can be achieved. In accordance with the invention, the cooperation between a plurality of electric eyes and a control unit is utilized to command a plurality of motors for controlling the moving direction of the apparatus. A wharf having a charging device and a position device is also provided so that the batteries in the apparatus can be charged after each navigational turn on the apparatus.

1989-01-24

152

33. INTERIOR VIEW OF FEEDER PREPARING TO INSERT THE FEEDER ...  

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

33. INTERIOR VIEW OF FEEDER PREPARING TO INSERT THE FEEDER ROD INTO ONE OF THE TWO LARGER NAIL OR SPIKE CUTTING MACHINES WHICH PRODUCE THE LARGER SIZE NAILS; NOTE THE OTHER SPIKE CUTTING MACHINE IN THE BACKGROUND - LaBelle Iron Works, Thirtieth & Wood Streets, Wheeling, Ohio County, WV

153

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

SciTech Connect

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

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

2013-03-29

154

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

1987-01-01

155

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

PubMed

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 all sizes, with the groove and ridge widths ranged from 1 to 6microm, but the same groove depth of about 1.6microm. In addition, actin cytoskeleton and nuclei became highly aligned in the microgrooves with and without 4% cyclic stretching. To further examine whether MC3T3-E1 osteoblasts proliferate in microgrooves with cyclic stretching, we grew the cells in six-well silicone dishes containing microgrooves in three wells and smooth surfaces in other three wells. After 4% cyclic stretching for 3, 4, and 7 days, we found that cell numbers in the microgrooves were not significantly different (p>0.05) from those on the smooth surface (p>0.05). Taken together, these results show that MC3T3-E1 osteoblasts can align and proliferate in microgrooves with 4% cyclic stretching. We suggest that the silicone microgrooves can be a useful tool to study the phenotype of MC3T3-E1 osteoblasts under controlled substrate strains. The silicone microgrooves can also be useful for delivering defined substrate strains to other adherent cells in cultures. PMID:10807994

Wang, J H; Grood, E S; Florer, J; Wenstrup, R

2000-06-01

156

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

PubMed Central

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

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

2014-01-01

157

Development of dry coal feeders  

NASA Technical Reports Server (NTRS)

Design and fabrication of equipment of feed coal into pressurized environments were investigated. Concepts were selected based on feeder system performance and economic projections. These systems include: two approaches using rotating components, a gas or steam driven ejector, and a modified standpipe feeder concept. Results of development testing of critical components, design procedures, and performance prediction techniques are reviewed.

Bonin, J. H.; Cantey, D. E.; Daniel, A. D., Jr.; Meyer, J. W.

1977-01-01

158

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

PubMed

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

2013-06-01

159

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

PubMed

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

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

2014-07-01

160

Identification of anti-adipogenic proteins in adult bovine serum suppressing 3T3-L1 preadipocyte differentiation.  

PubMed

Adipocyte differentiation is a complex developmental process forming adipocytes from various precursor cells. The murine 3T3-L1 preadipocyte cell line has been most frequently used in the studies of adipocyte differentiation. Differentiation of 3T3-L1 preadipocytes includes a medium containing fetal bovine serum (FBS) with hormonal induction. In this study, we observed that differentiation medium containing adult bovine serum (ABS) instead of FBS did not support differentiation of preadipocytes. Impaired adipocyte differentiation was due to the presence of a serum protein factor in ABS that suppresses differentiation of preadipocytes. Using a proteomic analysis, alpha-2-macroglobulin and paraoxonase/arylesterase 1, which were previously shown to suppress differentiation of preadipocytes, were identified as anti-adipogenic proteins. Although their functional mechanisms have not yet been elucidated, the anti-adipogenic effects of these proteins are discussed. PMID:24195790

Park, Jeongho; Park, Jihyun; Nahm, Sang-Soep; Choi, Inho; Kim, Jihoe

2013-12-01

161

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

PubMed

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

Hammer, Bruce E; Kidder, Louis S; Williams, Philip C; Xu, Wayne Wenzhong

2009-11-01

162

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

Microsoft Academic Search

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

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

2001-01-01

163

Restoration of adiponectin expression via the ERK pathway in TNF?-treated 3T3-L1 adipocytes.  

PubMed

Adiponectin and tumor necrosis factor?? (TNF??) exert opposite effects on obesity?associated inflammation and insulin signaling. The purpose of the present study was to investigate the effects of chronic TNF?? on adiponectin levels in 3T3?L1 adipocytes, as well as the potential reversal mechanisms. Differentiated 3T3?L1 adipocytes were exposed to TNF?? for three different incubation times and then to various wash?off periods with or without mitogen?activated protein kinase (MAPK) inhibitors. TNF?? significantly reduced adiponectin gene expression in a dose? and time?dependent manner and activated c?Jun N?terminal kinases (JNK), extracellular signal?regulated kinases (ERK) and p38 MAPK. A 16 h restoration period fully reversed the decrease in adiponectin levels following 16 h treatment with TNF??; however, 16 h withdrawal of TNF?? following 32 or 48 h treatment did not completely reverse the TNF???induced decrease in adiponectin levels. In 3T3?L1 adipocytes, 32 or 48 h wash?off periods were required following 32 or 48 h TNF?? treatments, respectively. The pattern of ERK activation following TNF?? exposure and removal was similar to the pattern of adiponectin expression. Furthermore, ERK1/2 inhibition accelerated the recovery of adiponectin levels compared with the levels in the untreated control adipocytes. Therefore, the inhibitory effects of TNF?? on adiponectin levels in differentiated 3T3?L1 cells were fully reversed following a wash?out period equivalent to the TNF?? treatment time, potentially through the ERK 1/2 pathway. PMID:24890319

Chang, Eugene; Choi, Jung Mook; Kim, Won Jun; Rhee, Eun-Jung; Oh, Ki Won; Lee, Won-Young; Park, Se Eun; Park, Sung Woo; Park, Cheol-Young

2014-08-01

164

Mammalian ste20-like kinase and SAV1 promote 3T3-L1 adipocyte differentiation by activation of PPAR?.  

PubMed

The mammalian ste20 kinase (MST) signaling pathway plays an important role in the regulation of apoptosis and cell cycle control. We sought to understand the role of MST2 kinase and Salvador homolog 1 (SAV1), a scaffolding protein that functions in the MST pathway, in adipocyte differentiation. MST2 and MST1 stimulated the binding of SAV1 to peroxisome proliferator-activated receptor ? (PPAR?), a transcription factor that plays a key role in adipogenesis. The interaction of endogenous SAV1 and PPAR? was detected in differentiating 3T3-L1 adipocytes. This binding required the kinase activity of MST2 and was mediated by the WW domains of SAV1 and the PPYY motif of PPAR?. Overexpression of MST2 and SAV1 increased PPAR? levels by stabilizing the protein, and the knockdown of SAV1 resulted in a decrease of endogenous PPAR? protein in 3T3-L1 adipocytes. During the differentiation of 3T3-L1 cells into adipocytes, MST2 and SAV1 expression began to increase at 2 days when PPAR? expression also begins to increase. MST2 and SAV1 significantly increased PPAR? transactivation, and SAV1 was shown to be required for the activation of PPAR? by rosiglitazone. Finally, differentiation of 3T3-L1 cells was augmented by MST2 and SAV1 expression and inhibited by knockdown of MST1/2 or SAV1. These results suggest that PPAR? activation by the MST signaling pathway may be a novel regulatory mechanism of adipogenesis. PMID:22292086

Park, Byoung Hee; Kim, Dae Soon; Won, Gun Woo; Jeon, Hyun Jeong; Oh, Byung-Chul; Lee, YoungJoo; Kim, Eung-Gook; Lee, Yong Hee

2012-01-01

165

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

Microsoft Academic Search

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

Corinth A. Auld; Robin G. Hopkins; Karishma M. Fernandes; Ron F.. Morrison

2006-01-01

166

Dominant-negative C\\/EBP disrupts mitotic clonal expansion and differentiation of 3T3-L1 preadipocytes  

Microsoft Academic Search

Hormonal induction of growth-arrested 3T3-L1 preadipocytes rapidly activates expression of CCAAT\\/enhancer-binding protein (C\\/EBP) . Acquisition of DNA-binding activity by C\\/EBP, however, is delayed until the cells synchronously enter the S phase of mitotic clonal expansion (MCE). After MCE, C\\/EBP activates expression of C\\/EBP and peroxisome proliferator-activated receptor , which then transcriptionally activate genes that give rise to the adipocyte phenotype.

Jiang-Wen Zhang; Qi-Qun Tang; Charles Vinson

2004-01-01

167

Osteoblastic Phenotype Expression of MC3T3-E1 Cultured on Electrospun Polycaprolactone Fiber Mats Filled with Hydroxyapatite Nanoparticles  

Microsoft Academic Search

Electrospun (e-spun) fiber mats of polycaprolactone (PCL; Mn ) 80 000 g mol-1) with or without the presence of hydroxyapatite (HAp) nanoparticles (at 1% w\\/v based on the volume of the PCL solution) were successfully fabricated. The potential for use of these e-spun fiber mats as bone scaffolds was assessed by mouse calvaria- derived pre-osteoblastic cells, MC3T3-E1, in terms of

Patcharaporn Wutticharoenmongkol; Prasit Pavasant; Pitt Supaphol

2007-01-01

168

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

MAYUMI ONO; KUMATO MIFUNE; AKIHIKO YOSHIMURA; SHUN-ICHI OHNISHI; MICHIHIKO KUWANO

1985-01-01

169

Dose-dependent effects of berberine on cell cycle pause and apoptosis in Balb\\/c 3T3 cells  

Microsoft Academic Search

In determining the morphological appearance of Balb\\/c 3T3 cells from berberine-treated (100 and 200 µg\\/ml) cultures by light microscopy demonstrated that the high berberine concentration (200 µg\\/ml) treatment was associated with the accumulation of numerous apoptotic cells, as identified by condensed nuclei and decrease in cell size. On the other hand, accumulation of cells in G2\\/M phase instead of induction

I. Wen Yang; Chih Chung Chou; Benjamin Yat Ming Yung

1996-01-01

170

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

Microsoft Academic Search

Adiponectin is a fat-derived cytokine with anti-diabetic and anti-atherogenic properties. In this study, effects of sulfonylureas (SUs) on adiponectin production and the action mechanism were evaluated using 3T3-L1 adipocytes. The cells were incubated with glimepiride, glibenclamide, gliclazide, pioglitazone, metformin and the medium only as the control. In the control, the adiponectin level evaluated as the production rate per 24h was

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

2008-01-01

171

Insulin regulation of lipoprotein lipase activity in 3T3-L1 adipocytes is mediated at posttranscriptional and posttranslational levels.  

PubMed

Insulin is a major regulator of lipoprotein lipase (LPL) activity. The molecular events associated with LPL regulation by insulin in 3T3-L1 adipocytes were studied by determining LPL enzyme activity, mRNA levels, protein synthetic rate, and transcription run-off activity. Adipocytes treated with insulin (10(-6) M for 48 h) had substantially higher LPL activity (mean difference compared to carrier-treated cells 146%) with little difference in LPL mRNA levels (mean level 109% of control). Insulin regulation of LPL activity was dose-dependent but changes in LPL mRNA were not. Within 2 h of hormone addition, LPL activity was higher in insulin-treated versus carrier-treated adipocytes although their LPL mRNA levels were similar. In [35S]methionine pulse-labeled adipocytes, insulin decreased LPL protein synthetic rate measured by immunoprecipitation 42-48%, although increases (75-340%) in heparin-releasable LPL activity were detected in the same cells. In contrast, during differentiation of 3T3-L1 fibroblasts to the adipocyte state, 5-80-fold increases of heparin-releasable LPL activity were closely associated with similar (8-60-fold) increases in LPL mRNA levels. LPL synthetic rate was 16-fold greater, and LPL gene transcription initiation measured by transcriptional run-off was 10-fold higher in adipocytes than in undifferentiated cells. Differentiation of 3T3-L1 fibroblasts increases transcription of the LPL gene leading to increased LPL mRNA, protein synthetic rate, and enzyme activity. Insulin regulation of LPL activity in 3T3-L1 adipocytes, however, is mediated entirely at posttranscriptional and posttranslational levels. PMID:2656693

Semenkovich, C F; Wims, M; Noe, L; Etienne, J; Chan, L

1989-05-25

172

Dynamic tracking and mobility analysis of single GLUT4 storage vesicle in live 3T3-L1 cells  

Microsoft Academic Search

Glucose transporter 4 (GLUT4) is responsible for insulin-stimulated glucose transporting into the insulin-sensitive fat and muscle cells. The dynamics of GLUT4 storage vesicles (GSVs) remains to be explored and it is unclear how GSVs are arranged based on their mobility. We examined this issue in 3T3-L1 cells via investigating the three-dimensional mobility of single GSV labeled with EGFP-fused GLUT4. A

Chen Hong LI; Li BAI; Dong Dong LI; Sheng XIA; Tao XU

2004-01-01

173

Grape-seed derived procyanidins interfere with adipogenesis of 3T3-L1 cells at the onset of differentiation  

Microsoft Academic Search

OBJECTIVE:Our group's previous results on the effects of a grape seed procyanidin extract (GSPE) on adipose metabolism showed that peroxisome proliferator-activated receptor-? (PPAR?) plays a central role in the lipolytic effects of GSPE on adipocytes. Since PPAR?2 is a main regulator of the differentiation process of adipocytes, we investigated whether GSPE affects the adipogenesis of 3T3-L1 cells.DESIGN:We performed a time

M Pinent; M C Bladé; M J Salvadó; L Arola; H Hackl; J Quackenbush; Z Trajanoski; A Ardévol

2005-01-01

174

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

PubMed Central

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

2013-01-01

175

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

PubMed

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

1996-05-01

176

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

Microsoft Academic Search

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

Joanne M. Reilly; Mary P. Thompson

2000-01-01

177

Visible Light Irradiation of [60]Fullerene Causes Killing and Initiation of Transformation in Balb\\/3T3 Cells  

Microsoft Academic Search

Cell transformation in vitro is a model of carcinogenesis in vivo. Two-stage transformation assay increases the sensitivity of cells to chemicals and permits detection of carcinogens acting as initiating agents. [60]Fullerene (C60) was cytotoxic in BALB\\/3T3 cells when it was irradiated by visible light, but not without light irradiation. Under conditions when C60 was cytotoxic, it acted as an initiating

Ayako Sakai; Yoko Yamakoshi; Naoki Miyata

1999-01-01

178

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

SciTech Connect

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

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

2012-08-03

179

Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells  

SciTech Connect

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)] [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan); Nishio, Hiroaki, E-mail: nishio@fupharm.fukuyama-u.ac.jp [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)] [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)

2010-05-28

180

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

PubMed

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

Lee, Young Soon; Choi, Eun Mi

2014-04-01

181

Pinctada fucata mantle gene 3 (PFMG3) promotes differentiation in mouse osteoblasts (MC3T3-E1).  

PubMed

Nacre is secreted from the mantle of pearl oysters. In vivo and in vitro experiments have demonstrated that water-soluble extracts of nacre stimulate osteoblast differentiation and matrix mineralization, but the component responsible for this activity is unclear. It was reported that Pinctada fucata mantle gene 3 (PFMG3) with an N-terminal signal peptide could be secreted into the nacre of P. fucata. Here we report that PFMG3 is specifically expressed at the outer fold of the mantle and could promote calcium carbonate crystal formation in vitro. Consistent with this observation, we found that matrix mineralization of MC3T3-E1 cells, a murine osteoblast cell line, is accelerated upon treatment with PFMG3. Intriguingly, we observed that alkaline phosphatase activity and cell viability are increased after treating MC3T3-E1 cell with PFMG3. mRNA levels of osteoblast-specific marker genes osteocalcin and osteopontin are also increased. We conclude that PFMG3 from the mantle of P. fucata promotes MC3T3-E1 osteoblast cell differentiation, matrix mineralization, and calcium carbonate deposition in vitro. Our findings provide new evidence that PFMG3 may be used as a potential therapeutic molecule for the treatment of osteoporosis. PMID:21109014

Wang, Xiaoyan; Liu, Shangfeng; Xie, Liping; Zhang, Rongqing; Wang, Zhao

2011-02-01

182

Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells.  

PubMed

Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3-4-fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes. PMID:24513287

Sano, Soichi; Izumi, Yasukatsu; Yamaguchi, Takehiro; Yamazaki, Takanori; Tanaka, Masako; Shiota, Masayuki; Osada-Oka, Mayuko; Nakamura, Yasuhiro; Wei, Min; Wanibuchi, Hideki; Iwao, Hiroshi; Yoshiyama, Minoru

2014-03-01

183

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.

1988-07-01

184

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))

1988-11-01

185

Anti-obesity effect of resveratrol-amplified grape skin extracts on 3T3-L1 adipocytes differentiation  

PubMed Central

Resveratrol (3,4,5-trihydroxy-trans-stilbene), a phytoalexin found in grape skin, grape products, and peanuts as well as red wine, has been reported to have various biological and pharmacological properties. The purpose of this study was to investigate the anti-obesity effect of resveratrol-amplified grape skin extracts on adipocytes. The anti-obesity effects of grape skin extracts were investigated by measuring proliferation and differentiation in 3T3-L1 cells. The effect of grape skin ethanol extracts on cell proliferation was detected by the MTS assay. The morphological changes and degree of adipogenesis of preadipocyte 3T3-L1 cells were measured by Oil Red-O staining assay. Treatment with extracts of resveratrol-amplified grape skin decreased lipid accumulation and glycerol-3-phosphate dehydrogenase activity without affecting 3T3-L1 cell viability. Grape skin extract treatment resulted in significantly attenuated expression of key adipogenic transcription factors, including peroxisome proliferator-activated receptor, CCAAT/enhancer-binding proteins, and their target genes (FAS, aP2, SCD-1, and LPL). These results indicate that resveratrol-amplified grape skin extracts may be useful for preventing obesity by regulating lipid metabolism.

Zhang, Xian-Hua; Huang, Bo; Choi, Soo-Kyong

2012-01-01

186

Proinflammatory cytokine production and insulin sensitivity regulated by overexpression of resistin in 3T3-L1 adipocytes  

PubMed Central

Resistin is secreted from adipocytes, and high circulating levels have been associated with obesity and insulin resistance. To investigate whether resistin could exert autocrine effects in adipocytes, we expressed resistin gene in 3T3-L1 fibroblasts using a lentiviral vector, and selected several stably-transduced cell lines under blasticidin selection. We observed that 3T3-L1 adipocytes expressing resistin have a decreased gene expression for related transcriptional factors (CCAAT/enhancer binding protein ?(C/EBP?) , peroxisome proliferator-activated receptor gamma (PPAR?), and adipocyte lipid binding protein (ALBP/aP2) which is one of target genes for the PPAR? during adipocyte differentiation,. Overexpression of resistin increased the levels of three proinflammatory cytokines, tumor necrosis factor alpha (TNF?), interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), which play important roles for insulin resistance, glucose and lipid metabolisms during adipogenesis. Furthermore, overexpressing resistin in adipocytes inhibits glucose transport 4 (GLUT4) activity and its gene expression, reducing insulin's ability for glucose uptake by 30 %. In conclusion, resistin overexpression in stably transduced 3T3-L1 cells resulted in: 1) Attenuation of programmed gene expression responsible for adipogenesis; 2) Increase in expression of proinflammatory cytokines; 3) Decrease in insulin responsiveness of the glucose transport system. These data suggest a new role for resistin as an autocrine/paracrine factor affecting inflammation and insulin sensitivity in adipose tissue.

Fu, Yuchang; Luo, Liehong; Luo, Nanlan; Garvey, W Timothy

2006-01-01

187

Artepillin C, as a PPAR? ligand, enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells.  

PubMed

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) ? plays an important role in adipocyte differentiation. Its ligands, including thiazolidinediones, improve insulin sensitivity in type 2 diabetes. We investigated the effects of artepillin C, an ingredient of Baccharis dracunculifolia, on adipogenesis and glucose uptake using 3T3-L1 cells. In PPAR? ligand-binding assays, artepillin C exhibited binding affinity toward PPAR?. Artepillin C dose-dependently enhanced adipocyte differentiation of 3T3-L1 cells. As a result of the artepillin C-induced adipocyte differentiation, the gene expression of PPAR? and its target genes, such as aP2, adiponectin and glucose transporter (GLUT) 4, was increased. These increases were abolished by cotreatment with GW9662, a PPAR? antagonist. In mature 3T3-L1 adipocytes, artepillin C significantly enhanced the basal and insulin-stimulated glucose uptake. These effects were decreased by cotreatment with a PI3K inhibitor. Although artepillin C had no effects on the insulin signaling cascade, artepillin C enhanced the expression and plasma membrane translocation of GLUT1 and GLUT4 in mature adipocytes. In conclusion, these findings suggest that artepillin C promotes adipocyte differentiation and glucose uptake in part by direct binding to PPAR?, which could be the basis of the pharmacological benefits of green propolis intake in reducing the risk of type 2 diabetes. PMID:21219874

Choi, Sun-Sil; Cha, Byung-Yoon; Iida, Kagami; Lee, Young-Sil; Yonezawa, Takayuki; Teruya, Toshiaki; Nagai, Kazuo; Woo, Je-Tae

2011-04-01

188

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

2005-01-01

189

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.

2013-01-01

190

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

PubMed

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

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

2012-01-01

191

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

SciTech Connect

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

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

2013-10-11

192

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

2000-01-01

193

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

SciTech Connect

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

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

1987-08-01

194

Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocytes.  

PubMed

Recently, it has been shown that adiponectin is an important insulin-sensitizing fat-derived protein which is downregulated in insulin resistance and obesity, and replenishment of which improves insulin sensitivity. In contrast, interleukin (IL)-6 appears as an adipocytokine serum concentrations of which are elevated in these states. However, it has not been determined whether IL-6 might impact on expression and secretion of adiponectin. To clarify this, 3T3-L1 adipocytes were treated with different concentrations of IL-6 for various periods of time. Adiponectin mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction and secretion was determined by radioimmunoassays. Interestingly, treatment of 3T3-L1 cells with 30 ng/ml IL-6 significantly decreased adiponectin secretion to 75% of control levels. Adiponectin secretion was also inhibited between 25% and 45% by chronic treatment with forskolin (50 microM), tumor necrosis factor alpha (100 ng/ml), and dexamethasone (100 nM). Furthermore, adiponectin mRNA expression was downregulated by up to 50% in a time- and dose-dependent manner, with significant inhibition detectable at concentrations as low as 3 ng/ml IL-6 and as early as 8h after effector addition. The inhibitory effect of IL-6 was partially reversed by pretreatment of 3T3-L1 cells with pharmacological inhibitors of a p44/42 mitogen-activated protein (MAP) kinase. Moreover, the negative effect of IL-6 on adiponectin mRNA expression could be reversed by withdrawal of the hormone for 24h. Taken together, our results suggest that adiponectin gene expression is reversibly downregulated by IL-6 and support the concept of adiponectin being an important selectively controlled modulator of insulin sensitivity. PMID:12589818

Fasshauer, Mathias; Kralisch, Susan; Klier, Margit; Lossner, Ulrike; Bluher, Matthias; Klein, Johannes; Paschke, Ralf

2003-02-21

195

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

NASA Astrophysics Data System (ADS)

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

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

2012-04-01

196

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

SciTech Connect

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

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

2013-06-28

197

Endothelin-1 regulates adiponectin gene expression and secretion in 3T3-L1 adipocytes via distinct signaling pathways.  

PubMed

Adiponectin, which is specifically and highly expressed in adipose tissue, has pleiotropic insulin-sensitizing effects. Endothelin-1 (ET-1) is a potent vasoconstrictive peptide mainly produced by endothelial cells. We previously showed that ET-1 can induce insulin resistance in vitro and in vivo and proposed that it might regulate adiponectin expression and secretion, thus affecting the homeostasis of whole-body energy metabolism. In the present study, we explored the regulatory effects of ET-1 on adiponectin expression and secretion and the underlying mechanisms in 3T3-L1 adipocytes using Northern blotting and ELISA. ET-1 was found to cause a significant time- and dose-dependent decrease in adiponectin expression, and this effect was inhibited by the ET type A receptor (ETAR) antagonist BQ-610 but not by the ETBR antagonist BQ-788. To explore the underlying mechanism, we examined the involvement of the cAMP-dependent protein kinase A-, phospholipase A2-, protein kinase C-, and MAPK-mediated pathways using inhibitors and found that only PD98059 and U0126, inhibitors that blocked MAPK/ERK kinase's ability to activate the ERKs, prevented ET-1-induced down-regulation of adiponectin. Furthermore, acute ET-1 treatment significantly stimulated adiponectin secretion by 3T3-L1 adipocytes, and this effect was inhibited by the ETAR antagonist BQ-610, the inositol-1,4,5-triphosphate receptor blocker 2-APB, and phospholipase C inhibitor U73122, showing that the release of adiponectin stimulated by ET-1 was mediated through the ETAR and the inositol-1,4,5-triphosphate pathway. In conclusion, ET-1 regulates adiponectin expression and secretion by two different signaling pathways in 3T3-L1 adipocytes. These findings suggested that the cardiovascular system affects adipocyte physiology by regulating the expression of adipocytokines and, consequently, energy homeostasis via vasoactive factors, such as ET-1. PMID:17194742

Juan, Chi-Chang; Chuang, Tung-Yueh; Chang, Chih-Ling; Huang, Seng-Wong; Ho, Low-Tone

2007-04-01

198

[Reduced expression of class I MHC antigens in NIH3T3 cells transformed with activated ras oncogenes].  

PubMed

For CTL-mediated lysis of tumor cells the existence of class I molecules is required, in addition to tumor specific antigens, on the cell surface. Therefore, it is likely that tumor cells with altered class I antigen expression might escape from immune surveillance by T-cells. There have been many reports that virally-or chemically-induced murine and human tumor cells and/or tissues expressed reduced number of cell surface class I molecules. However, it is unknown how the regulatory pathway of class I antigen expression and the intracellular oncogenic cascade are interrelated. In this report, class I antigen expressions in NIH3T3 cells transformed with activated ras genes are analyzed. Transformed cells were tumorigenic in allogeneic immunocompetent mice and less sensitive to allogeneic killer T-cells than untransformed NIH3T3. The levels of cell surface expression of H-2K and H-2D region products analyzed using a monoclonal antibodies and a flow-cytometer showed marked reduction of antigen expression in the transformants. Scatchard analysis of the binding of monoclonal antibodies to cell-surface class I molecules indicated that the affinity of the reaction was unchanged after transformation of NIH3T3 cells with activated N-ras oncogene, thus confirmed that the reduced expression observed by flow-cytometric analysis was due to decreased number of the antigen in the transformants. Northern hybridization analyses of H-2 class I and B2-microglobulin transcripts showed that the amounts of both class I and B2-microglobulin mRNAs paralleled with the levels of cell surface expression of the antigen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3500904

Matsushima, S

1987-09-01

199

Microarray analysis of 1alpha,25-dihydroxyvitamin D3-treated MC3T3-E1 cells.  

PubMed

The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], demonstrates potent antiproliferative actions on normal as well as on malignant cell types by blocking the transition from the G1- to the S-phase of the cell cycle. Key target genes for 1,25-(OH)(2)D(3) in this non-classic effect remain largely unknown. Therefore, this study aims to identify genes that, through changes in expression after 1,25-(OH)(2)D(3) treatment, contribute to the observed antiproliferative effect. cDNA microarrays containing 4600 genes were used to investigate changes in gene expression in MC3T3-E1 mouse osteoblasts at 6 and at 12h after treatment with 1,25-(OH)(2)D(3) (10(-8)M), preceding (6h) or coinciding with (12h) the G1/S block in these cells. Approximately one fifth of the genes that were significantly down-regulated after a 12h incubation period with 1,25-(OH)(2)D(3) were genes involved in the DNA replication process, a basic process for cell growth that starts at the end of G1-phase and continues in S-phase. Down-regulation of these genes by 1,25-(OH)(2)D(3) was confirmed by quantitative RT-PCR in MC3T3-E1. In conclusion, cDNA microarrays revealed that treatment of MC3T3-E1 cells with 1,25-(OH)(2)D(3) resulted in the down-regulation of DNA replication genes in parallel with the observed G1/S-arrest. PMID:15225810

Eelen, Guy; Verlinden, Lieve; Van Camp, Mark; Mathieu, Chantal; Carmeliet, Geert; Bouillon, Roger; Verstuyf, Annemieke

2004-05-01

200

Serial passage of MC3T3-E1 cells down-regulates proliferation during osteogenesis in vitro.  

PubMed

Generally, fibroblast-like cells and other types of human cells have been used to demonstrate the principles of replicative senescence in vitro and in vivo. These cells go through three stages of proliferation, including vigorous proliferation, declining proliferation and quiescence or no proliferation. Any variation of this process occurring in osteoprogenitor cells may offer insight into the mechanism of age-related osteopaenia that predisposes individuals to osteoporosis and bone fractures. We selected MC3T3-E1 cells derived from mouse calvaria to study the mechanism of replicative senescence of pre-osteogenic cells because: (i) these cells constitute a well-known model for studying osteogenesis in vitro; (ii) they undergo a developmental sequence of proliferation and differentiation similar to primary cells in culture; and (iii) they show signs of replicative senescence. These cells were aged by multiple passaging before their use for studying growth kinetics and the effects of population density, effect of extracellular matrix (ECM), size and phases of the cell cycle. Our results show that (i) MC3T3-E1 cells go through the first two stages of proliferation in a manner similar to human cells, but escape the quiescent phase; (ii) the rate of proliferation is similar for low passage (LP) and high passage (HP) cells, but is decreased in very high passage cells (VHP); (iii) growth inhibition is observed using HP cells seeded at high density; (iv) HP ECM stimulates proliferation of both LP and HP cells; (v) a small increase in cell size is observed in HP cells, but no change is seen in the distribution analysis of their cell cycle; (vi) distribution analysis of the cell cycle of VHP cells reveals a decreased and an increased frequency of cells in S and G2 + M phases of their cell cycle, respectively. These results suggest that the mouse MC3T3-E1 cell line exhibits many of the cellular and molecular markers associated with replicative senescence in culture as defined by human cells, such as fibroblast-like cells. Alteration in the sensitivity of MC3T3-E1 cells to intercellular contact and increase in cell size are the primary factors contributing to decreased proliferation of HP cells. PMID:15377332

Peterson, W J; Tachiki, K H; Yamaguchi, D T

2004-10-01

201

Endothelin-1 inhibits TNF alpha-induced iNOS expression in 3T3-F442A adipocytes.  

PubMed

1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha-induced iNOS expression. PMID:12839867

Mérial-Kieny, Christelle; Lonchampt, Michel; Cogé, Francis; Verwaerde, Patrick; Galizzi, Jean-Pierre; Boutin, Jean A; Lafontan, Max; Levens, Nigel; Galitzky, Jean; Félétou, Michel

2003-07-01

202

Endothelin-1 inhibits TNF?-induced iNOS expression in 3T3-F442A adipocytes  

PubMed Central

Endothelin-1 (ET-1) and tumor necrosis factor ? (TNF?) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNF? induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNF?-induced iNOS expression and to determine whether or not ET-1 could influence TNF?-induced iNOS expression and NO production. In differentiated 3T3-F442A, treatment with TNF? (20 ng ml?1) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription–polymerase chain reaction and Northern blot analysis. TNF?-induced iNOS expression requires nuclear factor ?B activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38–MAP kinase pathways. ET-1, but not ET-3, inhibited the TNF?-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA2 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNF?, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNF?-induced iNOS expression.

Merial-Kieny, Christelle; Lonchampt, Michel; Coge, Francis; Verwaerde, Patrick; Galizzi, Jean-Pierre; Boutin, Jean A; Lafontan, Max; Levens, Nigel; Galitzky, Jean; Feletou, Michel

2003-01-01

203

Analysis of the cytotoxicity of differentially sized titanium dioxide nanoparticles in murine MC3T3-E1 preosteoblasts  

Microsoft Academic Search

There is an increased use of nanophase titanium dioxide (TiO2) in bone implants and scaffolds. However, nano-debris is generated at the bone-biomaterial interface. Therefore, TiO2 nanoparticles (NPs) of many sizes were investigated for cytotoxic effects on murine MC3T3-E1 preosteoblasts. These TiO2 NPs induced a time- and dose-dependent decrease in cell viability. There was a significant increase in lactate dehydrogenase\\u000a (LDH)

Yilin Zhang; Weiqiang Yu; Xinquan Jiang; Kaige Lv; Shengjun Sun; Fuqiang Zhang

2011-01-01

204

Casein kinase 2 regulates the active uptake of the organic osmolyte taurine in NIH3T3 mouse fibroblasts  

Microsoft Academic Search

Inhibition of the constitutively active casein kinase 2 (CK2) with 2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidasole stimulates\\u000a the Na+-dependent taurine influx via the taurine transporter TauT in NIH3T3 cells. CK2 inhibition reduces the TauT mRNA level and\\u000a increases the localization of TauT to ER but has no detectable effect on TauT protein expression. On the other hand, CK2 inhibition\\u000a increases the affinity of TauT towards

Jack H. Jacobsen; Christian A. Clement; Martin B. Friis; Ian H. Lambert

2008-01-01

205

Influence of sodium hypochlorite treatment of electropolished and magnetoelectropolished nitinol surfaces on adhesion and proliferation of MC3T3 pre-osteoblast cells.  

PubMed

The influence of 6 % sodium hypochlorite (NaClO) treatment on adhesion and proliferation of MC3T3 pre-osteoblast cells seeded on electropolished (EP) and magnetoelectropolished (MEP) nitinol surfaces were investigated. The chemistry, topography, roughness, surface energy, wettability of EP and MEP nitinol surfaces before and after NaClO treatment were studied with X-ray photoelectron spectroscopy (XPS), profilometry, and contact angle meter. In vitro interaction of osteoblast cell and NaClO treated EP and MEP nitinol surfaces were assessed after 3 days of incubation by scanning electron microscopy. The XPS analysis shows that NaClO treatment increases oxygen content especially in subsurface oxide layer of EP and MEP nitinol. The changes of both basic components of nitinol, namely nickel and titanium in oxide layer, were negligible. The NaClO treatment did not influence physico-morphological surface properties of EP and MEP nitinol to a big extent. The osteoblast cells show remarkable adherence and proliferation improvement on NaClO treated EP and MEP nitinol surfaces. After 3 days of incubation they show almost total confluence on both NaClO treated surfaces. The present study shows that NaClO treatment of EP and MEP nitinol surfaces alters oxide layer by enriching it in oxygen and by this improves bone cell-nitinol interaction. PMID:22661249

Rokicki, Ryszard; Haider, Waseem; Hryniewicz, Tadeusz

2012-09-01

206

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: asorisky@ohri.ca

2009-02-01

207

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

PubMed

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

2013-06-28

208

Echinacoside promotes bone regeneration by increasing OPG/RANKL ratio in MC3T3-E1 cells.  

PubMed

Echinacoside (ECH), isolated from Cistanche tubulosa (Schrenk) R. Wight stems, was subjected to in vitro experiments to investigate its bioactivities on proliferation, differentiation and mineralization of the osteoblastic cell line MC3T3-E1. MTT assay, the alkaline phosphatase (ALP) activity and calcium deposition were determined, and the secretion of collagen I (COL I), osteocalcin (OCN), osteoprotegerin (OPG) and receptor activator of nuclear factor-?B ligand (RANKL) were also assayed by enzyme-linked immunosorbent assay (ELISA). The results showed that ECH caused a significant increase in cell proliferation, ALP activity, COL I contents, OCN levels and an enhancement of mineralization in osteoblasts at the concentration range from 0.01 to 10nmol·L(-1) (p<0.05), suggesting that ECH has a stimulatory effect on osteoblastic bone formation or has potential activity against osteoporosis. In addition, the ratio of OPG/RANKL also could be enhanced by ECH. These findings provide the potent evidence that ECH can promote bone regeneration in cultured osteoblastic MC3T3-E1 cells, which might be done by elevating the OPG/RANKL ratio, and potential evidence for echinacoside to be a promising drug or a lead compound in the development of disease-modifying drug to prevent osteoporosis. PMID:22951288

Li, Fei; Yang, Yanan; Zhu, Panpan; Chen, Weina; Qi, Dongli; Shi, Xiupu; Zhang, Chunfeng; Yang, Zhonglin; Li, Ping

2012-12-01

209

Protein kinase C activation enhances cAMP accumulation in Swiss 3T3 cells: inhibition by pertussis toxin.  

PubMed Central

Addition of phorbol 12,13-dibutyrate (PBt2) in the presence of forskolin or cholera toxin caused marked (6- to 8-fold) and rapid accumulation of cAMP in Swiss 3T3 cells. The effect of PBt2 is mediated by protein kinase C because the synthetic diacylglycerol 1-oleoyl-2 acetylglycerol substitutes for PBt2 in enhancing cAMP accumulation and because the enhancing effect of either PBt2 or 1-oleoyl-2-acetylglycerol was prevented by down-regulation of protein kinase C. Vasopressin, which activates protein kinase C but does not directly affect adenylate cyclase in 3T3 cells, also enhanced cAMP accumulation in cells treated with cholera toxin or forskolin. This effect was abolished by down-regulation of protein kinase C. Treatment with pertussis toxin blocked the enhancing effect of PBt2 in a concentration- and time-dependent manner. Pertussis toxin neither prevented protein kinase C activation by PBt2 nor other biologic responses elicited by PBt2. The results presented here suggest an unusual function for a pertussis toxin substrate--namely, coupling protein kinase C activation to cAMP production. Images

Rozengurt, E; Murray, M; Zachary, I; Collins, M

1987-01-01

210

The Mixed-Lineage Kinase DLK Is a Key Regulator of 3T3-L1 Adipocyte Differentiation  

PubMed Central

Background The mixed-lineage kinase (MLK) family member DLK has been proposed to serve as a regulator of differentiation in various cell types; however, its role in adipogenesis has not been investigated. In this study, we used the 3T3-L1 preadipocyte cell line as a model to examine the function of DLK in adipocyte differentiation. Methods and Findings Immunoblot analyses and kinase assays performed on 3T3-L1 cells showed that the expression and activity of DLK substantially increase as differentiation occurs. Interestingly, DLK appears crucial for differentiation since its depletion by RNA interference impairs lipid accumulation as well as expression of the master regulators of adipogenesis C/EBP? and PPAR?2 at both the mRNA and protein levels. In contrast, neither the expression nor the DNA binding activity of C/EBP?, an activator for C/EBP? and PPAR?, is affected by DLK loss. Conclusions Taken together, these results suggest that DLK is important for expression of mature adipocyte markers and that its action most likely takes place via regulation of C/EBP? transcriptional activity and/or initiation of C/EBP? and PPAR?2 gene transcription.

Couture, Jean-Philippe; Daviau, Alex; Fradette, Julie; Blouin, Richard

2009-01-01

211

Upregulation of the thioredoxin-dependent redox system during differentiation of 3T3-L1 cells to adipocytes.  

PubMed

Hydrogen peroxide acts as a signaling molecule in early adipogenesis. In differentiating adipocytes, elevated hydrogen peroxide generation is balanced through induction of antioxidant enzymes such as catalase and peroxiredoxins. Thioredoxin reductases (TrxR) and glutathione peroxidases (GPx) are selenoenzymes that constitute part of the major thiol-dependent antioxidant systems in cells. Here we show that the protein levels of cytoplasmic/nuclear TrxR1 and mitochondrial TrxR2 increase in the course of adipocyte differentiation of 3T3-L1 cells together with the TrxR2 substrate thioredoxin 2 (Trx2), resulting in elevated TrxR activity in mature adipocytes. Gene and protein expression of the GPx isoenzyme GPx4 was also stimulated during adipogenesis. Chronic exposure of 3T3-L1 cells to the anti-adipogenic factors tumor necrosis factor ? (TNF-?) or rapamycin during differentiation suppressed TrxR1 and Trx2 upregulation, concomitantly with inhibition of adipogenesis and lipogenesis. In contrast, TNF-? or rapamycin did not affect expression of TrxRs and their Trx substrates in mature adipocytes. These results indicate that upregulation of the thioredoxin-dependent redox system is linked to the development of an adipocyte phenotype. PMID:24516001

Rajalin, Ann-Marie; Micoogullari, Mustafa; Sies, Helmut; Steinbrenner, Holger

2014-06-01

212

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

SciTech Connect

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

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

2011-11-11

213

Resveratrol Metabolites Modify Adipokine Expression and Secretion in 3T3-L1 Pre-Adipocytes and Mature Adipocytes  

PubMed Central

Objective Due to the low bioavailability of resveratrol, determining whether its metabolites exert any beneficial effect is an interesting issue. Methods 3T3-L1 maturing pre-adipocytes were treated during differentiation with 25 µM of resveratrol or with its metabolites and 3T3-L1 mature adipocytes were treated for 24 hours with 10 µM resveratrol or its metabolites. The gene expression of adiponectin, leptin, visfatin and apelin was assessed by Real Time RT-PCR and their concentration in the incubation medium was quantified by ELISA. Results Resveratrol reduced mRNA levels of leptin and increased those of adiponectin. It induced the same changes in leptin secretion. Trans-resveratrol-3-O-glucuronide and trans-resveratrol-4?-O-glucuronide increased apelin and visfatin mRNA levels. Trans-resveratrol-3-O-sulfate reduced leptin mRNA levels and increased those of apelin and visfatin. Conclusions The present study shows for the first time that resveratrol metabolites have a regulatory effect on adipokine expression and secretion. Since resveratrol has been reported to reduce body-fat accumulation and to improve insulin sensitivity, and considering that these effects are mediated in part by changes in the analyzed adipokines, it may be proposed that resveratrol metabolites play a part in these beneficial effects of resveratrol.

Eseberri, Itziar; Lasa, Arrate; Churruca, Itziar; Portillo, Maria P.

2013-01-01

214

The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells.  

PubMed

To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells. PMID:19138663

Yang, Fang; Nakajima, Yoshihiro; Kumagai, Megumi; Ohmiya, Yoshihiro; Ikeda, Masaaki

2009-02-27

215

Tyrosine phosphorylation and morphological transformation induced by four vanadium compounds on MC3T3E1 cells.  

PubMed

The present study was performed to determine the phosphotyrosine-protein levels induced by insulin and by four vanadium derivatives in MC3T3E1 osteoblast-like cells. We have also attempted to associate these patterns with the vanadium-induced growth and morphological changes of such cells. Vanadate (Vi), vanadyl (VO), bis(maltolato)oxovanadium (IV) (BMOV) and bis(maltolato)dioxovanadium (V) (BMV) stimulate cell growth in a narrow range of concentration, but are also inhibitors for the cells at high concentrations. Vanadium-treated cells displayed clear changes in their morphology after overnight incubation. However, BMV was the least cytotoxic and the weakest inducer of morphological changes. All the compounds promote the phosphorylation of tyrosine residues in several proteins. This effect was more pronounced at low than at high doses. At low doses (10 microM), BMV showed a phosphorylation pattern similar to that of insulin, while Vi, VO and BMOV induced strong phosphorylation of cell proteins. The present findings suggest that the vanadium-induced growth regulation and morphological changes in MC3T3E1 osteoblast-like cells are associated with the ability of these agents to increase the phosphotyrosine protein levels and to inhibit phosphotyrosine phosphatases. These properties are dependent on the oxidation state as well as on the organic ligand which coordinates the vanadium atom. PMID:10497886

Sálice, V C; Cortizo, A M; Gómez Dumm, C L; Etcheverry, S B

1999-08-01

216

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

PubMed

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

2013-01-01

217

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.

2012-01-01

218

Gel microstructure regulates proliferation and differentiation of MC3T3-E1 cells encapsulated in alginate beads.  

PubMed

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 by CaCl(2) solutions, which supplied additional nutrients for the cells compared to water or buffer. The mesh size and shear modulus of the hydrogel were hypothesized to regulate proliferation and differentiation of osteoprogenitor cells. MC3T3-E1 cells demonstrated enhanced osteoblast differentiation when encapsulated in high-density alginate with smaller mesh size and more rigid mechanical properties, as confirmed by increased alkaline phosphatase activity and osteocalcin secretion. However, MC3T3-E1 cells encapsulated in low-density alginate beads with a larger mesh size and more compliant mechanical properties exhibited increased proliferation. These results demonstrate that the microstructure of alginate hydrogels can regulate the behavior of osteoprogenitor cells, thus suggesting that the tuning the properties of the gel may be a useful approach for enhancing new bone formation. PMID:22306825

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

2012-05-01

219

Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells  

SciTech Connect

Diacylglycerol and phorbol esters activate protein kinase C in intact cells. The authors report here that addition of the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) to quiescent cultures of Swiss 3T3 cells caused a marked increase in the rate of ouabain-sensitive YWRb uptake, a measure of the activity of the Na /K pump. The effect was dose-dependent and could be detected after 1 min of exposure to the diacylglycerol. OAG stimulated Na influx via an amiloride-sensitive pathway and increased intracellular pH by 0.15 pH unit. Phorbol 12,13-dibutyrate (PBt2) also enhanced ouabain sensitive YWRb uptake and amiloride-sensitive SSNa influx. Prolonged treatment (40 hr) of 3T3 cells with PBt2 at a saturating dose, which reduces the number of PBt2 binding sites and protein kinase C activity, abolished the ionic response of the cells to a subsequent addition of either OAG or PBt2. They suggest that activation of protein kinase C elicits, either directly or indirectly, enhanced Na /H antiport activity, which, in turn, leads to Na influx, intracellular pH modulation, and stimulation of the Na /K pump.

Vara, F.; Schneider, J.A.; Rozengurt, E.

1985-04-01

220

3T3-L1 Preadipocytes Exhibit Heightened Monocyte-Chemoattractant Protein-1 Response to Acute Fatty Acid Exposure  

PubMed Central

Preadipocytes contribute to the inflammatory responses within adipose tissue. Whilst fatty acids are known to elicit an inflammatory response within adipose tissue, the relative contribution of preadipocytes and mature adipocytes to this is yet to be determined. We aimed to examine the actions of common dietary fatty acids on the acute inflammatory and adipokine response in 3T3-L1 preadipocytes and differentiated mature adipocytes. Gene expression levels of key adipokines in 3T3-L1 preadipocytes and adipocytes were determined following incubation with palmitic acid, myristic acid or oleic acid and positive inflammatory control, lipopolysaccharide for 2 and 4 h. Inflammatory kinase signalling was assessed by analysis of nuclear factor-?B, p38-mitogen-activated protein kinase and c-jun amino-terminal kinase phosphorylation. Under basal conditions, intracellular monocyte chemoattractant protein-1 and interleukin-6 gene expression levels were increased in preadipocytes, whereas mature adipocytes expressed increased gene expression levels of leptin and adiponectin. Fatty acid exposure at 2 and 4 h increased both monocyte chemoattractant protein-1 and interleukin-6 gene expression levels in preadipocytes to greater levels than in mature adipocytes. There was an accompanying increase of inhibitor of ?B-? degradation and nuclear factor-?B (p65) (Ser536) phosphorylation with fatty acid exposure in the preadipocytes only. The current study points to preadipocytes rather than the adipocytes as the contributors to both immune cell recruitment and inflammatory adipokine secretion with acute increases in fatty acids.

Dordevic, Aimee L.; Konstantopoulos, Nicky; Cameron-Smith, David

2014-01-01

221

The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells.  

PubMed

Thrombomodulin (TM) is an anticoagulant endothelial cell surface glycoprotein containing six tandem epidermal growth factor (EGF)-like structures. We prepared a recombinant TM peptide (rTME1-6, from R214GHWA to DSGK466 of native TM) composed of these six EGF-like structures and investigated the effect of rTME1-6 peptide on the growth of the Swiss 3T3 fibroblast cell line. It was found that rTME1-6 induced proliferation of Swiss 3T3 cells and accelerated [3H]thymidine uptake into their DNA. [3H]Thymidine uptake increased in a dose-dependent manner, plateauing at 50 ng/mL rTME1-6, which was 1.8 times the control level. rTME1-6 peptide (50 ng/mL) also accelerated the DNA synthesis of human dermal fibroblasts (HDFs), A549 (a human lung cancer cell line), HepG2 (a human hepatocarcinoma cell line), and U937 cells (a human monocytic cell line) to 1.5, 1.6, 1.4, and 1.2 times the control level, respectively. The magnitude of the acceleration of DNA synthesis in Swiss 3T3 induced by rTME1-6 was approximately 20% of that of EGF on a molar basis. The uptake of [3H]thymidine was accelerated synergistically by coculture of the cells with rTME1-6 and insulin, similar to the coculture with EGF and insulin. The effects of rTME1-6 were abolished by addition of polyclonal antihuman TM IgG, whereas the actions of insulin and EGF were not influenced. Glucose uptake in Swiss 3T3 cells also increased 1.6 times over control levels by culture with 50 ng/mL rTME1-6 (1.25 nmol/L), compared with 2.7 times by 10 ng/mL EGF (1.66 nmol/L). Binding of [125I]EGF (0.5 ng/mL, 0.083 nmol/L) by the cells was inhibited by about 60% by addition of an eight-fold molar excess of nonlabeled EGF (0.664 nmol/L), whereas no inhibition of [125I]EGF binding was observed, even in the presence of a 1,000-fold molar excess (83 nmol/L) of rTME1-6. Specific binding of [125I]rTME1-6 on the cells showed a saturation curve, and the apparent concentration of rTME1-6 required for half maximum binding of the peptide on the cells was calculated to be 31.5 ng/mL. Thus, the overall results indicated that the rTME1-6 peptide had mitogenic activity for Swiss 3T3 cells, accelerated DNA synthesis and glucose uptake, and that the mitogenic activity might be mediated by binding of the peptide to a specific site different from the EGF receptor. PMID:7795228

Hamada, H; Ishii, H; Sakyo, K; Horie, S; Nishiki, K; Kazama, M

1995-07-01

222

Correlation of undermethylation of intracisternal A-particle genes with expression in murine plasmacytomas but not in NIH/3T3 embryo fibroblasts.  

PubMed

We report here comparative studies of DNA methylation and expression of intracisternal A-particle (IAP) genes in murine plasmacytomas, TEPC-15 and MOPC-315, in NIH/3T3 embryo fibroblasts, and in normal liver from young adult BALB/c mice. In Southern blot analysis using methylation-sensitive restriction enzymes, we observed hypomethylation of 4.7- and 5.3-kilobase pair IAP proviruses, and partial undermethylation of other IAP DNA fragments in TEPC-15, MOPC-315, and 3T3 cells, while most of these DNA fragments were hypermethylated in liver. The extent of undermethylation at 12 different sites including the long terminal repeat sequence region and 5'-flanking sequence within one specific IAP gene was found to be similar between 3T3 cells and plasmacytomas. Little or no undermethylation of these sites was found in liver. Hypomethylation of IAP genes, however, is not correlated with their expression in 3T3 cells (as it is found for plasmacytomas), since 3T3 cells, like liver cells, have no detectable transcripts. We also observed hypomethylation of the kappa-light chain gene in 3T3 cells, suggesting that undermethylation may be a generalized phenomenon in these cells. The relationships between gene undermethylation and the control of gene expression in 3T3 cells is discussed. PMID:6091872

Morgan, R A; Huang, R C

1984-11-01

223

Protein kinase C inhibitors enhance the synergistic mitogenic effects of ethanolamine analogues and insulin in NIH 3T3 fibroblasts.  

PubMed

Monomethylethanolamine (1 mM) and dimethylethanolamine (1 mM) stimulated DNA synthesis 10- and 15-fold, respectively, in NIH 3T3 fibroblasts. In addition, simultaneous treatments with insulin (500 nM) and methylated ethanolamine analogues (1 mM or less) resulted in synergistic activation of DNA synthesis. The order of mitogenic potency of ethanolamine analogues was dimethylethanolamine > monomethylethanolamine > ethanolamine. Choline (1-5 mM) alone had no effect on DNA synthesis, but it increased the combined effects of lower concentrations of ethanolamine analogues and insulin. The synergistic effects of ethanolamine analogues, choline and insulin were considerably (1.7- to 1.9-fold) enhanced by GF 109203X (3 microM), a specific inhibitor of protein kinase C. The results suggest that ethanolamine analogues enhance insulin-induced DNA synthesis by a mechanism which is inhibited by the protein kinase C system. PMID:8602830

Kiss, Z; Crilly, K S; Anderson, W B

1996-03-01

224

Biological activity of bovine placental lactogen in 3T3-F442A preadipocytes is mediated through a somatogenic receptor.  

PubMed

Bovine placental lactogen (bPL) exhibited antimitogenic differentiation-promoting biological activity in 3T3-F442A preadipocytes. Competitive binding studies and affinity labelling revealed bPL activity to be mediated through a somatogenic type of receptor that recognizes human growth hormone (hGH) and bovine GH, but not ovine prolactin or human PL. The bioactivity of bPL was sixfold lower than that of hGH despite that bPL is binding to the somatogenic receptors with fivefold higher affinity. This discrepancy may result from the relatively low ability of bPL to induce post-receptoral effects such as receptor dimerization. PMID:1618336

Vashdi, D; Elberg, G; Sakal, E; Gertler, A

1992-06-29

225

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

PubMed

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

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

2011-01-01

226

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

PubMed

White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation. PMID:24413028

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

2014-01-01

227

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

PubMed Central

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

Woll, P. J.; Rozengurt, E.

1988-01-01

228

Biphasic alteration of glucose transport in 3T3-L1 cells during differentiation to the adipocyte-like phenotype.  

PubMed

Glucose transport activity and subcellular distribution of glucose transporters, GLUT1 and GLUT4 were studied in non-confluent (NCF), confluent (CF), and differentiated 3T3-L1 cells (A). During growth of the fibroblasts to confluence, basal transport activity decreased to 20% of that in non-confluent cells. Corresponding with the reduction in transport activity, the abundance of GLUT1 in plasma membranes as normalized per cell decreased by 75% during growth of the cells to confluence. This effect was mainly due to a reduction of total cellular GLUT1. In addition, the portion of GLUT1 located in intracellular vesicles (low-density microsomes) was moderately increased in confluent cells, and was further increased in cells differentiated to the adipocyte-like phenotype (in NCF 11%, in CF 24.5%, and in A 60% of the total GLUT1). GLUT4, in contrast, was approximately 10-times more abundant in low-density microsomes than in the plasma membranes of the differentiated cells. Insulin failed to stimulate glucose transport activity in non-confluent cells but produced an approximately 2-fold stimulation in confluent cells, probably through translocation of the GLUT1 from the intracellular compartment to the plasma membrane. In the differentiated adipocytes, insulin stimulated a 10-fold increase in glucose transport activity, the maximum levels approaching basal transport rates of non-confluent cells; both GLUT1 and GLUT4 were translocated in response to insulin. These data indicate that insulin sensitivity in 3T3-L1 cells develops in a biphasic pattern. In confluent fibroblasts, a moderate effect of insulin conferred exclusively by GLUT1 is detectable, probably reflecting the small intracellular compartment of GLUT1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8458611

Ziehm, D; Schürmann, A; Weiland, M; Joost, H G

1993-02-01

229

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I  

PubMed Central

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly 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. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety.

Bernards, Matthew T.; Qin, Chunlin; Ratner, Buddy D.; Jiang, Shaoyi

2009-01-01

230

Regulation of lysyl oxidase by basic fibroblast growth factor in osteoblastic MC3T3-E1 cells.  

PubMed

Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking. A cross-linked collagenous extracellular matrix is required for bone formation. This study investigated whether lysyl oxidase, like its type I collagen substrate, is down-regulated by basic fibroblast growth factor (bFGF) in osteoblastic MC3T3-E1 cells and determined the degree of post-transcriptional control. Steady-state lysyl oxidase mRNA levels decreased to 30% of control after 24 h of treatment with 1 and 10 nm bFGF. This regulation was time-dependent. COL1A1 mRNA levels declined to less than 10% of control after 24 h of bFGF treatment. Media lysyl oxidase activity decreased consistent with steady-state mRNA changes in cultures that were refed after 24 h of growth factor treatment. Interestingly, treatment of MC3T3-E1 cells with 0.01-0.1 nm bFGF for 24 h and treatment with 1 nm bFGF for up to 12 h resulted in a modest stimulation of lysyl oxidase gene expression and enzyme activity. At least 50% of the down-regulation of lysyl oxidase was shown to be posttranscriptional. New protein synthesis was not required for the down-regulation by bFGF, but cycloheximide did increase constitutive lysyl oxidase mRNA levels 2.5-fold. We conclude that lysyl oxidase and COL1A1 are regulated similarly by bFGF in these osteoblastic cells, consistent with the in vivo effects of this growth factor on bone collagen metabolism. PMID:8626440

Feres-Filho, E J; Menassa, G B; Trackman, P C

1996-03-15

231

Ivy gourd (Coccinia grandis L. Voigt) root suppresses adipocyte differentiation in 3T3-L1 cells  

PubMed Central

Background Ivy gourd (Coccinia grandis L. Voigt) is a tropical plant widely distributed throughout Asia, Africa, and the Pacific Islands. The anti-obesity property of this plant has been claimed but still remains to be scientifically proven. We therefore investigated the effects of ivy gourd leaf, stem, and root on adipocyte differentiation by employing cell culture model. Methods Dried roots, stems, and leaves of ivy gourd were separately extracted with ethanol. Each extract was then applied to 3T3-L1 pre-adipocytes upon induction with a mixture of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone, for anti-adipogenesis assay. The active extract was further fractionated by a sequential solvent partitioning method, and the resulting fractions were examined for their abilities to inhibit adipogenesis in 3T3-L1 cells. Differences in the expression of adipogenesis-related genes between the treated and untreated cells were determined from their mRNA and protein levels. Results Of the three ivy gourd extracts, the root extract exhibited an anti-adipogenic effect. It significantly reduced intracellular fat accumulation during the early stages of adipocyte differentiation. Together with the suppression of differentiation, expression of the genes encoding PPAR?, C/EBP?, adiponectin, and GLUT4 were down-regulated. Hexane-soluble fraction of the root extract also inhibited adipocyte differentiation and decreased the mRNA levels of various adipogenic genes in the differentiating cells. Conclusions This is the first study to demonstrate that ivy gourd root may prevent obesity based mainly on the ability of its active constituent(s) to suppress adipocyte differentiation in vitro. Such an inhibitory effect is mediated by at least down-regulating the expression of PPAR?-the key transcription factor of adipogenesis in pre-adipocytes during their early differentiation processes.

2014-01-01

232

St. John's Wort Promotes Adipocyte Differentiation and Modulates NF-?B Activation in 3T3-L1 Cells.  

PubMed

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

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

2014-01-01

233

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.

WANG, YUFAN; DONG, WEIPING; DING, XIAOYING; WANG, FENG; WANG, YUFEI; CHEN, XINYA; YU, LONG; LI, XIAOHUA; ZHANG, AIFANG; PENG, YONGDE

2012-01-01

234

Helenalin-mediated Post-transcriptional Regulation of p21(Cip1) Inhibits 3T3-L1 Preadipocyte Proliferation  

PubMed Central

We have previously shown that post-transcriptional mechanisms involving the 26S proteasome regulate the cyclin-dependent kinase inhibitors (CKIs), p21(Cip1) and p27(Kip1) during preadipocyte proliferation. Earlier studies further demonstrated that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 accumulation, an F-box protein mediating SCF E3 ligase ubiquitylation and degradation of both CKIs during S phase progression. Data presented here demonstrate that helenalin dose-dependently induced G1 arrest of synchronously replicating 3T3-L1 preadipocytes. This effect occurred in the absence of discernable indices of cell toxicity or apoptosis under the conditions used in this study. Our results demonstrate that helenalin markedly increased p21 protein accumulation in both density-arrested and proliferating preadipocytes in a dose-dependent manner. This increase in p21 protein abundance occurred without change in mRNA transcript demonstrating that post-transcriptional mechanisms were involved. This notion was further supported by the modest accumulation of polyubiquitylated p21 following treatment with helenalin suggesting that suppression of targeted p21 proteolysis by the 26S proteasome contributed to helenalin-mediated p21 accumulation. The increase in p21 protein was compartmentalized to the nucleus where p21 is known to inhibit cell cycle progression. Finally, helenalin increased protein-protein interactions between p21 and cyclin-dependent kinase 2 (Cdk2) which may account in part for the anti-proliferative effect in 3T3-L1 preadipocytes.

Fernandes, Karishma M.; Auld, Corinth A.; Hopkins, Robin G.; Morrison, Ron F.

2008-01-01

235

1-Deoxynojirimycin isolated from a Bacillus subtilis stimulates adiponectin and GLUT4 expressions in 3T3-L1 adipocytes.  

PubMed

We have demonstrated that 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilis MORI could enhance the levels of adiponectin and its receptors in differentiated 3T3-L1 adipocytes, which has been shown to be effective in lowering blood glucose levels and enhancing insulin sensitivity. DNJ was not toxic to differentiated 3T3-L1 adipocytes for up to a concentration of 5 microM. In terms of expression levels of adiponectin and its receptors (AdipoR1 and AdipoR2), DNJ in concentrations as low as 0.5 microM elevated both mRNA and protein levels of adiponectin and transcript levels of AdipoR1 and AdipoR2. In addition, DNJ increased phosphorylation of 5' adenosine monophosphateactivated protein kinase (AMPK) in a statistically significant manner. Finally, treatment with DNJ resulted in increased mRNA expression of glucose transporter 4 (GLUT4), which encodes for a glucose transporter, along with a significant increase in glucose uptake into the adipocytes based on results of a 2-deoxy-D-[3H] glucose uptake assay. Our findings indicate that DNJ may greatly facilitate glucose uptake into adipose tissues by increasing the action of adiponectin via its up-regulated expression as well as its receptor genes. In addition, the glucose-lowering effects of DNJ may be achieved by an increased abundance of GLUT4 protein in the plasma membrane, as a consequence of the increased transcript levels of the GLUT4 gene and the activation of AMPK. PMID:23648852

Lee, Seung-Min; Do, Hyun Ju; Shin, Min-Jeong; Seong, Su-Il; Hwang, Kyo Yeol; Lee, Jae Yeon; Kwon, Ohsuk; Jin, Taewon; Chung, Ji Hyung

2013-05-01

236

Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.  

PubMed

Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), ?1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and ?-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)?2 and C/EBP?. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis. PMID:24626784

Zhang, Juan; Tang, Hongju; Zhang, Yuqing; Deng, Ruyuan; Shao, Li; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

2014-05-01

237

Antidiabetic screening of commercial botanical products in 3T3-L1 adipocytes and db/db mice.  

PubMed

Numerous botanicals are purported to improve glucose metabolism and diabetic risk factors with varying degrees of supportive evidence. We investigated 203 commercially available botanical products representing 90 unique botanical species for effects on lipogenic activity in differentiating 3T3-L1 adipocytes. Anti-inflammatory activity of 21 of these products was further assessed in tumor necrosis factor alpha (TNFalpha)-stimulated, mature 3T3-L1 adipocytes. From these results, rho-isoalpha acids, Acacia nilotica bark, fennel, and wasabi were tested in the db/db mouse model. Fifty-nine percent of the 90 unique botanicals increased adipogenesis as did the standard troglitazone relative to the solvent controls. Botanical species with the greatest percentage of positive products were Centella asiatica, Panax quinquefolius, and Phyllanthus amarus at 100%, Vitis vinifera at 80%, Humulus lupulus at 71%, Aloe barbadensis at 66%, and Momordica charantia, Phaseolus vulgaris, and Punica granatum at 60%. All 21 subset samples inhibited TNFalpha-stimulated free fatty acid release and attenuated TNFalpha inhibition of adiponectin secretion. Both rho-isoalpha acids and A. nilotica reduced nonfasting glucose in the db/db mouse model, whereas A. nilotica also decreased nonfasting insulin levels. A post hoc analysis of the screening results indicated that the positive predictive value of the lipogenesis assay alone was 72%, while adding the criterion of a positive response in the anti-inflammatory assays increased this figure to 82%. Moreover, this large-scale evaluation demonstrates that antidiabetic, in vitro efficacy of botanicals is more a function of manufacturing or quality control differences than the presence of marker compounds and further underscores the need to develop functional as well as analytical bases for standardization of dietary supplements. PMID:20521979

Babish, John G; Pacioretty, Linda M; Bland, Jeffrey S; Minich, Deanna M; Hu, Jeffrey; Tripp, Matthew L

2010-06-01

238

Characterization of human mast cells developed in vitro from fetal liver cells cocultured with murine 3T3 fibroblasts.  

PubMed

Cocultures of dispersed human fetal liver cells with murine Swiss 3T3 fibroblasts resulted in the development of human mast cells after 1 to 4 weeks of culture. Mast cells were detected by immunohistochemistry using a murine monoclonal anti-tryptase antibody, before metachromasia appeared with toluidine blue. When subjected to double immunohistochemistry using murine monoclonal anti-chymase and anti-tryptase antibodies, 94% +/- 10% (SD) of the mast cells seen at day 30 of culture were of the MCT type. These results contrast with those obtained with human mast cells derived from cord blood mononuclear cells cocultured with murine 3T3 fibroblasts which are comprised of substantially greater numbers of MCTC cells, averaging 48% +/- 31% (SD) at day 30 of culture. Mast cells developed in vitro from fetal liver cells or cord blood mononuclear cells contained similar amounts (+/- SD) of histamine (0.9 +/- 0.5 pg/cell and 1.1 +/- 1 pg/cell, respectively) and tryptase (1.7 +/- 0.4 pg/cell and 1.9 +/- 1.2 pg/cell, respectively) on day 30 of culture. Fetal-liver-derived mast cells from a 30-day-old culture were identified by immunoelectron microscopy using gold-labelled antitryptase antibody. Typically, these mast cells appeared immature as they had large nuclear to cytoplasmic ratio and a small number of ill-formed cytoplasmic granules. For both fetal-liver- and cord-blood-derived mast cells, there was no evidence of conversion of the MCT type into the MCTC type provided by this study. These results suggest that commitment to develop as an MCT or MCTC type of mast cell may have occurred in mast cell precursors present in fetal liver and cord blood mononuclear cells, prior to granulation. PMID:1398760

Irani, A A; Craig, S S; Nilsson, G; Ishizaka, T; Schwartz, L B

1992-09-01

239

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts  

SciTech Connect

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of (3H)thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of (3H)thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.

Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y. (Osaka Univ. Medical School (Japan))

1991-03-01

240

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

2003-01-01

241

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

SciTech Connect

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

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

2011-06-17

242

An angiotensin II AT 1 receptor antagonist, telmisartan augments glucose uptake and GLUT4 protein expression in 3T3-L1 adipocytes  

Microsoft Academic Search

Evidence has accumulated that some of the angiotensin II AT1 receptor antagonists have insulin-sensitizing property. We thus examined the effect of telmisartan on insulin action using 3T3-L1 adipocytes. With standard differentiation inducers, a higher dose of telmisartan effectively facilitated differentiation of 3T3-L1 preadipocytes. Treatment of both differentiating adipocytes and fully differentiated adipocytes with telmisartan caused a dose-dependent increase in mRNA

Muneya Fujimoto; Hiroaki Masuzaki; Tomohiro Tanaka; Shintaro Yasue; Tsutomu Tomita; Kayoko Okazawa; Junji Fujikura; Hideki Chusho; Ken Ebihara; Tatsuya Hayashi; Kiminori Hosoda; Kazuwa Nakao

2004-01-01

243

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation  

Microsoft Academic Search

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonprolifer- ating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose trans- porter 1

Amr K. El-Jack; Konstantin V. Kandror; Paul F. Pilch

1999-01-01

244

Murine 3T3-L1 Adipocyte Cell Differentiation Model: Validated Reference Genes for qPCR Gene Expression Analysis  

PubMed Central

Background Analysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL). Methodology/Principal Findings Using three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion. Conclusion Our results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.

Arsenijevic, Tatjana; Gregoire, Francoise; Delforge, Valerie; Delporte, Christine; Perret, Jason

2012-01-01

245

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 ..mu..M 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.

1988-01-01

246

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.

1986-01-01

247

Aurantio-obtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells.  

PubMed

Osteoporosis is one of the major metabolic bone diseases and is among the most challenging noncommunicable diseases to treat. Although there is an increasing interest in identifying bioactive molecules for the prevention and management of osteoporosis, such studies principally focus only on differentiation and mineralization of osteoblasts or inhibition of osteoclast activity. Stimulation of osteoblast migration must be a promising osteoanabolic strategy for improved metabolic bone disease therapy. In this study, we show that an anthraquinone derivative, aurantio-obtusin, stimulated chemotactic migration of MC3T3-E1 osteoblast cells in a concentration-dependent manner. The use of a real-time chemotaxis analyzing system, TAXIScan, facilitated the evaluation of both velocity and directionality of osteoblast migration in response to the compound. Besides migration, the compound stimulated osteoblast differentiation and mineralization. Taken together, the data presented in this paper demonstrate that aurantio-obtusin is a promising osteoanabolic compound of natural origin with potential therapeutic applications in the prevention of osteoporosis and other metabolic bone diseases. PMID:24841966

Vishnuprasad, Chethala N; Tsuchiya, Tomoko; Kanegasaki, Shiro; Kim, Joon Ho; Han, Sung Soo

2014-05-01

248

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

PubMed Central

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

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

2004-01-01

249

The yeast KEX-2-processing endoprotease is active in the Golgi apparatus of transfected NIH 3T3 fibroblasts.  

PubMed

Proteolytic processing of polyprotein precursors at pairs of basic amino acids is a prerequisite for the generation of bioactive peptide hormones. While the mammalian endoproteases responsible for these cleavages are yet to be identified, this function has been unequivocally assigned in yeast to the product of the KEX-2 gene. To study the molecular mechanisms involved in polyprotein processing, we have transfected the yeast KEX-2 gene into mouse NIH 3T3 fibroblasts and established a new cell line (called 2N-DK) where the KEX-2 endoprotease is permanently expressed. Immunofluorescence studies show that the KEX-2 enzyme is retained within the Golgi of the 2N-DK cells. The evidence for this cellular location is supported by measurement of intracellular and extracellular KEX-2 enzyme activity. In this permanently transfected cell line, KEX-2 activity is exclusively intracellular, in contrast to the situation previously described in transiently infected cell lines, where extracellular KEX-2 activity was detected. Furthermore, infection of 2N-DK cells with a recombinant retrovirus expressing a cDNA coding for porcine proopiomelanocortin (POMC) resulted in the synthesis of POMC and its efficient processing into beta-lipotropin and beta-endorphin, two of its physiologically authentic maturation products. These results suggest that in the fibroblast cell line 2N-DK, proteolytic processing of POMC by KEX-2 endoprotease occurs in the Golgi apparatus. PMID:2284001

Germain, D; Zollinger, L; Racine, C; Gossard, F; Dignard, D; Thomas, D Y; Crine, P; Boileau, G

1990-10-01

250

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: ron_morrison@uncg.edu

2006-07-21

251

Inhibition of mitotic clonal expansion mediates fisetin-exerted prevention of adipocyte differentiation in 3T3-L1 cells.  

PubMed

Adipocytes are the key player in adipose tissue inflammation and subsequent systemic insulin resistance and its development involves complex process of proliferation and differentiation of preadipocytes. Fistein, a polyphenol flavonoid, is known to exert anti-inflammatory, anti-carcinogenic and anti-diabetic effects. In this study, we aimed to investigate the effect of fisetin on adipocyte proliferation and differentiation in 3T3-L1 preadipocyte cell line and its mechanism of action. We found that fisetin inhibits adipocyte differentiation in a concentration dependent manner, which were evidenced by Oil Red O staining and the protein expression of mature adipocyte marker genes fatty acid synthase and peroxisome proliferator-activated receptor ?. Moreover, the proliferation of preadipocytes was also markedly suppressed by treatment of fisetin for 24 and 48 h in the differentiation medium. We also found that fisetin inhibition of adipocyte differentiation was largely due to the effect on mitotic clonal expansion. Fisetin suppression of preadipocyte proliferation at early stage of differentiation was accompanied by the changes of expression of a series of cell cycle regulatory proteins. Altogether, our results suggest that the inhibition of adipocyte differentiation by fisetin may be at least in part mediated by cell cycle arrest during adipogenesis. PMID:23918651

Lee, Youngyi; Bae, Eun Ju

2013-11-01

252

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

SciTech Connect

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

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

2008-10-15

253

Inhibition of preadipocyte differentiation and lipid accumulation by Orengedokuto treatment of 3T3-L1 cultures.  

PubMed

Obesity is a major cause of metabolic syndrome and is due to an increase in the number and hypertrophy of adipocytes. Accordingly, inhibition of the differentiation and proliferation of adipocytes may be used in the treatment and prevention of metabolic syndrome. This study investigated the effects of 50 commonly used Kampo medicines on the differentiation of 3T3-L1 preadipocytes to search for a drug with an antiobesity effect. Kampo medicines were screened, and the strongest differentiation-inhibitory effect was noted with Orengedokuto. To explore the active ingredients in Orengedokuto, the effects of four crude drug components of Orengedokuto were investigated. It was found that the differentiation-inhibitory effect of Orengedokuto was accounted for by Coptidis rhizome and Phellodendri cortex. Furthermore, berberine, a principal ingredient common to Coptidis rhizome and Phellodendri cortex, showed a differentiation-inhibitory effect. The effect of berberine involves an inhibition of the mRNA and protein expression of peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer binding protein ? (C/EBP?). Moreover, berberine inhibited lipid accumulation in adipocytes. These findings suggest that an antiobesity effect could be a new indication for Orengedokuto and that its active ingredient is berberine, with a mechanism involving the inhibition of PPAR? and C/EBP? expression. PMID:21557367

Ikarashi, Nobutomo; Tajima, Masataka; Suzuki, Kunihiro; Toda, Takahiro; Ito, Kiyomi; Ochiai, Wataru; Sugiyama, Kiyoshi

2012-01-01

254

Stimulation of osteoblastic differentiation and mineralization in MC3T3-E1 cells by yeast hydrolysate.  

PubMed

In a previous study, it was reported that yeast hydrolysate (YH) was effective in promoting bone growth in Sprague-Dawley (SD) rats. To further clarify the mechanism of YH, the effects of YH on proliferation, differentiation and gene expression in vitro were investigated using osteoblastic cell lines (MC3T3-E1). Cell proliferation increased significantly as much as 110% of the basal value when cells were treated with 100?µg/mL of YH. Alkaline phosphatase (ALP) activity increased significantly with a YH concentration of 25-100?µg/mL, and the activity increased 152% that of the control at 100?µg/mL. The calcium content increased as much as 129% at 100?µg/mL YH. The gene expression levels of ALP and collagen type II (COL II) significantly increased approximately 1.3-fold and 1.7-fold of control, respectively, at 100?µg/mL. YH increased significantly the mRNA level of bone sialoprotein (BSP) but not in a dose-dependent manner. The mRNA levels of bone morphogenetic proteins (BMP)-2, BMP-4, collagen type I (COL I) and osteonectin (ON) did not increase. In summary, YH increased the proliferation of osteoblasts and directly stimulated ALP and bone matrix proteins (e.g. BSP, COL II), and these increases trigger osteoblastic differentiation (e.g. mineralized nodule formation). PMID:21077261

Lee, Hyun-Sun; Jung, Eun-Young; Bae, Song Hwan; Kwon, Ki Han; Kim, Jin-Man; Suh, Hyung Joo

2011-05-01

255

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

PubMed

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

2010-11-23

256

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

PubMed

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

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

2013-01-01

257

A study of the Influence of mevalonic acid and its metabolites on the morphology of swiss 3T3 cells  

PubMed Central

We used two model systems to investigate the effect of compactin, a competitive inhibitor of beta-hydroxy beta-methylglutarylcoenzyme A reductase, on the shape of Swiss 3T3 cells. We maintained cells in a quiescent state in medium deficient in platelet-derived growth factor (PDGF), or we added PDGF to quiescent cells to initiate traverse through a single cell cycle. In both systems, the cells responded to compactin by acquiring a characteristic rounded shape. Cell rounding seemed to depend on an induced deficiency of mevalonic acid (MVA) since the response could be prevented or reversed by adding MVA to the culture medium. Compactin-induced rounding appeared in PDGF-stimulated cells concomitantly with a compactin-mediated inhibition of DNA synthesis, and both effects had similar sensitivities to exogenous compactin and MVA. However, cell rounding seemed to be unrelated to other, previously observed effects of MVA deficiency. Compactin did not influence the total content of cell cholesterol, and little cholesterol was formed when we added radioactive MVA to round cells to effect shape change reversal. Measurement of the dolichol-dependent glycosylation of cell protein revealed no evidence of dolichol deficiency. In addition, reversal of cell rounding by MVA was not prevented by concentrations of tunicamycin that effectively blocked the incorporation of radioactive mannose into cell protein or by concentrations of cycloheximide that blocked protein synthesis. Taken together, our results suggest a new role for MVA or its products in the maintenance of cell shape.

1982-01-01

258

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

PubMed

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

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

2008-07-01

259

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

2012-01-01

260

Effect of Zizyphus jujuba extract on the inhibition of adipogenesis in 3T3-L1 preadipocytes.  

PubMed

Obesity, the leading metabolic disease in the world, is a serious health problem in industrialized countries. The Zizyphus jujuba fruit has been used as traditional Chinese medicinal herb and considered to affect various physiological functions in the body for thousands of years. We investigated the anti-obesity effect of Z. jujuba on adipocyte differentiation of 3T3-L1 preadipocytes and found that treatment with an extract of Z. jujuba suppressed lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity without affecting cell viability. Further fractionation of the initial Z .jujuba extract with organic solvent revealed that the chloroform fraction (CHCl(3)-F) elicited the most inhibitory effect, which involved significant attenuation of the expression of key adipogenic transcription factors, including peroxisome proliferator-activated receptor (PPAR)gamma and CCAAT enhancer binding proteins (C/EBPs) at the protein level. These results suggest that CHCl(3)-F may block adipogenesis, at least in part, by decreasing the expression of PPARgamma, C/EBPalpha and beta. PMID:19606518

Kubota, Hiroaki; Morii, Risako; Kojima-Yuasa, Akiko; Huang, Xuedan; Yano, Yosihisa; Matsui-Yuasa, Isao

2009-01-01

261

A SCANNING ELECTRON MICROSCOPE STUDY OF SURFACE FEATURES OF VIRAL AND SPONTANEOUS TRANSFORMANTS OF MOUSE BALB/3T3 CELLS  

PubMed Central

Cells of the mouse line Balb/3T3 as well as three virus-induced transformants and two spontaneous transformants grown in vitro have been studied for their topography by scanning electron microscopy. The parent cell in confluent culture closely resembles an endothelial cell in its form and in the structure of its association with adjacent cells. The tumorigenic transformants produced by SV40, murine sarcoma virus, or polyoma viruses are fusiform to pleomorphic and distinctly different from the cell of origin. They show relatively smooth surfaces except for blebs and marginal microvilli. Perhaps most surprising is the similarity they bear to one another. This is made the more singular by the very different form shown by the tumorigenic transformants of spontaneous origin. One of these, S2-4, possesses a thickened rather than the lamellar form of the parent A31 cell and is covered by long microvilli and many spherical blebs. The other, TuT3, more closely resembles the cell of origin but shows extensive ruffling at its margins. All transformants grow without evidence of contact inhibition. The significance of the surface morphologies and the factors influencing cell form are discussed.

Porter, Keith R.; Todaro, George J.; Fonte, Virginia

1973-01-01

262

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

NASA Technical Reports Server (NTRS)

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

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

1998-01-01

263

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

PubMed

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

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

1995-10-01

264

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

PubMed Central

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

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

2013-01-01

265

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

PubMed

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

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

2012-01-01

266

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

PubMed

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

2004-02-01

267

NIH-3T3 fibroblast transplants enhance host regeneration and improve spatial learning in ventral subicular lesioned rats.  

PubMed

Transplants, besides providing neural replacement, also stimulate host regeneration, which could serve as a powerful means to establish functional recovery in CNS insults. Earlier, we have reported the H3-GFP transplant mediated recovery of cognitive functions in the ventral subicular lesioned rats. In the present study, we demonstrate the efficacy of a non-neural fibroblast transplants in mediating host regeneration and functional recovery in ventral subicular lesioned rats. Adult male Wistar rats were lesioned with ibotenic acid in the ventral subiculum (VSL) and were transplanted with NIH-3T3 fibroblast cells into CA1 region of the hippocampus. Ventral subicular lesioning impaired the spatial task performances in rats and produced considerable degree of dendritic atrophy of the hippocampal pyramidal neurons. Two months following transplantation, the transplants were seen in the dentate gyrus and expressed BDNF and bFGF. Further, the VSL rats with fibroblast transplants showed enhanced expression of BDNF in the hippocampus and enhanced dendritic branching and increased spine density in the CA1 hippocampal pyramidal neurons. Transplantation of fibroblast cells also helped to establish functional recovery and the rats with transplants showed enhanced spatial learning performances. We attribute the recovery of cognitive functions to the graft mediated host regeneration, although the mechanisms of functional recovery remain to be elucidated. PMID:21074573

Rekha, J; Veena, L R; Prem, Neethi; Kalaivani, P; Choudhury, Rupam; Alladi, Phalguni Anand; Agrahari, Maulishree; Raju, T R; Kutty, Bindu M

2011-04-15

268

Mevalonate deprivation mediates the impact of lovastatin on the differentiation of murine 3T3-F442A preadipocytes.  

PubMed

The statins competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and consequently the synthesis of mevalonate. The use of statins is associated with insulin resistance, presumably due to the impaired differentiation and diminished glucose utilization of adipocytes. We hypothesize that mevalonate is essential to adipocyte differentiation and adipogenic gene expression. Adipo-Red assay and Oil Red O staining showed that an eight-day incubation with 0-2.5?µmol/L lovastatin dose-dependently reduced the intracellular triglyceride content of murine 3T3-F442A adipocytes. Concomitantly, lovastatin downregulated the expression of peroxisome proliferator-activated receptor ? (Ppar?), leptin (Lep), fatty acid binding protein 4 (Fabp4), and adiponectin (AdipoQ) as measured by quantitative real-time polymerase chain reaction (real-time qPCR). The expression of sterol regulatory element binding protein 1 (Srebp-1), a transcriptional regulator of Ppar? and Lep genes, was also suppressed by lovastatin. Western-blot showed that lovastatin reduced the level of CCAAT/enhancer binding protein ? (C/EBP?) while inducing a compensatory over-expression of HMG CoA reductase. The impact of lovastatin on intracellular triglyceride content and expression of the adipogenic genes was reversed by supplemental mevalonate. Mevalonate-derived metabolites have essential roles in promoting adipogenic gene expression and adipocyte differentiation. PMID:24477821

Elfakhani, Manal; Torabi, Sheida; Hussein, Deema; Mills, Nathaniel; Verbeck, Guido F; Mo, Huanbiao

2014-03-01

269

Feeder bus route design problem  

SciTech Connect

The US transit industry is in a financial crisis, caused primarily by the rising operating cost and shrinking resources. Among the deficit-reducing strategies that have been proposed, a better integrated transit system has significant advantages in that it offers the possibility of simultaneously reducing cost and increasing revenue. This research develops an optimization-based methodology for the design of an integrated feeder bus/rail transit system. The proposed methodology includes two types of models, an analytic model and a network model. The study defines the Feeder Bus Route Design Problem (FBRDP) as that of locating bus routes and bus stops as well as determining the service characteristics of a feeder bus system, designed to access an existing rail system. The proposed analytic model provides approximate values for the optimal route density, operating headway and bus stop spacing. Within the network approach, the author considers demand with a single (many-to-one) and with multiple (many-to-many) destinations. The network optimization models for the FBRDP are routing-type models that cannot be solved exactly and need to be solved heuristically. A two-phase heuristic is developed that can be viewed as a generalization of the sequential-savings approach to vehicle routing.

Kuah, G.K.

1986-01-01

270

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.

2013-01-01

271

Ox-LDL Induces ER Stress and Promotes the adipokines Secretion in 3T3-L1 Adipocytes  

PubMed Central

Adipocytes behave as a rich source of adipokines, which may be the link between obesity and its complications. Endoplasmic reticulum (ER) stress in adipocytes can modulate adipokines secretion. The aim of this study is to evaluate the effect of oxidized low density lipoprotein?ox-LDL?treatment on ER stress and adipokines secretion in differentiated adipocytes. 3T3-L1 pre-adipocytes were cultured and differentiated into mature adipocytes in vitro. Differentiated adipocytes were incubated with various concentrations of ox-LDL (0-100 µg/ml) for 48 hours; 50µg/ml ox-LDL for various times (0-48 hours) with or without tauroursodeoxycholic acid (TUDCA) (0-400µM) pre-treatment. The protein expressions of ER stress markers, glucose regulated protein 78(GRP78) and CCAAT/enhancer binding protein [C/EBP] homologous protein (CHOP) in adipocytes were detected by Western blot. The mRNA expressions of visfatin and resistin were measured by real-time PCR and the protein release of visfatin and resistin in supernatant were determined by ELISA. Treatment with ox-LDL could increase the cholesterol concentration in adipocytes. Ox-LDL induced the expressions of GRP78 and CHOP protein in adipocytes and promoted visfatin and resistin secretion in culture medium in dose and time-dependent manner. TUDCA could attenuate the effect of ox-LDL on GRP78 and CHOP expressions and reduce visfatin and resistin at mRNA and protein level in dose-dependent manner. In conclusion, ox-LDL promoted the expression and secretion of visfatin and resistin through its activation of ER stress, which may be related to the increase of cholesterol load in adipocytes.

Chen, Yaqin; Chen, Mingjie; Wu, Zhihong; Zhao, Shuiping

2013-01-01

272

Ox-LDL induces ER stress and promotes the adipokines secretion in 3T3-L1 adipocytes.  

PubMed

Adipocytes behave as a rich source of adipokines, which may be the link between obesity and its complications. Endoplasmic reticulum (ER) stress in adipocytes can modulate adipokines secretion. The aim of this study is to evaluate the effect of oxidized low density lipoprotein (ox-LDL) treatment on ER stress and adipokines secretion in differentiated adipocytes. 3T3-L1 pre-adipocytes were cultured and differentiated into mature adipocytes in vitro. Differentiated adipocytes were incubated with various concentrations of ox-LDL (0-100 µg/ml) for 48 hours; 50 µg/ml ox-LDL for various times (0-48 hours) with or without tauroursodeoxycholic acid (TUDCA) (0-400 µM) pre-treatment. The protein expressions of ER stress markers, glucose regulated protein 78(GRP78) and CCAAT/enhancer binding protein [C/EBP] homologous protein (CHOP) in adipocytes were detected by Western blot. The mRNA expressions of visfatin and resistin were measured by real-time PCR and the protein release of visfatin and resistin in supernatant were determined by ELISA. Treatment with ox-LDL could increase the cholesterol concentration in adipocytes. Ox-LDL induced the expressions of GRP78 and CHOP protein in adipocytes and promoted visfatin and resistin secretion in culture medium in dose and time-dependent manner. TUDCA could attenuate the effect of ox-LDL on GRP78 and CHOP expressions and reduce visfatin and resistin at mRNA and protein level in dose-dependent manner. In conclusion, ox-LDL promoted the expression and secretion of visfatin and resistin through its activation of ER stress, which may be related to the increase of cholesterol load in adipocytes. PMID:24278099

Chen, Yaqin; Chen, Mingjie; Wu, Zhihong; Zhao, Shuiping

2013-01-01

273

Interaction of cinnamic acid derivatives with commercial hypoglycemic drugs on 2-deoxyglucose uptake in 3T3-L1 adipocytes.  

PubMed

Hydroxycinnamic acid derivatives are naturally occurring substances found in fruits, vegetables, and flowers and are consumed as dietary phenolic compounds. The effect of cinnamic acid, ferulic acid, p-coumaric acid, eugenol, chlorogenic acid, and caffeic acid, alone and in combination with two commercial oral hypoglycemic drugs (OHD), namely, thiazolidinedione (THZ) and metformin, on the uptake of 2-deoxyglucose (2DG) by 3T3-L1 adipocytes is studied. All of the phytochemicals other than cinnamic acid show synergistic interaction in 2DG uptake with both of the OHDs. THZ (20 ?M) in combination with ferulic acid (25 ?M) or p-coumaric acid (25 ?M) increases 2DG uptake by 7- or 6.34-fold, respectively, with respect to control, whereas metformin (20 ?M), along with ferulic acid (25 ?M) or cinnamic acid (25 ?M), increases 2DG uptake by 6.45- or 5.87-fold, respectively, when compared to control. Chlorogenic and cinnamic acids increased the expression of PPAR?, whereas other hydroxycinnamic acids enhanced the expression of PI3K, indicating different mechanisms of action between these compounds. These phytochemicals were able to reduce the expressions of the fatty acid synthase and HMG CoA reductase genes, indicating that they may be able to reduce the secondary complications caused by the accumulation of lipids. These studies suggest that hydroxycinnamic acid derivatives may be beneficial for the treatment of diabetes mellitus. They may act as a supplement with commercial drugs and may reduce the secondary complications caused by OHDs. PMID:21870829

Prabhakar, Pranav Kumar; Doble, Mukesh

2011-09-28

274

Characterization of a platelet-derived growth factor receptor on Swiss 3T3 cells by affinity crosslinking.  

PubMed

Crosslinking experiments with various bifunctional reagents were used to investigate the nature and fate of the platelet growth factor (PDGF) receptor on Swiss mouse 3T3 cells. With ethylene glycol bis succinimidyl succinate (EGS) two bands with Mr 205,000 and Mr 190,000 were labeled at equal intensity, while with disuccinimidyl suberate (DSS) and the photoactivatable p-azidophenylglyoxal (pAPG) almost exclusively the latter band was labeled, when analyzed by SDS polyacrylamide gel electrophoresis under reducing conditions. Evidence is presented that the Mr 190,000 band represents a Mr 175,000 receptor protein crosslinked to a single chain of the PDGF-dimer and the Mr 205,000 species the same Mr 175,000 protein crosslinked to both chains of PDGF. Pretreatment of cells with tunicamycin generated a third labeled band with Mr 150,000, while pretreatment with neuraminidase resulted in a shift of the Mr 205,000 and 190,000 bands by 5,000. This shows that the PDGF receptor is a sialoglycoprotein, consisting of a Mr approximately 135,000 proteinaceous core and a Mr approximately 40,000 carbohydrate moiety containing sialic acid. The virtually unchanged labeling intensity seen with tunicamycin and neuraminidase pretreated cells further suggests that the carbohydrate portion of the receptor is not required for PDGF binding. Finally, the crosslinking technique was used to show that at 37 degrees C performed 125I-PDGF receptor complexes disappear from the cell surface with a t1/2 approximately 8 min. PMID:2838625

Hosang, M

1988-01-01

275

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.

1995-08-01

276

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors  

PubMed Central

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38? cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 ± 5.2 and 50.5 ± 8.0 pmol/mg protein). P2Y receptor stimulation of CD38? cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave.

Bruzzone, Santina; Kunerth, Svenja; Zocchi, Elena; De Flora, Antonio; Guse, Andreas H.

2003-01-01

277

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

PubMed Central

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

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

2014-01-01

278

Visfatin is involved in TNF?-mediated insulin resistance via an NAD+/Sirt1/PTP1B pathway in 3T3-L1 adipocytes  

PubMed Central

Tumor necrosis factor ? (TNF?) is a well-known mediator of inflammation in the context of obesity in adipose tissue. Its action appears to be directly linked to perturbations of the insulin pathway, leading to the development of insulin resistance. Visfatin has been suspected to be linked to insulin sensitivity, but the mechanism involved is still partly unknown. The aim of this study was to evaluate the role of visfatin in the impairment of the insulin pathway by TNF? activity in 3T3-L1 adipocytes and to unveil the mechanisms involved in such impairment. We demonstrated in 3T3-L1 adipocytes that visfatin was involved in TNF?-mediated insulin resistance in adipocytes. Indeed, after TNF? treatment in 3T3-L1 cells, visfatin was downregulated, leading to decreased nicotinamide adenine dinucleotide (NAD+) concentrations in cells. This decrease was followed by a decrease in Sirt1 activity, which was linked to an increase in PTP1B expression. The modulation of PTP1B by visfatin was likely responsible for the observed decreases in glucose uptake and Akt phosphorylation in 3T3-L1 adipocytes. Here, we demonstrated a complete pathway involving visfatin, NAD+, Sirt1, and PTP1B that led to the perturbation of insulin signaling by TNF? in 3T3-L1 adipocytes.

Gouranton, Erwan; Romier, Beatrice; Marcotorchino, Julie; Tourniaire, Franck; Astier, Julien; Peiretti, Franck; Landrier, Jean-Francois

2014-01-01

279

Effect of miR-205 on 3T3-L1 preadipocyte differentiation through targeting to glycogen synthase kinase 3 beta.  

PubMed

MiR-205 plays an important role during adipogenesis by modulating the Wnt signaling pathway. Here, we report that miR-205 can regulate the differentiation of 3T3-L1 preadipocyte cells by targeting glycogen synthase kinase 3 beta (GSK-3?), which is a negative regulatory factor of Wnt signaling. When transiently overexpressed in 3T3-L1 cells, miR-205 suppressed the translation of GSK-3?, resulting in increased expression of ?-catenin, which can promote cell proliferation by facilitating the transcription of the Wnt target genes cyclin D1 and c-Myc. However, stable overexpression of miR-205 in 3T3-L1 cells did not show any apparent inhibitory effect on adipogenic differentiation. While endogenous miR-205 was inhibited in 3T3-L1 cells, the adipogenesis marker gene, C/EBP?, was significantly activated and more lipid droplets appeared in differentiated adipocytes. However, systemic silencing of miR-205 in mice by using a locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) did not lead to any observable increase in adipose tissue differentiation, implying that, as opposed to the findings from 3T3-L1 cells, miR-205 is dispensable for adipose tissue development in mice. PMID:24563321

Yu, Jingwei; Chen, Yaosheng; Qin, Limei; Cheng, Luxi; Ren, Guangcai; Cong, Peiqing; Mo, Delin; He, Zuyong

2014-06-01

280

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.

2011-01-01

281

MicroRNA-344 inhibits 3T3-L1 cell differentiation via targeting GSK3? of Wnt/?-catenin signaling pathway.  

PubMed

Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of complex events in which microRNAs might play an essential role. In this study, we found that the overexpression of microRNA-344 (miR-344) inhibits 3T3-L1 cell differentiation and decreases triglyceride accumulation after MDI stimulation. We demonstrated that miR-344 directly targets the 3' UTR of GSK3? (Glycogen synthase kinase 3 beta). Knockdown of GSK3? with siRNA results in inhibiting 3T3-L1 differentiation, while its overexpression restores the effect of miR-344. In addition, miR-344 elevates the level of active ?-catenin, which is the downstream effector of GSK3? in the Wnt/?-catenin signaling pathway. These data indicate that miR-344 inhibits adipocyte differentiation via targeting GSK3? and subsequently activating the Wnt/?-catenin signaling pathway. PMID:24333578

Chen, Hu; Wang, Siqi; Chen, Luxi; Chen, Yaosheng; Wu, Ming; Zhang, Yun; Yu, Kaifan; Huang, Zheng; Qin, Lijun; Mo, Delin

2014-01-31

282

Is your bird feeder safe?  

USGS Publications Warehouse

Bird feeding is a popular activity for millions of Americans. Some of our favorite bird species commonly visit bird feeders and these stations may be an important factor in their well-being during some segments of their life-cycle. However, poorly maintained feeding stations may contribute to the occurrence of infectious disease and mortality. In recent years there have been unprecedented reports of songbird mortality events and the occurrence of a previously unreported disease in songbirds. The National Wildlife Health Center of the U.S. Geological Survey conducts research on diseases in wildlife, their causes, and means of preventing or reducing disease outbreaks.

Ruth, J.; Friend, M.

1997-01-01

283

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

SciTech Connect

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

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

2012-11-23

284

Disruption of the vimentin intermediate filament system during adipose conversion of 3T3-L1 cells inhibits lipid droplet accumulation.  

PubMed

During the differentiation of 3T3-L1 pre-adipocytes, vimentin intermediate filaments are reorganized to form cage-like structures around the nascent lipid droplets. Initial studies with 3T3-L1 cells indicated that aggregation of vimentin filaments by nocodazole treatment during or shortly after induction of adipose conversion dramatically reduced the lipid droplet content of 3T3-L1 cells 96-120 hours after induction. Specific but transient disruption of vimentin following anti-IFA antibody injection also resulted in a decrease in lipid droplet formation in differentiating cells. To specifically and stably affect filament organization, 3T3-L1 cells lines were established by transfection with a glucocorticoid-regulatable, dominant negative mutant vimentin cDNA expression plasmid. Treatment of these cells (83 delta C) with dexamethasone resulted in expression of vimentin with a carboxyl-terminal deletion, which led to the disruption of the endogenous filament network. Induction of adipose conversion in 83 delta C cells lead to the formation of lipid droplets comparable to those seen in untransfected 3T3-L1 cells. Addition of dexamethasone during the adipose conversion of 83 delta C cells did not affect the induction of the marker enzyme glycerol-3-phosphate dehydrogenase or the incorporation of [14C]palmitate into triglycerides during a 10 minute pulse label. There was, however, a failure to form prominent lipid droplets and to accumulate [14C]palmitate-labeled triglycerides. Pulse-chase experiments indicated that the failure of these cells to accumulate triglyceride was associated with an increased rate of turnover. These studies indicate that vimentin filaments provide a function that influences lipid stability during adipose conversion of 3T3-L1 cells. PMID:9004039

Lieber, J G; Evans, R M

1996-12-01

285

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

2009-01-01

286

Real-time monitoring of 3T3-L1 preadipocyte differentiation using a commercially available electric cell-substrate impedance sensor system.  

PubMed

Real-time analysis offers multiple benefits over traditional end point assays. Here, we present a method of monitoring the optimisation of the growth and differentiation of murine 3T3-L1 preadipocytes to adipocytes using the commercially available ACEA xCELLigence Real-Time Cell Analyser Single Plate (RTCA SP) system. Our findings indicate that the ACEA xCELLigence RTCA SP can reproducibly monitor the primary morphological changes in pre- and post-confluent 3T3-L1 fibroblasts induced to differentiate using insulin, dexamethasone, 3-isobutyl-1-methylxanthine and rosiglitazone; and may be a viable primary method of screening compounds for adipogenic factors. PMID:24388983

Kramer, Adam H; Joos-Vandewalle, Julia; Edkins, Adrienne L; Frost, Carminita L; Prinsloo, Earl

2014-01-24

287

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

288

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

2002-01-01

289

Evaluation and Test on Flat Conductor Feeders.  

National Technical Information Service (NTIS)

The test was conducted to determine if flat conductor cable (FCC) could meet the electrical requirements of a 3 phase AC power feeder system. Flat cable feeders were designed, procured and tested for capability of transmitting 90KVA power in an installati...

J. P. Morris

1970-01-01

290

Lipopolysaccharides reduce adipogenesis in 3T3-L1 adipocytes through activation of NF-?B pathway and downregulation of AMPK expression.  

PubMed

Lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria serve as endotoxin to exert potent immune responses. However, the effect of LPS on adipogenesis has not been elucidated. The present study was designed to examine the effect of LPS on adipogenesis in 3T3-L1 preadipocytes and possible mechanism(s) of action involved. Our results revealed that LPS challenge significantly suppressed adipogenesis in 3T3-L1 preadipocytes mainly through downregulated expression of the late adipogenic markers PPAR? and aP2 as well as AMP-activated protein kinase (AMPK) expression and activity. As an inflammatory factor, LPS was found to lead to an overt reduction in I?B? levels compared with the time-matched controls, consolidating its pro-inflammatory property in 3T3-L1 preadipocytes. Our data also revealed that LPS retarded adipogenesis, the effect of which was partially reversed by the selective inhibitor of IKK?. I?B? was found to be involved in the anti-adipogenic effect of LPS. In conclusion, LPS is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through activation of NF-?B and inhibition of AMPK. With the activation of NF-?B pathway and inhibition of AMPK, LPS suppresses C/EBP ? DNA-binding activity and the expression of late adipogenic markers PPAR? and aP2. PMID:23686584

Wang, Lifeng; Li, Linlin; Ran, Xinjian; Long, Mei; Zhang, Minfang; Tao, Yicun; Luo, Xin; Wang, Ye; Jiao, Yi; Mao, Xinmin; Ren, Jun

2013-12-01

291

BALB/3T3 cells infected by the ts3 mutant of polyoma virus fail to accumulate virus-specific early RNA at the nonpermissive temperature.  

PubMed Central

We measured the accumulation of virus-specific early RNA in BALB/3T3 cells infected by the ts3 mutant of polyoma virus by annealing cytoplasmic RNA from infected cells to purified, radiolabeled, "early" strand of polyoma DNA. Cells infected by the ts3 mutant fail to accumulate virus-specific early RNA at the nonpermissive temperature.

Cogen, B; Eckhart, W

1977-01-01

292

Tissue Inhibitor of Metalloproteinase-1 Promotes NIH3T3 Fibroblast Proliferation by Activating p-Akt and Cell Cycle Progression  

PubMed Central

Tissue inhibitor of metalloproteinase-1 (TIMP-1) plays various roles in cell growth in different cell types. However, few studies have focused on TIMP-1’s effect on fibroblast cells. In this study, we investigated the effects of TIMP-1 overexpression on NIH3T3 fibroblast proliferation and potential transduction signaling pathways involved. Overexpression of TIMP-1, by transfection of the pLenti6/ V5-DEST-TIMP-1 plasmid, significantly promoted NIH3T3 proliferation as determined by the BrdU array. Neither 5 nor 15 nM GM6001 (matrix metalloproteinase system inhibitor) affected NIH3T3 proliferation, but 45 nM GM6001 inhibited proliferation. TIMP-1 overexpression activated the p-Akt pathway, but not the p-ERK or p-p38 pathway. In TIMP-1-transfected cells, cyclinD1 was upregulated and p21CIP1 and p27KIP1 were downregulated, which promoted cell entry into the S and G2/M phases. The PI3-K inhibitor LY294002 abolished the TIMP-1-induced effects. Overex-pression of intracellular TIMP-1 stimulated NIH3T3 fibroblast proliferation in a matrix metalloproteinase (MMP)-indepen-dent manner by activating the p-Akt pathway and related cell cycle progression.

Lu, Yang; Liu, Shuxin; Zhang, Shujia; Cai, Guangyan; Jiang, Hongwei; Su, Huabin; Li, Xiaofan; Hong, Quan; Zhang, Xueguang; Chen, Xiangmei

2011-01-01

293

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

PubMed Central

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

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

2013-01-01

294

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

PubMed

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

2013-09-01

295

GPER mediates the inhibitory actions of estrogen on adipogenesis in 3T3-L1 cells through perturbation of mitotic clonal expansion.  

PubMed

G-protein-coupled estrogen receptor 1 (GPER) mediates non-genomic signaling of estrogenic events. Here we showed for the first time that Gper/GPER is expressed in Swiss 3T3 mouse embryo preadipocytes 3T3-L1, and that Gper/GPER is up-regulated during differentiation of the cells induced by monocyte differentiation-inducing (MDI) cocktail. Activation of GPER by the natural ligand 17?-estradiol (E2), and the specific agonist G1, was shown to inhibit lipid accumulation in 3T3-L1 cells, while such inhibition was reversed upon knockdown of GPER using specific siRNA. GPER was also found to mediate perturbation of mitotic clonal expansion (MCE) in these cells by inhibiting cell cycle arrest during MDI cocktail-induced differentiation. Persistent activation of cell cycle regulating factors cyclin-dependant kinase (CDK) 4, CDK6 and cyclin D1, and phosphorylation of retinoblastoma (Rb) protein at serine 795 was observed in the G1-treated cells. Taken together, our results indicate that E2-GPER signaling leads to an inhibition of adipogenesis in 3T3-L1 cells via perturbation of MCE. PMID:23871778

Zhu, Pei; Yuen, Jacky M L; Sham, Kathy W Y; Cheng, Christopher H K

2013-11-01

296

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

SciTech Connect

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

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

2012-03-02

297

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))

1990-01-01

298

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.

JIN, LIFANG; LI, FAN; WANG, HUIPING; LI, YUNHUA; WEI, FANG; DU, LIANFANG

2013-01-01

299

Stimulatory effects of extract prepared from the bark of Cinnamomum cassia blume on the function of osteoblastic MC3T3-E1 cells.  

PubMed

The ethanol extract from the bark of Cinnamomum cassia Blume (CCE) was tested for estrogenic activity. CCE (4-60 microg/mL) significantly induced the growth of MCF-7 cells, an ER-positive human breast cancer cell line, over that of untreated control cells (p < 0.05). In the ER competitive binding assay, CCE showed higher affinity with ERbeta compared with ERalpha. To investigate the bioactivities of CCE, which act on bone metabolism, the effects of CCE on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were studied. CCE (4-60 microg/mL) dose-dependently increased the survival of MC3T3-E1 cells. In addition, CCE (10 and 50 microg/mL) increased alkaline phosphatase (ALP) activity, collagen synthesis and osteocalcin secretion in MC3T3-E1 cells. Treatment with CCE (10 and 50 microg/mL) prevented apoptosis induced by TNF-alpha (10(-10) m) in osteoblastic cells. In the presence of TNF-alpha, culture with CCE (10-100 microg/mL) for 48 h inhibited the production of IL-6 and nitric oxide in osteoblastic MC3T3-E1 cells. These results suggest that Cinnamomum cassia has a direct stimulatory effect on bone formation in vitro and may contribute to the prevention of osteoporosis and inflammatory bone diseases. PMID:16906639

Lee, Kyung-Hee; Choi, Eun-Mi

2006-11-01

300

Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes.  

PubMed

Fatty acid binding protein 3 (H-FABP, FABP3) has been significantly associated with intramuscular fat (IMF) content in pigs, which is positively correlated with palatability of pork. However, its underlying function is not fully elucidated. We have investigated the effects of overexpression of the FABP3 gene on differentiation and adipogenesis of 3T3-L1 preadipocytes in the fat Banna mini-pig inbred line (fBMIL). Eukaryotic vectors that expressed the FABP3 protein were constructed, and stably established in the 3T3-L1 preadipocytes cell line. Cells were induced in a standard differentiation cocktail. Morphological changes and the degree of adipogenesis were measured by Oil Red O staining assay and triacylglycerol content measurement, respectively. mRNA expression levels of triacylglycerol metabolism-related genes were measured by qPCR. FABP3 significantly promoted differentiation of 3T3-L1 cells and enhanced triacylglycerol levels (P?3T3-L1 preadipocytes primarily by upregulating lipogenic PPAR?, 422/aP2 and GPDH genes. PMID:24737696

Yi, Bao; Wang, Jigui; Wang, Shuang; Yuan, Daoli; Sun, Jiazeng; Li, Zhili; Mao, Yaping; Hou, Qiang; Liu, Weiquan

2014-08-01

301

Modulation of adipogenesis, lipolysis and glucose consumption in 3T3-L1 adipocytes and C2C12 myotubes by hydroxytyrosol acetate: a comparative study.  

PubMed

Hydroxytyrosol acetate (Hd-Ac) is a polyphenol that is present in the olive fruit and oil at a concentration similar to that of hydroxytyrosol (Hd). The effects of Hd-Ac on adipogenesis, lipolysis, and glucose consumption in 3T3-L1 cells were investigated. Treatment with Hd-Ac at concentrations of 0-75 ?mol/L inhibited 3T3-L1 differentiation and lipid accumulation in a dose-dependent manner. At the same concentration range, no effect on cell viability was observed in the MTT assay. Inhibition of adipogenesis was associated with the downregulation of PPAR?, C/EBP?, SREBP-1c, and their downstream target genes (GLUT4, CD36, and FAS) as revealed by qRT-PCR. On the other hand, Hd-Ac dose dependently activated glycerol release in fully differentiated 3T3-L1 adipocytes, indicating lipolysis. This stimulation of lipolysis was mediated via the activation of hormone-sensitive lipase (HSL) by phosphorylation at Ser563 and Ser660, and the phosphorylation of perilipin. Further investigation of the in vitro activities of this polyphenol showed that Hd-Ac has the capability to increase glucose consumption in 3T3-L1 adipocytes and C2C12 myotubes. PMID:24099771

Drira, Riadh; Sakamoto, Kazuichi

2013-11-01

302

Inhibitory effects of the constituents of Hippophae rhamnoides on 3T3-L1 cell differentiation and nitric oxide production in RAW264.7 cells.  

PubMed

Three new flavonol glycosides, hippophaeosides A-C (1-3), together with 27 known constituents, were isolated from Hippophae rhamnoides L. leaves. Their structures were determined by spectroscopic analyses. Their inhibitory activities on 3T3-L1 preadipocyte differentiation and triglyceride accumulation in maturing adipocytes, and nitric oxide production in RAW264.7 cells were examined. PMID:23449196

Yang, Zhi-Gang; Wen, Xiu-Feng; Li, Yong-Hai; Matsuzaki, Keiichi; Kitanaka, Susumu

2013-01-01

303

Inhibitory effects of baicalin in the early stage of 3T3-L1 preadipocytes differentiation by down-regulation of PDK1/Akt phosphorylation.  

PubMed

Baicalin is a flavonoid derived from the root of Scutellaria baicalensis and exhibits a broad spectrum of biological activities including anti-adipogenesis. However, the inhibitory role of baicalin in the early stage of 3T3-L1 adipocyte differentiation relevant to the signaling up-stream of peroxisome proliferator-activated receptor-? (PPAR-?) and CCAAT/enhancer binding proteins (C/EBPs) expression is unclear, and is the subject of the present investigation. We used 3T3-L1 preadipocytes for adipocyte differentiation, Oil Red-O staining for the intracellular lipid accumulation assay, and real-time polymerase chain reaction (RT-PCR) for assaying the expression of major adipocyte transcription factors. We found that baicalin markedly suppressed the Akt phosphorylation in early stage of adipocytes differentiation. In addition, we observed that baicalin and LY294002 (as an inhibitor of Akt phosphorylation) significantly inhibited adipocyte differentiation by down-regulating several adipocyte-specific transcription factors, including PPAR-? and C/EBPs in 3T3-L1 preadipocytes. Furthermore, we observed that baicalin significantly suppressed the Akt phosphorylation by inhibiting phosphoinositide-dependent kinase 1 (PDK1). These results indicate that the anti-adipogenesis effect of baicalin involves down-regulation of major transcription factors in 3T3-L1 adipocyte differentiation including PPAR-?, C/EBP-?, and C/EBP-? through the down-regulation of PDK1/Akt phosphorylation. PMID:24091917

Kwak, Dong Hoon; Lee, Ji-Hye; Song, Kwang Hoon; Ma, Jin Yeul

2014-01-01

304

In vitro influence of sublethal hypoxia on differentiation of the 3T3-L1 preadipose cell line and its physiological implications  

Microsoft Academic Search

Exposure of cultures of 3T3-L1 preadipose cells to nitrogen for 16 hours kills almost all of the cells, but after exposure to 5% oxygen for 16 hours most of the cells survive, and recover when culture is continued in 20% oxygen. The extent of recovery depends on the insulin concentration of the medium. Isotope incorporation and flow cytometry experiments show

Campbell M. Macfarlane

1997-01-01

305

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

PubMed

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

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

2009-04-30

306

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

SciTech Connect

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

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

2012-08-15

307

Characteristics of dental school feeder institutions.  

PubMed

A major challenge faced by all dental schools is the need to attract highly qualified student applicants. The purpose of this study was to use 2002-03 AADSAS data to identify and characterize feeder colleges and universities that are the major source of applicants to U.S. dental schools. Feeder schools were defined as any institutions with five or more applicants, and minority-feeder schools as those with two or more minority applicants. Feeder schools were ranked by their total numbers of applicants (Category 1) and by their ratio of applicants to total undergraduate enrollment (Category 2). Feeder institutions were compared using total enrollment, degree status, geographic distribution, religious affiliation, numbers of minority applicants, and college admissions selectivity criteria. The top fifty Category 1 schools had an average enrollment of over 19,000 students and an average of sixty-seven applicants. The top fifty Category 2 schools had an average enrollment of approximately 8,500 students and an average of forty-nine applicants. Less than 1 percent of applicants from the top feeder institutions attended the nation's most competitive schools. California and Utah accounted for 28 percent of the total applicants from feeder institutions, followed by Florida (6.2 percent) and New York (5.7 percent). Seventeen of the top twenty-five Category 2 schools (68 percent) were affiliated with or had student bodies associated with a particular religion, with the Seventh-Day Adventist and Mormon institutions accounting for 544 applicants. The majority of all applicants from feeder institutions attended schools in the Southwest. The majority of black and Hispanic feeder institutions were in Florida, Tennessee, Louisiana, and Puerto Rico. Results suggest that factors such as school size, geographic location, religious affiliation, and admissions selectivity criteria of colleges and universities may have a direct impact on the dental applicant pool. PMID:15342655

Thibodeau, Edward A; Mentasti, Lauren E

2004-09-01

308

In vitro cytotoxic and cell transforming activities exerted by the pesticides cyanazine, dithianon, diflubenzuron, procymidone, and vinclozolin on BALB/c 3T3 cells.  

PubMed

Cytotoxic and cell transforming activities of the pesticides cyanazine, diflubenzuron, dithianon, procymidone, and vinclozolin were investigated in vitro by utilizing the BALB/c 3T3 cell transformation test performed in the presence or in the absence of S-9 mix as an exogenous bioactivation system for the chemicals. All the assayed pesticides were cytotoxic in the absence of S-9 mix, whereas only dithianon exerted cytotoxic effects in the presence of metabolic activation. All the chemicals tested did induce BALB/c 3T3 cell transformation, to a various extent, in the absence of S-9 mix. Cell transforming ability of cyanazine and diflubenzuron was not detectable in the presence of S-9. PMID:8419158

Perocco, P; Colacci, A; Grilli, S

1993-01-01

309

Identification of benzophenone C-glucosides from mango tree leaves and their inhibitory effect on triglyceride accumulation in 3T3-L1 adipocytes.  

PubMed

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

2011-11-01

310

Adaptive neural network controller for the flush material Belt Weigh Feeder  

Microsoft Academic Search

The flush material belt weigh feeder (BWF) is used in many material handling plants. The stability and the performance of the layer control system will affect the quality of the production. In general, the behavior of the flush material on the BWF is non-linear, time-lag, and disturbance character. The layer of the flush material on the belt is hard to

Tsung-Ying Sun; Ming-Chin Yang; Shang-Jeng Tsai; Jyun-Sian He

2009-01-01

311

An improved flush material belt weigh feeder system via fuzzy logic controller and adaptive neural networks  

Microsoft Academic Search

The Flush Material Belt Weigh Feeder (FMBWF) has used in many material handling plants. The stability and the performance of the layer control system will affect the quality of the production. In general, the behavior of the flush material on the BWF is non-linear, time-lag, and disturbance character. The layer of the flush material on the belt is hard to

Tsung-Ying Sun; Ming-Chin Yang; Shang-Jeng Tsai; Jyun-Sian He

2009-01-01

312

Thyroid-Stimulating Hormone Stimulates Interleukin6 Release from 3T3-L1 Adipocytes through a cAMP-Protein Kinase A Pathway  

Microsoft Academic Search

Objective: Thyroid-stimulating hormone (TSH) is a novel modulator of adipokine release from human and mouse adipocytes. The aim of our study was to identify the signal transduction pathways activated by TSH that stimulate interleukin (IL)-6 production.Research Methods and Procedures: Mouse 3T3-L1 preadipocyte and differentiated adipocyte cell cultures were studied. The effect of 0 to 1 ?M TSH on IL-6 protein

Tayze T. Antunes; AnneMarie Gagnon; Andrea Bell; Alexander Sorisky

2005-01-01

313

An extract of Lagerstroemia speciosa L. has insulin-like glucose uptake-stimulatory and adipocyte differentiation-inhibitory activities in 3T3-L1 cells.  

PubMed

The effects of extracts isolated from Lagerstroemia speciosa L. (banaba) on glucose transport and adipocyte differentiation in 3T3-L1 cells were studied. Glucose uptake-inducing activity of banaba extract (BE) was investigated in differentiated adipocytes using a radioactive assay, and the ability of BE to induce differentiation in preadipocytes was examined by Northern and Western blot analyses. The hot water BE and the banaba methanol eluent (BME) stimulated glucose uptake in 3T3-L1 adipocytes with an induction time and a dose-dependent response similar to those of insulin. Furthermore, there were no additive or synergistic effects found between BE and insulin on glucose uptake, and the glucose uptake activity of insulin could be reduced to basal levels by adding increasing amounts of BE. Unlike insulin, BE did not induce adipocyte differentiation in the presence of 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone (DEX). BE inhibited the adipocyte differentiation induced by insulin plus IBMX and DEX (IS-IBMX-DEX) of 3T3-L1 preadipocytes in a dose-dependent manner. The differences in the glucose uptake and differentiation inhibitory activities between untreated cells and those treated with BE were significant (P < 0.01). The inhibitory activity was further demonstrated by drastic reductions of peroxisome proliferator-activated receptor gamma2 (PPARgamma2) mRNA and glucose transporter-4 (GLUT4) protein in cells induced from preadipocytes with IS-IBMX-DEX in the presence of BE. The unique combination of a glucose uptake stimulatory activity, the absence of adipocyte differentiation activity and effective inhibition of adipocyte differentiation induced by IS-IBMX-DEX in 3T3-L1 cells suggest that BE may be useful for prevention and treatment of hyperglycemia and obesity in type II diabetics. PMID:11533261

Liu, F; Kim, J; Li, Y; Liu, X; Li, J; Chen, X

2001-09-01

314

Glutathione plays different roles in the induction of the cytotoxic effects of inorganic and organic arsenic compounds in cultured BALB\\/c 3T3 cells  

Microsoft Academic Search

The cytotoxicity of arsenic compounds towards BALB\\/c 3T3 cells in culture was investigated, together with the role of glutathione (GSH) in the induction of the cytotoxic effects. The rank order of cytotoxicity was as follows: arsenite (As3+)>arsenate (As5+)>dimethylarsinic acid (DMAA)>methylarsonic acid (MAA)>trimethylarsine oxide (TMAO). Arsenobetaine, arsenocholine and the tetramethylarsonium ion were less toxic. Depletion of GSH enhanced the cytotoxic effects

T. Ochi; T. Kaise; Y. Oya-Ohta

1994-01-01

315

Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells  

Microsoft Academic Search

Diosgenin, a steroid saponin extracted from the root of wild yam (Dioscorea villossa) is claimed to have osteogenic property. However, detailed studies providing evidence to this claim have not been fully undertaken. In this study, we investigated the effect of diosgenin on the osteogenesis of murine MC3T3-E1 osteoblastic cells. Cells were cultured with varying levels of diosgenin (0–10 ?M) within

Ethel H. Alcantara; Mee-Young Shin; Ho-Yong Sohn; Youn-Moon Park; Taewan Kim; Jae-Hwan Lim; Hyung-Jin Jeong; Soon-Tae Kwon; In-Sook Kwun

2011-01-01

316

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

1995-01-01

317

Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters  

Microsoft Academic Search

Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane frac- tion and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with correspond-

Laura I. Tanner; Gustav E. Lienhard

1989-01-01

318

Bitter melon ( Momordica charantia) triterpenoid extract reduces preadipocyte viability, lipid accumulation and adiponectin expression in 3T3-L1 cells  

Microsoft Academic Search

Bitter melon (Momordica charantia) contains a variety of potentially bioactive compounds which includes two classes of saponins known as cucurbitane and oleanane-type triterpenoids. A bitter melon (BM) extract was investigated for the potential to reduce cell viability, reduce lipid accumulation in differentiating 3T3-L1 cells and affect subsequent adiponectin expression. BM extract contained at least five different triterpenoids and reduced preadipocyte

David G. Popovich; Lu Li; Wei Zhang

2010-01-01

319

Stable release of BDNF from the fibroblast cell line NIH3T3 grown on silicone elastomers enhances survival of spiral ganglion cells in vitro and in vivo.  

PubMed

The treatment of choice for profound sensorineural hearing loss (SNHL) is direct electrical stimulation of spiral ganglion cells (SGC) via a cochlear implant (CI). The number and excitability of SGC seem to be critical for the success that can be achieved via CI treatment. However, SNHL is associated with degeneration of SGC. Long-term drug delivery to the inner ear for improving SGC survival may be achieved by functionalisation of CI electrodes with cells providing growth factors. Therefore, the capacity of brain-derived neurotrophic factor (BDNF)-secreting NIH3T3 cells grown on cylindrically shaped silicone elastomers (SE) to exert local and sustained neuroprotective effects was assessed in vitro and in vivo. An in vitro model to investigate adhesion and cell growth of lentivirally modified NIH3T3 cells synthesising BDNF on SE was established. The bioactivity of BDNF was characterised by co-cultivation of SGC with cell-coated SE. In addition, cell-coated SE were implanted into deafened guinea pigs. The recombinant NIH3T3 cells proliferated on silicone surfaces during 14 days of cultivation and expressed significantly increasing BDNF levels. Enhanced survival rates and neurite outgrowth of SGC demonstrated the bioactivity of BDNF in vitro. Implantation of SE with adhering BDNF-secreting NIH3T3 cells into the cochleae of systemically deafened guinea pigs induced a significant increase in SGC survival in comparison to SE without cell coating. Our data demonstrate a novel approach of cell-based long-term drug delivery to support SGC survival in vitro and in vivo. This therapeutic strategy--once transferred to cells suitable for clinical application--may improve CI performance. PMID:22564255

Warnecke, Athanasia; Sasse, Susanne; Wenzel, Gentiana I; Hoffmann, Andrea; Gross, Gerhard; Paasche, Gerrit; Scheper, Verena; Reich, Uta; Esser, Karl-Heinz; Lenarz, Thomas; Stöver, Timo; Wissel, Kirsten

2012-07-01

320

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

KO KOTANI; WATARU OGAWA; MICHIHIRO MATSUMOTO; TADAHIRO KITAMURA; HIROSHI SAKAUE; YASUHISA HINO; KAZUAKI MIYAKE; WATARU SANO; KAZUNORI AKIMOTO; SHIGEO OHNO; MASATO KASUGA

1998-01-01

321

Role of c-myc in Simian Virus 40 Large Tumor Antigen-Induced DNA Synthesis in Quiescent 3T3-L1 Mouse Fibroblasts  

Microsoft Academic Search

Stably transfected NIH 3T3-L1 mouse fibroblasts (L1 cells) expressing the simian virus 40 large tumor antigen (LTAg) maintain c-myc expression and proliferation in low serum, whereas cells expressing the mutant form LTAg-K1, defective in binding of the retinoblastoma suppressor gene product pRb, showed reduced levels of c-myc RNA and only background levels of DNA synthesis in low serum. The role

Heiko Hermeking; Dieter A. Wolf; Franz Kohlhuber; Achim Dickmanns; Marc Billaud; Ellen Fanning; Dirk Eick

1994-01-01

322

Growth control in cultured 3T3 fibroblasts. V. Purification of an Mr 13,000 polypeptide responsible for growth inhibitory activity  

Microsoft Academic Search

A growth regulatory factor, which reversibly inhibits DNA synthesis and proliferation of fibro- blasts, has been isolated from medium conditioned by exposure to density-inhibited mouse 3T3 cells. This factor, termed FGR-s (13K), yielded a single polypep- tide (Mr 13,000) when analyzed by SDS PAGE under both reducing and nonreducing conditions. The dose- response curve of growth inhibition by FGR-s (13K)

Yen-Ming Hsu; John L. Wang

1986-01-01

323

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: kkatoh@tsurumi.beppu.kyushu-u.ac.jp; 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)

2005-05-01

324

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

325

Effect of embryonic mouse cells BALB/c-3T3 on the proliferation of the human mammary cancer cell line T-47D.  

PubMed

In the present study, we explore the effect of the cellular extracts and culture medium of the embryonic mouse cell line BALB/c-3T3 (clone A31) on the proliferation and DNA content of the human T-47D breast cancer cell line. These effects were also studied in the presence of the potent anti-estrogen ICI 164,384. All experiments were prepared in MEM medium containing 5% fetal calf serum treated with dextran charcoal, as well as the homogenization of the BALB/c-3T3 cells to obtain the cellular extract. Aliquots of cellular extracts (2%) corresponding to 2 x 10(6) cells, or culture medium (16%), are incubated with the T-47D cells. After 9 days of culture, cellular extracts and culture medium provoke an intense proliferative effect corresponding respectively to 2 and 5 times the control value of T-47D cells. These effects on cell proliferation are correlated with DNA content. Although the anti-estrogen ICI 164,384 (5 x 10(-8) M) alone decreases the proliferation of T-47D cells by half, the presence of the culture medium from the BALB/c-3T3 cells abolishes this effect and, on the contrary, increases the cell proliferation 4-fold. It is concluded that mouse embryonic cells (BALB/c-3T3) contain factor(s) which stimulate very intensively the proliferation of hormone-dependent T-47D mammary cancer cells. This factor(s) is present in both the cell and the culture medium and can antagonize the anti-proliferative effect of the anti-estrogen ICI 164,384. PMID:1562526

Chetrite, G; Pasqualini, J R

1992-03-01

326

ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts  

Microsoft Academic Search

Focal adhesion assembly and actin stress fi- ber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Perme- abilization in the presence of GTP g S stimulated rho- dependent formation of stress fibers, and the redis- tribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTP g S at 8 min after permeabilization

J. C. Norman; D. Jones; S. T. Barry; M. R. Holt; S. Cockcroft; D. R. Critchley

1998-01-01

327

Pituitary adenylate cyclase polypeptide (PACAP) stimulates cyclic AMP formation in pituitary fibroblasts and 3T3 tumor fibroblasts: lack of enhancement by protein kinase C activation.  

PubMed

A number of neuropeptides were shown to produce potent mitogenic effects on Swiss 3T3 fibroblasts by activating the phospholipase C pathway. Here we provide evidence for the activation by PACAP of the adenylate cyclase pathway in 3T3, as well as in non-tumoral pituitary fibroblasts, similarly to what was seen in pituitary endocrine cells. In these cells, PACAP triggered elevation of both intracellular and extracellular contents of cAMP and the effect was time- and dose-dependent, with half-maximal stimulations being induced with about 0.1 nM. Following activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA), PACAP-induced cAMP production was amplified in pituitary endocrine cells, but was either unchanged or dampened in 3T3 and pituitary fibroblasts, respectively. Pretreatment of cells with pertussis toxin (PT) failed to change the effect of PMA on PACAP-stimulated adenylate cyclase activity, irrespective of the cell type being used. However, PT dramatically reduced the potentiation by PMA of cAMP production enhanced by forskolin in 3T3 cells. These results provide new evidence pointing to the presence in fibroblasts of receptors for PACAP, coupled to cAMP production, which may play a role in the modulation of the mitogenic signal. They also indicate that, compared with pituitary endocrine cells, PKC activation in fibroblasts differentially affected PACAP-induced cAMP formation and that these effects were unaltered upon inhibition by PT of Gi-like proteins. PMID:1280235

Koch, B; Lutz-Bucher, B

1992-09-01

328

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

PubMed

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

2012-08-15

329

Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes  

Microsoft Academic Search

A strong correlation between intramyocellular lipid concentrations and the severity of insulin resistance has fueled speculation that lipid oversupply to skeletal muscle, fat, or liver may desensitize these tissues to the anabolic effects of insulin. To identify free fatty acids (FFAs) capable of inhibiting insulin action, we treated 3T3-L1 adipocytes or C2C12 myotubes with either the saturated FFA palmitate (C16:0)

Jose Antonio Chavez; Scott A Summers

2003-01-01

330

Diacylglycerol Stimulates DNA Synthesis and Cell Division in Mouse 3T3 Cells: Role of Ca2+-sensitive Phospholipid-Dependent Protein Kinase  

Microsoft Academic Search

The synthetic diacylglycerol 1-oleoyl-2-acetylglycerol competes directly with [3H]phorbol 12,13-dibutyrate for common binding sites in monolayer cultures of Swiss 3T3 cells and rapidly stimulates the phosphorylation of a Mr 80,000 cellular protein that has recently been shown to reflect the activation of protein kinase C in intact cells. Thus, this diacylglycerol provided a useful tool to determine whether exogenously added diacylglycerols

Enrique Rozengurt; Angeles Rodriguez-Pena; Maurice Coombs; James Sinnett-Smith

1984-01-01

331

Screening of Dried Plant Seed Extracts for Adiponectin Production Activity and Tumor Necrosis Factor-Alpha Inhibitory Activity on 3T3-L1 Adipocytes  

Microsoft Academic Search

To search for dried plant seeds with potent anti-diabetes activity, we conducted a large scale screening for inhibitory activity\\u000a on tumor necrosis factor-alpha and facilitating activity on adiponectin production in vitro. These activities in 3T3-L1 adipocytes were screened from ethanol extracts of 20 kinds of dried plant seed marketed in Japan.\\u000a komatsuna (Brassica rapa var. perviridis), common bean (Phaseolus vulgaris

Yoshinori Okada; Mizue Okada; Yumi Sagesaka

2010-01-01

332

The ?-SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1  

PubMed Central

Silicon carbide (SiC), a compound of silicon and carbon, with chemical formula SiC, the beta modification (?-SiC), with a zinc blende crystal structure (similar to diamond), is formed at temperature below 1700°C. ?-SiC will be the most suitable ceramic material for the future hard tissue replacement, such as bone and tooth. The in vitro cytotoxicity of ?-SiC nanowires was investigated for the first time. Our results indicated that 100?nm long SiC nanowires could significantly induce the apoptosis in MC3T3-E1 cells, compared with 100??m long SiC nanowires. And 100?nm long SiC nanowires increased oxidative stress in MC3T3-E1 cells, as determined by the concentrations of MDA (as a marker of lipid peroxidation) and 8-OHdG (indicator of oxidative DNA damage). Moreover, transmission electron microscopy (TEM) was performed to evaluate the morphological changes of MC3T3-E1 cells. After treatment with 100?nm long SiC nanowires, the mitochondria were swelled and disintegrated, and the production of ATP and the total oxygen uptake were also decreased significantly. Therefore, ?-SiC nanowires may have limitations as medical material.

Xie, Weili; Xie, Qi; Jin, Meishan; Huang, Xiaoxiao; Zhang, Xiaodong; Shao, Zhengkai; Wen, Guangwu

2014-01-01

333

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.

1999-01-01

334

Ellagic Acid Reduces Adipogenesis through Inhibition of Differentiation-Prevention of the Induction of Rb Phosphorylation in 3T3-L1 Adipocytes.  

PubMed

Ellagic acid (EA) present in many fruits and nuts serves as antiproliferation, anti-inflammatory, and antitumorigenic properties. However, the effect of EA on preadipocytes adipogenesis and its mechanism(s) have not been elucidated. The present study was designed to examine the effect of EA on adipogenesis in 3T3-L1 preadipocytes and underlying mechanism(s) of action involved. Data show that EA administration decreased the accumulation of lipid droplets. The inhibition was diminished when the addition of EA was delayed to days 2-4 of differentiation. Clonal expansion was reduced in the presence of EA. FACS analysis showed that EA blocked the cell cycle at the G1/S transition. EdU incorporation also confirmed that EA refrained cell from entering S phase. Our data also revealed that the differentiation-induced protein expression of Cyclin A and phosphorylation of the retinoblastoma protein (Rb) were impaired by EA. Differentiation-dependent expression and DNA-binding ability of C/EBP ? were also inhibited by EA. Alterations in cell cycle-associated proteins may be important with respect to the antiadipogenic action of EA. In conclusion, EA is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through reduction of Cyclin A protein expression and Rb phosphorylation. With the blocking of G1/S phase transition, EA suppresses terminal differentiation and lipid accumulation in 3T3-L1 adipocytes. PMID:24302962

Wang, Lifeng; Li, Linlin; Ran, Xinjian; Long, Mei; Zhang, Minfang; Tao, Yicun; Luo, Xin; Wang, Ye; Ma, Xiaoli; Halmurati, Upur; Mao, Xinmin; Ren, Jun

2013-01-01

335

Delivering MC3T3-E1 cells into injectable calcium phosphate cement through alginate-chitosan microcapsules for bone tissue engineering*  

PubMed Central

Objective: To deliver cells deep into injectable calcium phosphate cement (CPC) through alginate-chitosan (AC) microcapsules and investigate the biological behavior of the cells released from microcapsules into the CPC. Methods: Mouse osteoblastic MC3T3-E1 cells were embedded in alginate and AC microcapsules using an electrostatic droplet generator. The two types of cell-encapsulating microcapsules were then mixed with a CPC paste. MC3T3-E1 cell viability was investigated using a Wst-8 kit, and osteogenic differentiation was demonstrated by an alkaline phosphatase (ALP) activity assay. Cell attachment in CPC was observed by an environment scanning electron microscopy. Results: Both alginate and AC microcapsules were able to release the encapsulated MC3T3-E1 cells when mixed with CPC paste. The released cells attached to the setting CPC scaffolds, survived, differentiated, and formed mineralized nodules. Cells grew in the pores concomitantly created by the AC microcapsules in situ within the CPC. At Day 21, cellular ALP activity in the AC group was approximately four times that at Day 7 and exceeded that of the alginate microcapsule group (P<0.05). Pores formed by the AC microcapsules had a diameter of several hundred microns and were spherical compared with those formed by alginate microcapsules. Conclusions: AC microcapsule is a promising carrier to release seeding cells deep into an injectable CPC scaffold for bone engineering.

Qiao, Peng-yan; Li, Fang-fang; Dong, Li-min; Xu, Tao; Xie, Qiu-fei

2014-01-01

336

Antiproliferative activity of flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the MCF7, KB, and NIH/3T3 cell lines.  

PubMed

This study assessed the antiproliferative effect in vitro of the flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the human breast adenocarcinoma (MCF-7), human mouth epidermal carcinoma (KB), and mouse embryonic fibroblast (NIH 3T3) cell lines, using sulforhodamine B (SRB) assay. A cell density of 2×10(4)/well was seeded in 96-well plates, and samples at different concentrations ranging from 10 to 500?mg/mL were tested. The optical density was determined in an ELISA multiplate reader (Thermo Plate TP-Reader). Results demonstrated that the hexane extract presented antiproliferative activity against both the tumor cell lines KB and MCF-7, presenting a GI(50) (MCF-7=13.09?mg/mL), TGI (KB=37.76?mg/mL), and IL(50) (KB=291.07?mg/mL). Also, the hexane extract presented antiproliferative activity toward NIH 3T3 cells GI(50) (183.65?mg/mL), TGI (280.54?mg/mL), and IL(50) (384.59?mg/mL). The results indicate that the flower hexane extract obtained from M. spicata associated with M. rotundifolia presents an antineoplastic activity against KB and MCF-7, although an antiproliferative effect at a high concentration of the extract was observed toward NIH 3T3. PMID:23066647

Nedel, Fernanda; Begnini, Karine; Carvalho, Pedro Henrique de Azambuja; Lund, Rafael Guerra; Beira, Fátima T A; Del Pino, Francisco Augusto B

2012-11-01

337

Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation  

SciTech Connect

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 PPAR{gamma} 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 PPAR{gamma} 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 PPAR{gamma} 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.

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 [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)] [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)] [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)

2009-12-25

338

Transport of L-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells.  

PubMed

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 ?M rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate L-[(3)H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H(+) gradient-stimulated uptake of L-[(3)H]proline. Uptake was rapid, independent of a Na(+) gradient but stimulated by an inwardly directed H(+) gradient with maximal uptake occurring at pH 6.0. L-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K(t)) of 130 ± 10 ?M and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min(-1 )mg of protein(-1). Glycine, L-alanine, and L-tryptophan strongly inhibited L-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H(+)-amino acid cotransport system PAT2. PMID:22711289

Zebisch, Katja; Brandsch, Matthias

2013-02-01

339

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.

1986-05-01

340

cis9, trans11-Conjugated Linoleic Acid Differentiates Mouse 3T3-L1 Preadipocytes into Mature Small Adipocytes through Induction of Peroxisome Proliferator-activated Receptor ?  

PubMed Central

Dietary conjugated linoleic acid (CLA) has been reported to exhibit a number of therapeutic effects in animal models and patients, such as anti-hypertensive, anti-hyperlipidemic, anti-arteriosclerotic, anti-carcinogenic, and anti-diabetic effects. However, the underlying mechanism is not well-characterized. In the present study, the effects of cis(c)9, trans(t)11-CLA on the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes were examined. Treatment with c9, t11-CLA in the presence of insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (differentiation cocktail) significantly stimulated the accumulation of triacylglycerol. The microscopic observation of cells stained by Oil Red O demonstrated that c9, t11-CLA increases the amount and proportion of small mature adipocytes secreting adiponectin, a benign adipocytokine, when compared to the differentiation cocktail alone. Furthermore, c9, t11-CLA increased bioactive peroxisome proliferator-activated receptor ? (PPAR?) levels in a nuclear extract of 3T3-L1 cells, suggesting the enhancing effect of this fatty acid on the nuclear transmission of PPAR?, a master regulator of adipocyte differentiation, in 3T3-L1 cells. These results suggest that the therapeutic effects of c9, t11-CLA on lifestyle-related diseases are partially due to the enhanced formation of small adipocytes from preadipocytes via PPAR? stimulation.

Sakuma, Satoru; Nishioka, Yuki; Imanishi, Ryohta; Nishikawa, Kenji; Sakamoto, Hirotada; Fujisawa, Junji; Wada, Koichiro; Kamisaki, Yoshinori; Fujimoto, Yohko

2010-01-01

341

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

PubMed Central

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

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

2014-01-01

342

Momordica charantia seed extract reduces pre-adipocyte viability, affects lactate dehydrogenase release, and lipid accumulation in 3T3-L1 cells.  

PubMed

A triterpenoid containing bitter melon (Momordica charantia) seed (BMS) extract was found to reduce cultured 3T3-L1 cell viability. The 50% lethal concentration values were determined to be 0.78?±?0.01?mg/mL at 24 hours, 0.69?±?0.01?mg/mL at 48 hours, and 0.56?±?0.02?mg/mL at 72 hours. 3T3-L1 cells were utilized as models of pre-adipocyte to adipocyte differentiation. BMS extract also caused a G(2)/M arrest in the cell cycle reducing cells by 23.9%, 37.7%, and 34.7% compared with the control after 72 hours of treatment at concentrations of 0.4, 0.5, and 0.6?mg/mL respectively. BMS extract did not increase the release of lactate dehydrogenase from 3T3-L1 cells, which was unexpected. Furthermore, BMS extract reduced lipid accumulation during differentiation from pre-adipocyte to adipocyte corresponding to reduction in overall triglyceride of 32.4% after 72 hours compared with untreated control cells. BMS is an underutilized agricultural commodity that may have potential for nutraceutical and functional food development. PMID:21332398

Popovich, David G; Lee, Yiyu; Li, Lu; Zhang, Wei

2011-03-01

343

Ellagic Acid Reduces Adipogenesis through Inhibition of Differentiation-Prevention of the Induction of Rb Phosphorylation in 3T3-L1 Adipocytes  

PubMed Central

Ellagic acid (EA) present in many fruits and nuts serves as antiproliferation, anti-inflammatory, and antitumorigenic properties. However, the effect of EA on preadipocytes adipogenesis and its mechanism(s) have not been elucidated. The present study was designed to examine the effect of EA on adipogenesis in 3T3-L1 preadipocytes and underlying mechanism(s) of action involved. Data show that EA administration decreased the accumulation of lipid droplets. The inhibition was diminished when the addition of EA was delayed to days 2–4 of differentiation. Clonal expansion was reduced in the presence of EA. FACS analysis showed that EA blocked the cell cycle at the G1/S transition. EdU incorporation also confirmed that EA refrained cell from entering S phase. Our data also revealed that the differentiation-induced protein expression of Cyclin A and phosphorylation of the retinoblastoma protein (Rb) were impaired by EA. Differentiation-dependent expression and DNA-binding ability of C/EBP? were also inhibited by EA. Alterations in cell cycle-associated proteins may be important with respect to the antiadipogenic action of EA. In conclusion, EA is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through reduction of Cyclin A protein expression and Rb phosphorylation. With the blocking of G1/S phase transition, EA suppresses terminal differentiation and lipid accumulation in 3T3-L1 adipocytes.

Wang, Lifeng; Li, Linlin; Ran, Xinjian; Long, Mei; Zhang, Minfang; Tao, Yicun; Luo, Xin; Wang, Ye; Ma, Xiaoli; Halmurati, Upur; Ren, Jun

2013-01-01

344

Myricetin, a naturally occurring flavonoid, prevents 2-deoxy-D-ribose induced dysfunction and oxidative damage in osteoblastic MC3T3-E1 cells.  

PubMed

Myricetin, a naturally occurring flavonoid, was investigated to determine whether it could protect osteoblasts from 2-deoxy-d-ribose induced dysfunction and oxidative damage in the MC3T3-E1 cells. MC3T3-E1 cells were incubated with 2-deoxy-d-ribose and/or myricetin, and markers of osteoblast function and oxidative damage were examined. Compared with control incubation, 2-deoxy-d-ribose significantly (P<0.05) inhibited alkaline phosphatase (ALP) activity, collagen content, and calcium deposition at the concentration of 20 mM. Cellular malondialdehyde (MDA), protein carbonyl, and advanced oxidation protein products contents were significantly (P<0.05) increased in the presence of 2-deoxy-d-ribose (20 mM). Myricetin significantly (P<0.05) increased cell survival, ALP activity, collagen, osteocalcin, osteoprotegerin, and calcium deposition and decreased MDA, protein carbonyl, and advanced oxidation protein products contents of osteoblastic MC3T3-E1 cells in the presence of 20 mM 2-deoxy-d-ribose. These results demonstrate that myricetin attenuates 2-deoxy-d-ribose induced damage, suggesting that myricetin may be a useful dietary supplement for minimizing oxidative injury in diabetes related bone diseases. PMID:18599037

Lee, Kyung-Hee; Choi, Eun-Mi

2008-09-01

345

Effect of 635 nm irradiation on high glucose-boosted inflammatory responses in LPS-induced MC3T3-E1 cells.  

PubMed

Hyperglycemia occurs in patients with poorly controlled diabetes mellitus and contributes to bone resorption and increased susceptibility to bacterial infections. Hyperglycemia can incite low-grade inflammation that can contribute to the resorption of bone, especially the periodontal bone. The increased susceptibility to periodontal infections can contribute to bone resorption through the activation of osteoclasts. In this study, the osteoblastic, clonal cell line, MC3T3-E1, was used in an in vitro model of hyperglycemia and lipopolysaccharide-induced reactive oxygen species generation to determine the potential anti-inflammatory effect of 635 nm light-emitting diode (LED) irradiation or whether 635 nm LED irradiation can be a potential anti-inflammatory treatment. LED irradiation of MC3T3-E1 cells stimulated with lipopolysaccharide in a high glucose-containing medium decreased the level of cyclooxygenase gene and protein expression and reduced the level of prostaglandin E2 expression by decreasing the amount of reactive oxygen species generation. LED irradiation also inhibited the osteoclastogenesis in MC3T3-E1 cells by regulating the receptor activator of nuclear factor kappa-B ligand and osteoprotegerin. These findings reveal the mechanisms which are important in the pathogenesis of diabetic periodontitis and highlight the beneficial effects of 635 nm LED irradiation in reducing the adverse effects of diabetic periodontitis. PMID:22699799

Kwon, HyukIl; Lim, WonBong; Kim, JiSun; Jeon, SangMi; Kim, SangWoo; Karna, Sandeep; Cha, HyunRok; Kim, OkJoon; Choi, HongRan

2013-05-01

346

Peanut sprout ethanol extract inhibits the adipocyte proliferation, differentiation, and matrix metalloproteinases activities in mouse fibroblast 3T3-L1 preadipocytes  

PubMed Central

3T3-L1 preadipocyte were differentiated to adipocytes, and then treated with 0, 10, 20, and 40 µg/mL of peanut sprout ethanol extract (PSEE). The main component of PSEE is resveratrol which contained 5.55 mg/mL of resveratrol. The MTT assay, Oil-Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, and the triglyceride concentration were determined in 3T3-L1 cells. MMP-2 and MMP-9 activities as well as mRNA expressions of C/EBP ? and C/EBP ? were also investigated. As the concentration of PSEE in adipocytes increased, the cell proliferation was decreased in a dose-dependent manner from 4 days of incubation (P < 0.05). The GDPH activity (P < 0.05) and the triglyceride concentration (P < 0.05) were decreased as the PSEE treatment concentration increased. The mRNA expression of C/EBP? in 3T3-L1 cells was significantly low in groups of PSEE-treated, compared with control group (P < 0.05). The MMP-9 (P < 0.05) and MMP-2 (P < 0.05) activities were decreased in a dose-dependent manner as the PSEE concentration increased from 20 µg/mL. In conclusion, it was found that PSEE has an effect on restricting proliferation and differentiation of adipocytes.

Kim, Woo Kyoung; Kang, Nam E; Kim, Myung Hwan

2013-01-01

347

12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells  

SciTech Connect

The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodomine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.

Shiba, Yoshiki; Sasaki, Yasuto; Kanno, Yoshinobu (Hiroshima Univ. School of Dentistry (Japan))

1988-10-01

348

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

1997-01-01

349

Protective effect of tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells.  

PubMed

Oxidative stress can induce apoptosis and decrease activities of osteoblasts. 2,3,5,4'-tetrahydroxystilbene-2-O-?-D-glucoside (TSG), is a potent antioxidant derived from a Chinese herb Polygonum multiflorum Thunb. To evaluate the protective effect provided by TSG to osteoblastic MC3T3-E1 cells, the cells were pretreated with TSG for 24h before being treated with 0.3mM hydrogen peroxide (H(2)O(2)) for 24 h, then some markers of osteoblast function and oxidative damage of the cells were examined. Our data demonstrated that TSG significantly (P< 0.05) increased cell survival, alkaline phosphatase (ALP) activity, calcium deposition, and the mRNA expression of ALP, collagen I (COL-I) and osteocalcin (OCN) in the presence of H(2)O(2). In addition, TSG decreased the production of receptor activator of nuclear factor-?B ligand (RANKL), interleukin-6 (IL-6), intracellular reactive oxygen species and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells induced by H(2)O(2). Taken together, these results demonstrated that the protective effect provided by TSG to osteoblastic MC3T3-E1 cells was mediated, at least in part, via inhibition of the release of bone-resorbing mediators and oxidative damage of the cells. Our results indicated that TSG may be effective in providing protection against osteoporosis associated with oxidative stress. PMID:22683865

Zhang, Jin-Kang; Yang, Liu; Meng, Guo-Lin; Fan, Jing; Chen, Jian-Zong; He, Qi-Zhen; Chen, Shi; Fan, Jin-Zhu; Luo, Zhuo-Jing; Liu, Jian

2012-08-15

350

Melanocortins induce interleukin 6 gene expression and secretion through melanocortin receptors 2 and 5 in 3T3-L1 adipocytes  

PubMed Central

Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. ?-Melanocyte-stimulating hormone (?-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and I?B kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the ?-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, ?-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that ?-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production.

Jun, Dong-Jae; Na, Kyung-Yoon; Kim, Wanil; Kwak, Dongoh; Kwon, Eun-Jeong; Yoon, Jong Hyuk; Yea, Kyungmoo; Lee, Hyeongji; Kim, Jaeyoon; Suh, Pann-Gill; Ryu, Sung Ho; Kim, Kyong-Tai

2010-01-01

351

The ? -SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1.  

PubMed

Silicon carbide (SiC), a compound of silicon and carbon, with chemical formula SiC, the beta modification ( ? -SiC), with a zinc blende crystal structure (similar to diamond), is formed at temperature below 1700°C. ? -SiC will be the most suitable ceramic material for the future hard tissue replacement, such as bone and tooth. The in vitro cytotoxicity of ? -SiC nanowires was investigated for the first time. Our results indicated that 100?nm long SiC nanowires could significantly induce the apoptosis in MC3T3-E1 cells, compared with 100? ? m long SiC nanowires. And 100?nm long SiC nanowires increased oxidative stress in MC3T3-E1 cells, as determined by the concentrations of MDA (as a marker of lipid peroxidation) and 8-OHdG (indicator of oxidative DNA damage). Moreover, transmission electron microscopy (TEM) was performed to evaluate the morphological changes of MC3T3-E1 cells. After treatment with 100?nm long SiC nanowires, the mitochondria were swelled and disintegrated, and the production of ATP and the total oxygen uptake were also decreased significantly. Therefore, ? -SiC nanowires may have limitations as medical material. PMID:24967352

Xie, Weili; Xie, Qi; Jin, Meishan; Huang, Xiaoxiao; Zhang, Xiaodong; Shao, Zhengkai; Wen, Guangwu

2014-01-01

352

Plumbagin activates ERK1/2 and Akt via superoxide, Src and PI3-kinase in 3T3-L1 cells.  

PubMed

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

2010-07-25

353