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1

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

PubMed Central

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

1982-01-01

2

Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium  

Microsoft Academic Search

Summary  An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells\\u000a or conditioned medium. The use of medium 199, supplemented with 0.4 ?g\\/ml hydrocortisone (HC) and 20% (v\\/v) whole fetal bovine\\u000a serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells\\u000a for HK

Donna M. Peehl; Richard G. Ham

1980-01-01

3

Culture of Human Limbal Epithelial Stem Cells on Tenon's Fibroblast Feeder-Layers: A Translational Approach.  

PubMed

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

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

2015-01-01

4

A novel culture system for porcine odontogenic epithelial cells using a feeder layer.  

PubMed

The growth of cells in vitro can provide useful models for investigating their behaviour and improving our understanding of their function in vivo. Although the developmental regulation of enamel matrix formation has been comprehensively analysed, the detailed cellular characteristics of ameloblasts remain unclear because of the lack of a system of long-term in vitro culture. Therefore, the establishment of odontogenic epithelial cell lines has taken on a new significance. Here, we report on a novel porcine odontogenic epithelial cell-culture system, which has permitted serial culture of these cells. Epithelial cells were harvested from third molar tooth buds in the fresh mandibles of 6-month-old pigs, and seeded on dishes in D-MEM containing 10% FBS. Before the cells reached confluence, the medium was changed to LHC-9 to select the epithelial cells. When trypsinized epithelial cells were plated together with 3T3-J2 cells as a feeder layer, the epithelial cells grew from single cells into colonies. The colonies then expanded and became confluent, and could be sub-cultured for up to 20 passages. The long-term culture cells expressed mRNA for amelogenin and ameloblastin, as well as enamelysin (MMP-20), which is a tissue-specific gene product unique to ameloblasts. These results show that the system is capable of sustaining the multiplication of odontogenic epithelial cells with the characteristics of ameloblasts. PMID:16257386

Honda, M J; Shimodaira, T; Ogaeri, T; Shinohara, Y; Hata, K; Ueda, M

2006-04-01

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

Feeder for particulate material  

SciTech Connect

Feeder apparatus for feeding at a controlled variable rate particulate solid material, such as coal, from a supply conduit located above a gravimetric feeder to a discharge chute located below the feeder. The particulate material flows to the feeder from the supply conduit along a path having a generally vertical center line and the discharge chute provides a flow path having a generally vertical center line closely laterally spaced relative to the center line of flow to the feeder. The feeder apparatus has a horizontally extending housing with an upwardly facing inlet through which the material flows to the feeder and a downwardly facing outlet forming part of the discharge chute through which the material flows from the feeder. Within the housing is a main feeder conveyor including an endless belt with a generally horizontal span adapted to receive at one end the particulate material flowing to the feeder and to convey in a layer a measured quantity of material a horizontal distance across weighing means to an exit zone at the opposite end of the belt, the horizontal distance being substantially greater than the spacing between the center lines of flow to the feeder and through such discharge chute. Associated with the main feeder conveyor is a gravimetric control system for controlling the amount of material deposited on the belt. Particulate material that drops off the main conveyor belt at the exit zone is received on a return belt conveyor and carried in a reverse direction a sufficient distance to bring the material to the discharge chute. Cooperation between the main feeder conveyor and the return conveyor permits gravimetric feeding of particulate material to the discharge chute that is closely laterally spaced to the flow path of material to the main conveyor by a distance that is substantially less than the length of travel of the material on the main conveyor.

Christofer, D.E.; Stock, A.J.

1981-03-24

7

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

8

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. PMID:23521774

2013-01-01

9

Phosphatidylcholine induces apoptosis of 3T3-L1 adipocytes  

PubMed Central

Background Phosphatidylcholine (PPC) formulation is used for lipolytic injection, even though its mechanism of action is not well understood. Methods The viability of 3T3-L1 pre-adipocytes and differentiated 3T3-L1 cells was measured after treatment of PPC alone, its vehicle sodium deoxycholate (SD), and a PPC formulation. Western blot analysis was performed to examine PPC-induced signaling pathways. Results PPC, SD, and PPC formulation significantly decreased 3T3-L1 cell viability in a concentration-dependent manner. PPC alone was not cytotoxic to CCD-25Sk human fibroblasts at concentrations <1 mg/ml, whereas SD and PPC formulation were cytotoxic. Western blot analysis demonstrated that PPC alone led to the phosphorylation of the stress signaling proteins, such as p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, and activated caspase-9, -8, -3 as well as cleavage of poly(ADP-ribose) polymerase. However, SD did not activate the apoptotic pathways. Instead, SD and PPC formulation induced cell membrane lysis, which may lead to necrosis of cells. Conclusions PPC results in apoptosis of 3T3-L1 cells. PMID:22145579

2011-01-01

10

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.

11

Organelle relationships in cultured 3T3-L1 preadipocytes  

PubMed Central

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha- naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol. PMID:7191426

1980-01-01

12

Precision powder feeder  

DOEpatents

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

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

2001-07-10

13

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

14

Aspartame downregulates 3T3-L1 differentiation.  

PubMed

Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. Since aspartame is 200 times sweeter than traditional sugar, it can give the same level of sweetness with less substance, which leads to lower-calorie food intake. There are reports that consumption of aspartame-containing products can help obese people lose weight. However, the potential role of aspartame in obesity is not clear. The present study investigated whether aspartame suppresses 3T3-L1 differentiation, by downregulating phosphorylated peroxisome proliferator-activated receptor ? (p-PPAR?), peroxisome proliferator-activated receptor ? (PPAR?), fatty acid-binding protein 4 (FABP4), CCAAT/enhancer-binding protein ? (C/EBP?), and sterol regulatory element-binding protein 1 (SREBP1), which are critical for adipogenesis. The 3T3-L1 adipocytes were cultured and differentiated for 6 d in the absence and presence of 10 ?g/ml of aspartame. Aspartame reduced lipid accumulation in differentiated adipocytes as evidenced by Oil Red O staining. qRT-PCR analysis showed that the PPAR?, FABP4, and C/EBP? mRNA expression was significantly reduced in the aspartame-treated adipocytes. Western blot analysis showed that the induction of p-PPAR?, PPAR?, SREBP1, and adipsin was markedly reduced in the aspartame-treated adipocytes. Taken together, these data suggest that aspartame may be a potent substance to alter adipocyte differentiation and control obesity. PMID:24961835

Pandurangan, Muthuraman; Park, Jeongeun; Kim, Eunjung

2014-10-01

15

Feeder for particulate material  

Microsoft Academic Search

Feeder apparatus for feeding at a controlled variable rate particulate solid material, such as coal, from a supply conduit located above a gravimetric feeder to a discharge chute located below the feeder. The particulate material flows to the feeder from the supply conduit along a path having a generally vertical center line and the discharge chute provides a flow path

D. E. Christofer; A. J. Stock

1981-01-01

16

Project Feeder Watch  

NSDL National Science Digital Library

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

2004-01-01

17

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

18

Comparison of the Ex Vivo Expansion of UCB-Derived CD34+ in 3D DBM/MBA Scaffolds with USSC as a Feeder Layer  

PubMed Central

Objective(s): Ex vivo expansion of hematopoitic stem cells is an alternative way to increase umbilical cord blood (UCB)-CD34+ cells for bone marrow transplantation. For this purpose demineralized bone matrix (DBM) and mineralized bone allograft (MBA) as two scaffolds based on bone matrix and stem cell niche, were simultaneously used to enhance the effect of human mesenchymal progenitor cells (MPCs) - unrestricted somatic stem cells (USSCs) - as a feeder layer. Materials and Methods: USSCs were isolated and characterized by morphological and immunological analysis then seeded on both scaffolds as a feeder layer. UCB-CD34+ were isolated by MACS method and were co-culture expanded by USSC in 3D and 2D environments. After 3 weeks expansion, cells were counted and were assessed by karyotype, flow cytometry, clonogenic activity, and long-term culture-initiating cells (LTC-IC). Results: Co-culture expansion in DBM and MBA was 29.22-fold and 27.77-fold, no significant differences in colony and LTC-IC were obtained. Maximum number of colonies belonged to the day 14 with the 73% CFU-GM (Colony Forming Unit- Granulocyte/Macrophage) in contrast to the day 0 which was BFU-E/CFU-E (Burst/Colony Forming Unit-Erythroid). Flow cytometry indicated that the percentage of CD34+ marker was decreased in USSC co-culture and the highest percentage was observed in simple 2D culture. Conclusion: Because of acid extraction in the DBM production process, mineral materials were removed and the protein background that was more flexible was presented. Therefore these results suggest that USSC-DBM can be a suitable ex vivo mimicry niche by intensifying of surface/volume ratio and supporting the stem cell differentiation and expansion. PMID:24379965

Sadat Hashemi, Zahra; Forouzandeh Moghadam, Mahdi; Soleimani, Masoud

2013-01-01

19

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

20

Effects of Eucommia ulmoides Oliver leaf extract on 3T3-L1 differentiation into adipocytes  

Microsoft Academic Search

The extract prepared from roasted Eucommia ulmoides Oliver leaves, Du-Zhong tea, was examined to explore its effect on differentiation of mouse 3T3-L1 preadipocyte cells into adipocytes. The boiling water extract of Du-Zhong tea inhibited lipid accumulation in 3T3-L1. The HPLC analysis of the extract identified catechin, protocatechuic acid, pyrogallol, and chlorogenic acid. Catechin weakly inhibited lipid accumulation after 3T3-L1 differentiation,

Eriko Matsuda; Yuko Yoshizawa; Yuki Yokosawa; Naomi Watanabe; Satoru Kawaii; Noboru Murofushi

2006-01-01

21

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

22

Feederism in a woman.  

PubMed

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

Terry, Lesley L; Vasey, Paul L

2011-06-01

23

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

24

Fucoxanthin and its metabolite, fucoxanthinol, suppress adipocyte differentiation in 3T3-L1 cells.  

PubMed

Fucoxanthin is a major carotenoid found in edible seaweed such as Undaria pinnatifida and Hijikia fusiformis. We investigated the suppressive effects of fucoxanthin and its metabolite, fucoxanthinol, on the differentiation of 3T3-L1 preadipocytes to adipocytes. Fucoxanthin inhibited intercellular lipid accumulation during adipocyte differentiation of 3T3-L1 cells. Furthermore, fucoxanthin was converted to fucoxanthinol in 3T3-L1 cells. Fucoxanthinol also exhibited suppressive effects on lipid accumulation and decreased glycerol-3-phosphate dehydrogenase activity, an indicator of adipocyte differentiation. The suppressive effect of fucoxanthinol was stronger than that of fucoxanthin. In addition, in 3T3-L1 cells treated with fucoxanthin and fucoxanthinol, peroxisome proliferator-activated receptor gamma (PPARgamma), which regulates adipogenic gene expression, was down-regulated in a dose-dependent manner. These results suggest that fucoxanthin and fucoxanthinol inhibit the adipocyte differentiation of 3T3-L1 cells through down-regulation of PPARgamma. Fucoxanthinol had stronger suppressive effects than fucoxanthin on adipocyte differentiation in 3T3-L1 cells. PMID:16786166

Maeda, Hayato; Hosokawa, Masashi; Sashima, Tokutake; Takahashi, Nobuyuki; Kawada, Teruo; Miyashita, Kazuo

2006-07-01

25

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

PubMed Central

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

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

2014-01-01

26

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

NASA Astrophysics Data System (ADS)

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

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

2014-08-01

27

Apoptosis observed in BALB/3T3 cells having ingested Staphylococcus aureus.  

PubMed

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 microg/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I-infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 +/- 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron-dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2- to 6-hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell. PMID:10529106

Murai, M; Sakurada, J; Seki, K; Shinji, H; Hirota, Y; Masuda, S

1999-01-01

28

Cranberries (Oxycoccus quadripetalus) inhibit adipogenesis and lipogenesis in 3T3-L1 cells.  

PubMed

Cranberries (Oxycoccus quadripetalus) are a valuable source of bioactive substances with high antioxidant potential and well documented beneficial health properties. In the present study, the activity of cranberries, in terms of the inhibiting effects of adipogenesis, was investigated using the 3T3-L1 cell line. The obtained results showed that cranberries reduced proliferation and viability of 3T3-L1 preadipocytes in a dose-dependent manner. Treatment with cranberries decreased the number of adipocytes and reduced lipid accumulation in maturing 3T3-L1 preadipocytes, demonstrating an inhibitory effect on lipogenesis. Moreover, it was found that cranberries directly induced lipolysis in adipocytes and down-regulated the expression of major transcription factors of the adipogenesis pathway, such as PPAR?, C/EBP? and SREBP1. These findings indicate that cranberries are capable of suppressing adipogenesis and therefore they seem to be natural bioactive factors effective in adipose tissue mass modulation. PMID:24262553

Kowalska, Katarzyna; Olejnik, Anna; Rychlik, Joanna; Grajek, W?odzimierz

2014-04-01

29

Inhibitory Effect of Deep-sea Water on Differentiation of 3T3-L1 Adipocytes  

Microsoft Academic Search

Currently, the utilization of deep-sea water (DSW) is receiving much attention due to its high productivity, large quantity,\\u000a and potential for biological application. The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess\\u000a adipocyte differentiation. Over the course of several days, confluent 3T3-L1 cells can be converted to adipocytes in the presence\\u000a of an adipogenic

Hee Sun Hwang; Seon Hwa Kim; Yung Geun Yoo; Yong Shik Chu; Yun Hee Shon; Kyung Soo Nam; Jong Won Yun

2009-01-01

30

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

31

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

32

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

33

EFFECT OF UNCOUPLING PROTEIN–1 EXPRESSION ON 3T3-L1 ADIPOCYTE GENE EXPRESSION  

PubMed Central

The mitochondrial respiratory uncoupling protein 1 (UCP1) partially uncouples substrate oxidation and oxidative phosphorylation to promote the dissipation of cellular biochemical energy as heat in brown adipose tissue. We have recently shown that expression of UCP1 in 3T3-L1 white adipocytes reduces the accumulation of triglycerides. Here, we investigated the molecular basis underlying UCP1 expression in 3T3-L1 adipocytes. Gene expression data show that forced UCP1 expression down-regulated several energy metabolism pathways; but ATP levels were constant. A metabolic flux analysis model was used to reflect the gene expression changes onto metabolic processes and concordance was observed in the down-regulation of energy consuming pathways. Our data suggest that adipocytes respond to long-term mitochondrial uncoupling by minimizing ATP utilization. PMID:18061577

Senocak, Fatih S.; Si, Yaguang; Moya, Colby; Russell, William K.; Russell, David H.; Lee, Kyongbum; Jayaraman, Arul

2008-01-01

34

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

35

Phagocytosis of Latex Particles in Relation to the Cell Cycle in 3T3 Cells  

Microsoft Academic Search

Cultures of 3T3 cells, synchronised by serum deprivation, were shown to phagocytose latex beads of 0.81 ?mdiameter more avidly in the G1 phase of the cell cycle. The maximum rate of about 0.12 particles\\/cell\\/hour was recorded 12 h after stimulation with 10% fetal bovine serum. After 24 h, when mitosis was beginning, the uptake fell to just above the unstimulated

P. A. Riley; R. T. Dean

1978-01-01

36

Stimulatory effect of daidzein in osteoblastic MC3T3-E1 cells  

Microsoft Academic Search

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

Emi Sugimoto; Masayoshi Yamaguchi

2000-01-01

37

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

38

Molecular Mechanisms of Apoptosis Induced by Ajoene in 3T3-L1 Adipocytes  

Microsoft Academic Search

Objective: Determine the biochemical pathways involved in induction of apoptosis by ajoene, an organosulfur compound from garlic.Research Methods and Procedures: Mature 3T3-L1 adipocytes were incubated with ajoene at concentrations up to 200 ?M. Viability and apoptosis were quantified using an MTS-based cell viability assay and an enzyme-linked immunosorbent assay for single-stranded DNA (ssDNA), respectively. Intracellular reactive oxygen species (ROS) production

Jeong-Yeh Yang; Mary Anne Della-Fera; Cass Nelson-Dooley; Clifton A. Baile

2006-01-01

39

Induction of Calcification in MC3T3-E1 Cells by Inorganic Polyphosphate  

Microsoft Academic Search

Relatively large amounts of inorganic polyphosphate [poly(P)] (400 ?M) have been found in normal osteoblasts. The effect of poly(P) with an average chain length of 65 phosphate residues on cell calcification was therefore investigated with the use of MC3T3-E1 cells. Expression of both osteopontin and osteocalcin was induced by poly(P) (0.1 ~ 1 mM), and cells treated with poly(P) were

Y. Kawazoe; T. Shiba; R. Nakamura; A. Mizuno; K. Tsutsumi; T. Uematsu; M. Yamaoka; M. Shindoh; T. Kohgo

2004-01-01

40

Zinc deficiency induces oxidative stress and AP1 activation in 3T3 cells  

Microsoft Academic Search

It has been postulated that one mechanism underlying zinc deficiency–induced tissue alterations is excessive cellular oxidative damage. In the present study we investigated if zinc deficiency can induce oxidative stress in 3T3 cells and trigger select intracellular responses that have been associated to oxidative stress. Cells were exposed to control media or to chelated media containing 0.5, 5, or 50

Patricia I Oteiza; Michael S Clegg; M. Paola Zago; Carl L Keen

2000-01-01

41

Inhibitory Effects of Fucoidan in 3T3-L1 Adipocyte Differentiation  

Microsoft Academic Search

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

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

2009-01-01

42

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

43

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. PMID:24288561

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

44

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. PMID:24795772

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

2014-01-01

45

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. PMID:24391675

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

2013-01-01

46

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

47

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. PMID:23661997

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

2013-01-01

48

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

PubMed

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

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

2012-12-01

49

Exogenous MC3T3 Preosteoblasts Migrate Systemically and Mitigate the Adverse Effects of Wear Particles  

PubMed Central

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

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

2012-01-01

50

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

PubMed

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

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

2013-10-15

51

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

52

Biochem.J. (1995)305. 621~26 (Printedin GreatBritain) Phorbolesterstimulatescholineuptakein Swiss3T3fibroblastsfollowing  

E-print Network

specificity of this response, a study was undertaken in Swiss 3T3 fibroblast cells, which normally express very low levels of PKCa.. A retroviral expression system was used to introduce the genes for PKCa into phosphatidylcholine (PtdCho) in a time- and concentration-dependent manner in wild-type NIH 3T3 fibroblasts

Wurtman, Richard

53

Weighing feeder retrofits and upgrades  

Microsoft Academic Search

The purpose of this paper is to provide the reader with a detailed overview of important application points when considering a weighing feeder retrofit. The focus is on weighing feeders used for both weighing and control where the feed rate is controlled by varying the belt speed. The information is also applicable to noncontrolled constant speed weigh belts. This paper

P. Farley

1994-01-01

54

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

55

Anti-adipogenic constituents from Dioscorea opposita in 3T3-L1 cells.  

PubMed

We previously reported the lipase inhibitory activity of the n-BuOH fraction of Dioscorea opposita (DOB) and its isolates. This study sought to evaluate their anti-adipogenic activity in terms of their effects on the adipogenic transcription factors peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer binding protein ? (C/EBP?) as well as phosphorylated AMP-activated protein kinase (p-AMPK) and carnitine palmitoyl transferase-1 (CPT-1). DOB apparently attenuated 3T3-L1 adipocyte differentiation (33.6% decrease at 20 µg/mL). In addition, a marked decrease (90.4%) in the expression of PPAR? was observed in the DOB-treated 3T3-L1 cells. Four isolates from DOB: (4E,6E)-1,7-bis(4-hydroxyphenyl)-4,6-heptadien-3-one (1), (3R,5R)-1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanediol (2), batatasin I (3), and (1E,4E,6E)-1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one (4), suppressed adipocyte differentiation by inhibiting PPAR? at 20 µM (85.9%, 68.6%, 76.2%, and 90.2% decrease, respectively) and C/EBP? (51.7%, 3.1%, 20.9%, and 59.8% decrease, respectively). Batatasin I was found to increase p-AMPK and CPT-1 at a concentration of 20 µM in 3T3-L1 adipocytes, resulting in inhibiting adipogenesis. Taken together, batatasin I might be responsible for the anti-adipogenic effect of DOB via inhibition of PPAR? and C/EBP? and activation of p-AMPK and CPT-1. PMID:25273391

Yang, Min Hye; Chin, Young-Won; Chae, Hee-Sung; Yoon, Kee Dong; Kim, Jinwoong

2014-01-01

56

Insulin Increases Tristetraprolin and Decreases VEGF Gene Expression in Mouse 3T3–L1 Adipocytes  

Microsoft Academic Search

Objectives:Tristetraprolin (TTP) family proteins (TTP\\/ZFP36; ZFP36L1, ZFP36L2, ZFP36L3) destabilize adenylate uridylate–rich element–containing mRNAs encoding cytokines, such as tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Little is known about the expression and insulin regulation of TTP and related genes in adipocytes. We analyzed the relative abundance of TTP family mRNAs in 3T3-L1 adipocytes compared to RAW264.7 macrophages and

Heping Cao; Joseph F. Urban; Richard A. Anderson

2008-01-01

57

Freeze-fracture of 3T3 cells for high-resolution scanning electron microscopy.  

PubMed

Triton-extracted, freeze-fractured 3T3 cells have been examined in the Hitachi S-900 field-emission SEM, after light platinum coating, at low beam voltage to evaluate the performance of the microscope under these conditions. For unstained material fixed in glutaraldehyde alone, high-resolution images can be obtained, at accelerating voltages of 1.5-5kV, after rotary deposition of platinum to an average thickness of 1.5-3 nm. Comparisons are made between these results and those of studies by TEM of deep-etch replicas of similar material previously published. PMID:3172183

Haggis, G H; Pawley, J B

1988-06-01

58

CTRP3 modulates the expression and secretion of adipokines in 3T3-L1 adipocytes.  

PubMed

The objective of this study was to investigate the impact of C1q/TNF related protein 3 (CTRP3), a novel adipokine, on the expression and secretion of adiponectin, leptin, visfatin, and apelin in 3T3-L1 adipocytes. The effect of insulin resistance on the impact was also investigated. 3T3-L1 adipocytes were treated with different concentrations (0, 10, 50, 250, 1250 ng/ml) CTRP3 for 12 h, and with 250 ng/ml CTRP3 for different times (0, 6, 12, 24, 48 h). The expression of adipokines between normal and insulin resistant adipocytes, as well as between the adipocytes pre-treated with and without Compound C were compared. The secretion and gene expression of the adipokines were detected by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR), respectively. The relative expression of AMPK (thr172) was detected by western blot analysis. With the increase in CTRP3 concentration or the duration of the treatment, the secretion of adiponectin, leptin, visfatin and apelin were all increased accordingly, which was significant under the treatment with 250 ng/ml and 1250 ng/ml CTRP3 for 12 h as well as 250 ng/ml CTRP3 for 12 h, 24 h and 48 h. Gene expression showed a similar trend. The secretion and gene expression of adipokines in insulin resistant adipocytes were all decreased significantly in comparison with that of normal adipocytes. The secretion secretion and gene expression of adiponectin, and the relative expression of AMPK (thr172) in adipocytes pre-treated with Compound C were decreased significantly in comparison with that in adipocytes without Compound C pretreatment. Thus, CTRP3 increased the expression and secretion of adiponectin, leptin, visfatin, and apelin in 3T3-L1 adipocytes, while insulin resistance inhibited the effects. CTRP3 up-regulated the expression of adiponectin in 3T3-L1 adipocytes through AMPK signaling pathway. PMID:25168658

Li, Xin; Jiang, Li; Yang, Miao; Wu, Yu-Wen; Sun, Su-Xin; Sun, Jia-Zhong

2014-08-27

59

Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media  

PubMed Central

Purpose: An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as nonhuman sera and feeder cells. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions when transplanted into the de-epithelialized limbal corneal surface. Methods: Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low-calcium medium and set in an air-liquid interface culture for 14 days. The resulting multilayered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry. Results: At graft time the cultured epithelial sheet consisted of 6–8 layers of properly stratified epithelium that displayed a CK19+/MUC5AC+/ CK3 -/CK12- phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (?4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival goblet cell mucin (MUC5AC) was not visible. The engrafted epithelium stained positively for the anti-human nuclei antibody, confirming that the epithelial cells on the rabbit corneas were of human origin. Conclusions: Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment. PMID:24357922

Jeon, Sohee; Choi, Seong Hyun; Wolosin, J. Mario; Joo, Choun-Ki

2013-01-01

60

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

PubMed

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

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

2014-11-01

61

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. PMID:24049664

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

2013-01-01

62

Physiological induction and reversal of focus formation and tumorigenicity in NIH 3T3 cells.  

PubMed Central

NIH 3T3 cells undergo morphological transformation in response to conditions of constrained growth, such as occur in low serum concentrations or at confluence. Transformation is expressed in a small fraction of the cells by the appearance of discrete foci of multiplying cells on a confluent monolayer of quiescent cells. We isolated and expanded cell populations from three dense and three light foci. Cells from each of these populations efficiently reproduced foci of the same morphotype when grown on a background of nontransformed NIH 3T3 cells. Using cultures derived from one of the dense foci (subline D/2), we found that the number of focus-forming units was stable and the cells remained tumorigenic when they were subjected to repeated thrice-weekly passage in 2% calf serum. However, equivalent passage in 10% calf serum eventually rendered the cells incapable of both focus production and tumor formation. The results show that the capacity to produce tumors as well as morphological transformation are produced as a response to physiological constraints of growth and/or metabolism in the absence of carcinogens and that both properties can be reversed by lifting the constraints. This behavior is typical of an adaptational response and, taken together with other supporting evidence, shows that tumorigenesis does not require conventional genetic alteration. Images PMID:2263601

Rubin, A L; Arnstein, P; Rubin, H

1990-01-01

63

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

PubMed Central

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

Yang, Soo Jin; Park, Na-Young

2014-01-01

64

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

PubMed

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

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

2014-03-25

65

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells.  

PubMed

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N(1)-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis. PMID:21809076

Ishii, Ikumi; Ikeguchi, Yoshihiko; Mano, Hiroshi; Wada, Masahiro; Pegg, Anthony E; Shirahata, Akira

2012-02-01

66

Umbelliferone increases the expression of adipocyte-specific genes in 3?t3-l1 adipocyte.  

PubMed

Umbelliferone (UMB), a natural product of coumarin family, has been shown to reduce blood glucose and to improve lipid profiles in streptozotocin (STZ)-induced diabetic rats. Our objective was to examine the effect of UMB on adipogenesis by investigating its stimulatory effect on lipid accumulation and mRNA expression of adipogenic transcription factors and adipocyte-specific genes in 3?T3-L1 preadipocyte culture. An Oil Red O staining was used to monitor lipid accumulation, and we found that UMB treatment at concentration range of 10-100??M significantly increased lipid accumulation of differentiating 3?T3-L1 cells. At the molecular level of adipogenesis, we examined the mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor ?, CCAAT/enhancer-binding protein ?, and sterol regulatory element-binding protein 1c. Those transcription factors were increased by UMB at 10-100??M. Interestingly, UMB also stimulated the mRNA expression of adipocyte-specific genes, adipocyte fatty acid-binding protein, lipoprotein lipase, fatty acid synthase, fatty acid translocase, and adiponectin. Our findings indicate that the stimulatory effect of UMB on adipocyte differentiation likely occurs through up-regulation of adipogenic transcription factors and downstream adipocyte-specific gene expression. PMID:24853372

Naowaboot, Jarinyaporn; Chung, Choon Hee; Choi, Ran; Pannangpetch, Patchareewan

2014-11-01

67

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

PubMed Central

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

1992-01-01

68

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

69

Lactoferrin suppress the adipogenic differentiation of MC3T3-G2/PA6 cells.  

PubMed

Lactoferrin accelerates the differentiation of osteogenic and chondrogenic lineage cells, whereas it inhibits the myogenic and adipogenic differentiation of pluripotent mesenchymal cells; however, the effect of lactoferrin on the differentiation of preadipocytes is unknown. In this study, we examined the effect of lactoferrin on adipogenic differentiation using a mouse preadipocyte cell line, MC3T3-G2/PA6. The cells were cultured in differentiation medium with or without lactoferrin to induce cellular differentiation. The cell lineage was then determined by Oil Red O staining, real-time PCR screening for the mRNA expression of phenotype-specific markers, and Western blot analysis. The number of Oil Red O-positive lipid droplets decreased following treatment with lactoferrin, as did the mRNA expression of C/EBPalpha, PPARgamma, aP2, and adiponectin. Furthermore, our Western blot data revealed a decrease in PPARgamma expression attributable to lactoferrin exposure. These results suggest that lactoferrin suppresses the adipogenic differentiation of MC3T3-G2/PA6 cells. PMID:19106469

Yagi, Motohiko; Suzuki, Naoto; Takayama, Tadahiro; Arisue, Masatoshi; Kodama, Takuya; Yoda, Yasushi; Numasaki, Hikaru; Otsuka, Kichibee; Ito, Koichi

2008-12-01

70

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

PubMed Central

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

2014-01-01

71

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

72

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. PMID:23506355

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

73

Light-microscopic studies of 3T3 cell plasma membrane alterations mediated by melittin.  

PubMed

Various light microscopic techniques were used to study the effect of melittin, a major toxic constituent of honey bee venom, on plasma membranes of 3T3 mouse fibroblasts. Bright-field light microscopy and Trypan Blue dye exclusion were used to demonstrate changes in membrane permeability after exposure to melittin. Differential interference contrast (DIC) microscopy showed that membrane vesiculation induced by melittin was dose dependent. Using both fluorescent lipid and glycoprotein markers, we found that membrane vesicles were primarily composed of lipids. A sequence of events associated with vesicle formation was depicted by DIC and fluorescence microscopy. Confocal laser scanning fluorescence microscopy demonstrated a translocation of membrane glycoproteins from the plasma membrane to the cytosol following melittin treatment. The significance of membrane vesiculation and translocation of membrane glycoproteins in damaged cells is discussed. PMID:9028005

Lo, W C; Henk, W G; Enright, F M

1997-01-01

74

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

75

MORPHOLOGICAL CHANGES OF THE 3T3-L1 FIBROBLAST PLASMA MEMBRANE UPON DIFFERENTIATION TO THE ADIPOCYTE FORM  

Microsoft Academic Search

SUMMARY By quantitative evaluation carried out on freeze-fracture replicas, we have investigated the changes in plasma membrane organization as the 3T3-L1 fibroblast phenotype differentiates into the 3T3-L1 adipocyte form. As differentiation takes place there is a dramatic change in overall appearance of the cell as it acquires large lipid-laden vacuoles. On freeze-fracture replicas we find: (1) a ninefold increase in

JING YU FAN; TEAN-LOUIS CARPENTIER; EMMANUEL VAN OBBERGHEN; CARL GRUNFELD; PHILLIP GORDEN; LELIO ORCI

1983-01-01

76

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

PubMed

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

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

2008-01-01

77

The expression profile of PKC isoforms during MC3T3-E1 differentiation.  

PubMed

Protein kinase C (PKC) is a family of kinases whose isoforms show subtle differences in physiological and biochemical responses, with their expression being cell- specific. We hypothesize that there may be a specific profile of expression of PKC isoforms in differentiating osteoblastic cells (OBC) with individual isoforms having specific functions. Herein, the MC3T3-E1 cell line was used as a differentiating model, which was induced from the pre-osteoblast stage to mature osteoblast and characterized with several phenotypic markers, including alkaline phosphatase activity, osteocalcin and bone sialoprotein. The expression of PKC isoforms was monitored using Western blot analysis. Upon induction of osteogenesis, the intracellular localization of PKC eta and theta was determined using immunofluorescence. Lastly, the effect of P38 MAP kinase inhibition was determined using SB203580. Results show 1) PKC alpha, delta, lambda were all highly expressed in MC3T3-E1 osteoblastic cells, 2) the expression of PKC theta was significantly down-regulated upon induction of osteoblastic differentiation; 3) PKC eta was non-detectable at certain cell culture days; however, was up-regulated as the cells transit from each differentiation phase. The increased expression of PKC eta correlated with increases in OC, BSP levels and alkaline phosphatase activity. Immunofluorescence procedure confirmed the Western blot results with an increase in PKC eta and a decrease in PKC theta upon osteogenic stimulation. The inhibition of p38 resulted in a marked down-regulation of PKC eta. The data demonstrate that there is a specific profile of expression of PKC isoforms in differentiating osteoblasts; the different expression pattern of individual isoforms may be either a consequence of the differentiation itself or plays a role in the regulatory mechanism of osteoblastic differentiation. This study has provided primary information on the temporal pattern of expression of PKC isoforms in the differentiating osteoblast and further insight into their possible role in osteoblastic cell maturation. PMID:16685425

Lampasso, J D; Chen, Wen; Marzec, N

2006-06-01

78

Online monitoring of BALB/3T3 metabolism and adhesion with multiparametric chip-based system.  

PubMed

A multiparametric chip-based system was employed to measure cell adhesion, metabolism, and response to metal compounds previously classified as cytotoxic in immortalized mouse fibroblasts (BALB/3T3 cell line). The system measures in parallel, online, and in label-free conditions the extracellular acidification rates (with pH-sensitive field effect transistors [ISFETs]), the cellular oxygen consumption (with amperometric electrode structures [Clark-type sensors]), and cell adhesion (with impedimetric interdigitated electrode structures [IDESs]). The experimental protocol was optimized to monitor metabolism and adhesion of the BALB/3T3 cell line. A total of 70,000 cells and a bicarbonate buffer-free running low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal clone serum III and 1mM Hepes were selected to maintain cells in good conditions on the chip during the measurements performed under perfusion conditions. Cells were exposed to sodium arsenite, cadmium chloride, and cis-platinum at concentrations ranging from 1 to 100 microM. The kinetics of cell response to these compounds was analyzed and suggests that the Clark-type sensors can be more sensitive than IDESs and ISFETs in detecting the presence of high chemical concentration when short exposure times (i.e., 2h) are considered. The cytotoxicity data obtained from the online measurements of acidification, respiration, and adhesion at 24h compare well, in terms of half-inhibition concentration values (IC(50)), with the ones obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and colony-forming efficiency (CFE) assay. The results show a good sensitivity of the system combined with the advantages of the online and label-free detection methods that allow following cell status before, during, and after the treatment in the same experiment. PMID:17709091

Ceriotti, L; Kob, A; Drechsler, S; Ponti, J; Thedinga, E; Colpo, P; Ehret, R; Rossi, F

2007-12-01

79

Toxicity of silver nanoparticle in rat ear and BALB/c 3T3 cell line.  

PubMed

BackgroundSilver nanoparticles (AgNPs) displayed strong activities in anti-bacterial, anti-viral, and anti-fungal studies and was reportedly efficient in treating otitis media .The potential impact of AgNPs on the inner ear was missing.ObjectiveAttempted to evaluate the potential toxicity of AgNPs in the inner ear, middle ear, and external ear canal after transtympanic injection in rats.ResultsIn in vitro studies, the IC50 for AgNPs in neutral red uptake assay was lower than that in NAD(P)H-dependent cellular oxidoreductase enzyme assay (WST-1) and higher than that in total cellular ATP and nuclear membrane integrity (propidium iodide) assessments. In in vivo experiments, magnetic resonance imaging (MRI) showed that significant changes in the permeability of biological barriers occurred in the middle ear mucosa, the skin of the external ear canal, and the inner ear at 5 h post-transtympanic injection of AgNPs at concentrations ranging from 20 ¿g/ml to 4000 ¿g/ml. The alterations in permeability showed a dosage-response relationship, and were reversible. The auditory brainstem response showed that 4000 ¿g/ml AgNPs induced hearing loss with partial recovery at 7 d, whereas 20 ¿g/ml caused reversible hearing loss. The functional change in auditory system was in line with the histology results. In general, the BALB/c 3T3 cell line is more than 1000 times more sensitive than the in vivo studies. Impairment of the mitochondrial function was indicated to be the mechanism of toxicity of AgNPs.ConclusionThese results suggest that AgNPs caused significant, dose-dependent changes in the permeability of biological barriers in the middle ear mucosa, the skin of the external ear canal, and the inner ear. In general, the BALB/c 3T3 cell line is more than 1000 times more sensitive than the in vivo studies. The rat ear model might be expended to other engineered nanomaterials in nanotoxicology study. PMID:25467963

Zou, Jing; Feng, Hao; Mannerström, Marika; Heinonen, Tuula; Pyykkö, Ilmari

2014-12-01

80

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

81

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

PubMed Central

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

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

2013-01-01

82

Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts.  

PubMed Central

Mechanical interactions between cell and substrate are involved in vital cellular functions from migration to signal transduction. A newly developed technique, traction force microscopy, makes it possible to visualize the dynamic characteristics of mechanical forces exerted by fibroblasts, including the magnitude, direction, and shear. In the present study such analysis is applied to migrating normal and transformed 3T3 cells. For normal cells, the lamellipodium provides almost all the forces for forward locomotion. A zone of high shear separates the lamellipodium from the cell body, suggesting that they are mechanically distinct entities. Timing and distribution of tractions at the leading edge bear no apparent relationship to local protrusive activities. However, changes in the pattern of traction forces often precede changes in the direction of migration. These observations suggest a frontal towing mechanism for cell migration, where dynamic traction forces at the leading edge actively pull the cell body forward. For H-ras transformed cells, pockets of weak, transient traction scatter among small pseudopods and appear to act against one another. The shear pattern suggests multiple disorganized mechanical domains. The weak, poorly coordinated traction forces, coupled with weak cell-substrate adhesions, are likely responsible for the abnormal motile behavior of H-ras transformed cells. PMID:11259288

Munevar, S; Wang , Y; Dembo, M

2001-01-01

83

MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization  

PubMed Central

The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0?nm) and sandblasting (Ra: approximately 1.0??m), and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells. PMID:22675359

Hayakawa, Tohru; Yoshida, Eiji; Yoshimura, Yoshitaka; Uo, Motohiro; Yoshinari, Masao

2012-01-01

84

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

PubMed

Microgravity decreases the differentiation of osteoblast. However, as this process is multistage and complex, the mechanism by which microgravity inhibits osteoblast differentiation is still unclear. We have previously found that 24?h acute treatment of simulated microgravity (SM) in a random positioning machine (RPM) significantly inhibited the differentiation of preosteoblasts and have explored whether osteoblasts show different response to microgravity condition at other stages, such as the mineralizing-stage. Murine MC3T3-E1 preosteoblasts induced for osteogenic differentiation for 7 days were cultured either under normal gravity or SM conditions for 24?h. SM treatment significantly suppressed mineralized nodule formation. Alkaline phosphatase (ALP) activity was dramatically decreased, and the expression of ALP gene was downregulated. Expression of well-known markers and regulators for osteoblasts differentiation, including osteocalcin (OC), type I collagen ?1 (Col I?1), dentin matrix protein 1 (DMP1) and runt-related transcription factor 2 (Runx2), were downregulated. Western blot analysis showed that the phosphorylated extracellular signal-regulated kinase (p-ERK) level was lower under SM condition. Thus, the initiation of osteoblast mineralization is suppressed by SM condition, and the suppression may be through the regulation of ALP activity and the osteogenic gene expression. ERK signaling might be involved in this process. These results are relevant to the decrease of osteoblast maturation and bone formation under microgravity condition. PMID:25318973

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

2014-10-16

85

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

86

Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells.  

PubMed Central

Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size-fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 A in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 A, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes. PMID:3474634

Luby-Phelps, K; Castle, P E; Taylor, D L; Lanni, F

1987-01-01

87

Puerarin enhances adipocyte differentiation, adiponectin expression, and antioxidant response in 3T3-L1 cells.  

PubMed

Puerarin, a major isoflavone glycoside from Kudzu root (Pueraria lobata), has been reported to exert antihyperglycemic and antioxidant effects and thus have pharmacological actions in the treatment of diabetes and cardiovascular diseases. We investigated the effects of puerarin on the changes of key gene expression associated with adipocyte differentiation and insulin sensitivity and link to cellular antioxidant response pathways. Puerarin treatment significantly enhanced differentiation of 3T3-L1 preadipocytes accompanying increased lipid accumulation and glucose-6-phosphate dehydrogenase (G6PDH) activity. At a molecular level, puerarin upregulated mRNA expression of peroxisome proliferator-activated receptor ? (PPAR?) and its target genes, an adipocyte-specific fatty acid binding protein (aP2) and GLUT4. Puerarin also caused a significant increase in mRNA level of adiponectin, an important insulin-sensitizing adipocytokine that is downregulated in insulin-resistant and diabetic states. In addition, treatment with puerarin was found to upregulate mRNA levels of G6PDH, glutathione reductase, and catalase, all of which are important for endogenous antioxidant responses. These data suggest that the hypoglycemic effects of puerarin can be attributed to the upregulation of PPAR? and its downstream target genes, GLUT4 and adiponectin expression, leading to increased glucose utilization. Puerarin may also be effective in preventing the rise of oxidative stress during adipocyte differentiation by increasing endogenous antioxidant responses. PMID:20806284

Lee, Ok-Hwan; Seo, Dong-Ho; Park, Cheon-Seok; Kim, Young-Cheul

2010-01-01

88

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. PMID:24722056

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

2014-01-01

89

Dual function of IL-33 on proliferation of NIH-3T3 cells.  

PubMed

The interleukin-33 (IL-33)-ST2L signaling pathway has been shown to play important roles in the field of immunology, especially as a trigger for allergic reactions such as bronchial asthma. However, coming back to the original finding that the ST2 gene is induced during initiation of the cell cycle of fibroblastic cell lines, the possible functions of the ST2 gene products and their specific ligand, IL-33, in the field of cell growth regulation are still interesting problems to be solved. In this study, we used NIH-3T3 mouse cell line and added IL-33 before and after cell proliferation assay, which revealed the dual function of IL-33. When IL-33 was added to the confluent cells before the start of cell proliferation, it suppressed the cell growth concentration-dependently. On the other hand, if IL-33 was added after the start of cell proliferation, it enhanced the cell growth. The negative effect of IL-33 on cell proliferation is a novel finding and would provide an important clue to the roles of IL-33 and ST2/ST2L in growth regulation. PMID:25573803

Tominaga, Shin-Ichi; Tago, Kenji; Tsuda, Hidetoshi; Komine, Mayumi

2015-03-01

90

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

91

TR4 promotes fatty acid synthesis in 3T3-L1 adipocytes by activation of pyruvate carboxylase expression.  

PubMed

We show that testicular orphan nuclear receptor 4 (TR4) increases the expression of pyruvate carboxylase (PC) gene in 3T3-L1 adipocytes by direct binding to a TR4 responsive element in the murine PC promoter. While TR4 overexpression increased PC activity, oxaloacetate (OAA) and glycerol levels with enhanced incorporation of (14)C from (14)C-pyruvate into fatty acids in 3T3-L1 adipocytes, PC knockdown by short interfering RNA (siRNA) or inhibition of PC activity by phenylacetic acid (PAA) abolished TR4-enhanced fatty acid synthesis. Moreover, TR4 microRNA reduced PC expression with decreased fatty acid synthesis in 3T3-L1 adipocytes, suggesting that TR4-mediated enhancement of fatty acid synthesis in adipocytes requires increased expression of PC gene. PMID:25240193

Park, Sung-Soo; Kim, Seung-Jin; Choi, Hojung; Chang, Chawnshang; Kim, Eungseok

2014-11-01

92

Enhancement of ajoene-induced apoptosis by conjugated linoleic acid in 3T3-L1 adipocytes  

Microsoft Academic Search

Ajoene has been shown to induce apoptosis in 3T3-L1 adipocytes. In this report the effects on apoptosis of combinations of\\u000a ajoene and trans-10, cis-12 conjugated linoleic acid (t10,c12CLA) in 3T3-L1 adipocytes were investigated. Although t10,c12CLA alone had no effect, ajoene\\u000a plus t10,c12CLA reduced cell viability more than ajoene alone at 24 h (59.1 vs. 85.9% of control, respectively; ppp<0.01). Immunoblotting analysis

Jeong-Yeh Yang; Mary Anne Della-Fera; Dorothy B. Hausman; Clifton A. Baile

2007-01-01

93

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

94

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. PMID:24917384

2014-01-01

95

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

PubMed

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

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

2015-01-01

96

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

Microsoft Academic Search

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

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

1989-01-01

97

Gene Expression Profiling of 3T3-L1 Adipocytes Expressing the Mitochondrial Uncoupling Protein 1 (UCP1)  

E-print Network

adipocytes. Murine 3T3-L1 preadipocytes, either expressing UCP1 or control (i.e., no UCP1) were cultured to confluence. On day 2 post confluence, the preadipocytes were induced to differentiate using a standard adipogenic cocktail consisting of insulin...

Senocak, Fatih

2006-07-11

98

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

PubMed Central

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

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

2015-01-01

99

Effects of microgravity on c-fos gene expression in osteoblast-like MC3T3-E1 cells  

NASA Astrophysics Data System (ADS)

The paper summarizes the data on proliferation and gravity-related gene expression of osteoblasts that were obtained from an experiment conducted under simulated and real microgravity conditions.Simulated microgravity conditions obtained in a clinostat depress proliferation of both osteoblast-like MC3T3-E1 and HeLa carcinoma cells. This depression of proliferation occurs in a collagen gel culture in which the flow of culture medium by rotation may be reduced. Interestingly, MC3T3-E1 cells which are probably one of target cells to microgravity are more sensitive than the HeLa cells. Simulated microgravity inhibited the epidermal growth factor (EGF)-induced c-fos gene expression in the MC3T3-E1 cells. To examine in detail the effect of real microgravity on the EGF signal transduction cascade in osteoblasts, MC3T3-E1 cells were cultured in the Cell Culture Experiment Module of the sounding rocket TR-1A6. The EGF-induced c-fos expression in cells was depressed under short-term microgravity conditions in the sounding rocket, while the phosphorylation of mitogen-activated protein kinase (MAPK) was not affected compared with the controls grown on the ground. These results suggest that an action site of microgravity in the signal transduction pathway may be downstream of MAPK.

Sato, A.; Hamazaki, T.; Oomura, T.; Osada, H.; Kakeya, M.; Watanabe, M.; Nakamura, T.; Nakamura, Y.; Koshikawa, N.; Yoshizaki, I.; Aizawa, S.; Yoda, S.; Ogiso, A.; Takaoki, M.; Kohno, Y.; Tanaka, H.

1999-01-01

100

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

Microsoft Academic Search

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

KEITH R. PORTER; GEORGE J. TODARO; VIRGINIA FONTE

1973-01-01

101

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

102

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

SciTech Connect

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

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

2013-11-01

103

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

PubMed

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

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

2010-03-01

104

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

SciTech Connect

Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the size distribution of the lipid droplets in immature and mature cells. • We used the Monte Carlo simulation approach, simulating 10 thousand of fusion events. • Four different interaction models between the lipid droplets were tested. • The best model which mimics the experimental measures was selected.

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

2014-02-15

105

Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures  

PubMed Central

Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we show in osteoblast cultures producing an abundant collagenous matrix that normally shows robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and ?-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10 ?M PolyP. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as measured by Pi release. Importantly, at the concentrations of polyphosphates used in this study which inhibited bone cell culture mineralization, the polyphosphates competitively saturated TNAP, thus potentially interfering with its ability to hydrolyze mineralization-inhibiting pyrophosphate (PPi) and mineralizing-promoting ?-glycerophosphate (in cell culture). In the biological setting, TNAP may regulate mineralization by shielding the essential inhibitory substrate pyrophosphate from TNAP degradation, and in the same process, delay the release of phosphate from this source. In conclusion, the inhibition of mineralization by polyphosphates is shown to occur via direct binding to apatitic mineral and by mixed inhibition of TNAP. PMID:23337041

Hoac, Betty; Kiffer-Moreira, Tina; Millán, José Luis; McKee, Marc D.

2013-01-01

106

Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures.  

PubMed

Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we demonstrate in osteoblast cultures producing an abundant collagenous matrix that normally show robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and ?-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10?M. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as measured by Pi release. Importantly, at the concentrations of polyphosphates used in this study which inhibited bone cell culture mineralization, the polyphosphates competitively saturated TNAP, thus potentially interfering with its ability to hydrolyze mineralization-inhibiting pyrophosphate (PPi) and mineralizing-promoting ?-glycerophosphate (in cell culture). In the biological setting, polyphosphates may regulate mineralization by shielding the essential inhibitory substrate pyrophosphate from TNAP degradation, and in the same process, delay the release of phosphate from this source. In conclusion, the inhibition of mineralization by polyphosphates is shown to occur via direct binding to apatitic mineral and by mixed inhibition of TNAP. PMID:23337041

Hoac, Betty; Kiffer-Moreira, Tina; Millán, José Luis; McKee, Marc D

2013-04-01

107

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

PubMed

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

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

2014-10-01

108

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. PMID:24926875

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

2014-01-01

109

Response of osteoblast-like MC3T3-E1 cells on bioactive titanium fabricated by a chemical treatment process using a calcium-phosphate slurry.  

PubMed

We recently developed a chemical treatment process using a calcium-phosphate slurry for fabricating new layers consisting of hydroxyapatite and titanium dioxide (TiO2) on titanium (Ti) substrate. In this study, the response of osteoblast-like MC3T3-E1 cells on Ti substrate treated with a calcium-phosphate slurry was investigated to elucidate its behavior in a biological environment. The cellular adhesiveness and proliferation capacity did not differ significantly between the treated and untreated Ti substrates, suggesting that the slurry treatment did not cause cytotoxicity. The slurry treatment did not affect the increase in alkaline phosphatase activity after the induction of cell differentiation, whereas it was found to be significantly advantageous for the calcification behavior on the slurry-treated Ti substrate. In consequence, the hard-tissue compatibility of Ti is expected to be improved by the chemical treatment process using a calcium-phosphate slurry. PMID:24307316

Ohtsu, Naofumi; Hirano, Mitsuhiro; Arai, Hirofumi

2014-11-01

110

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

PubMed Central

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. [BMB Reports 2013; 46(12): 582-587] PMID:24195790

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

2013-01-01

111

Effects of orexin A on proliferation, survival, apoptosis and differentiation of 3T3-L1 preadipocytes into mature adipocytes.  

PubMed

Metabolic activities of orexin A (OXA) in mature adipocytes are mediated via PI3K/PKB and PPAR?. However, the effects of OXA on preadipocytes are largely unknown. We report here that OXA stimulates the proliferation and viability of 3T3-L1 preadipocytes and protects them from apoptosis via ERK1/2, but not through PKB. OXA reduces proapoptotic activity of caspase-3 via ERK1/2. Inhibition of ERK1/2 prevents the differentiation of preadipocytes into adipocytes. Unlike insulin, neither short-term nor prolonged exposure of 3T3-L1 preadipocytes to OXA induces preadipocyte differentiation to adipocytes, despite increased ERK1/2 phosphorylation. Unlike insulin, OXA fails to activate PKB, which explains its inability to induce the differentiation of preadipocytes. PMID:23123090

Skrzypski, M; Kaczmarek, P; Le, T T; Wojciechowicz, T; Pruszy?ska-Oszmalek, E; Szczepankiewicz, D; Sassek, M; Arafat, A; Wiedenmann, B; Nowak, K W; Strowski, M Z

2012-11-30

112

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

PubMed Central

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

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

2009-01-01

113

Citrus aurantium flavonoids inhibit adipogenesis through the Akt signaling pathway in 3T3-L1 cells  

PubMed Central

Background Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF) on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells. Methods During adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 ?g/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation. Results The insulin-induced expression of C/EBP? and PPAR? mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBP?, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3? (Ser9), which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes. Conclusions In the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBP? and subsequently inhibits the activation of PPAR? and C/EBP?. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3? phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation. PMID:22471389

2012-01-01

114

A Proteomic Approach for Identification of Secreted Proteins during the Differentiation of 3T3-L1 Preadipocytes to Adipocytes  

Microsoft Academic Search

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-reg- ulated during this

Irina Kratchmarova; Dario E. Kalume; Blagoy Blagoev; Philipp E. Scherer; Alexandre V. Podtelejnikov; Henrik Molina; Perry E. Bickel; Jens S. Andersen; Minerva M. Fernandez; Jacob Bunkenborg; Peter Roepstorff; Karsten Kristiansen; Harvey F. Lodish; Matthias Mann; Akhilesh Pandey

2002-01-01

115

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

Microsoft Academic Search

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

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

116

Stimulation by bone sialoprotein of calcification in osteoblast-like MC3T3-E1 cells  

Microsoft Academic Search

Bone sialoprotein (BSP) containing an Arg-Gly-Asp cell-binding sequence was purified from bovine bone 4 M guanidine-HCl extract after HCl demineralization by a series of chromatographic procedures. When this protein was coated on culture dishes in the presence of type I collagen, it increased both DNA content and alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells, and stimulated calcification in the

H.-Y. Zhou; H. Takita; R. Fujisawa; M. Mizuno; Y. Kuboki

1995-01-01

117

Differential regulation of the stearoyl-CoA desaturase genes by thiazolidinediones in 3T3-L1 adipocytes  

Microsoft Academic Search

Two stearoyl-CoA desaturase (SCD) isoforms can be expressed during the differentiation of 3T3-L1 preadi- pocytes into adipocytes. Here we report on the effects of the peroxisome proliferator-activated receptor g ligand tro- glitazone (TRO) on scd1 and scd2 mRNA levels as deter- mined by Northern blotting, on SCD protein expression as determined by Western blotting, and on total lipid composi- tion

Young-Cheul Kim; F. Enrique Gomez; Brian G. Fox; James M. Ntambi

118

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

119

Egg yolk soluble protein stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells.  

PubMed

We determined the effects of yolk water-soluble protein (YSP) on bone formation in pre-osteoblastic MC3T3-E1 cells. YSP (50-5,000 microg/ml) increased cell proliferation and collagen content. Alkaline phosphatase (ALP) activity was also increased by YSP treatment. After enhancement of ALP activity, significant augmentation of calcification was observed. These results suggest that YSP is a promising agent for the prevention and treatment of bone loss. PMID:17485841

Ji, MinYoung; Leem, Kang-Hyun; Kim, Mujo; Kim, Hye Kyung

2007-05-01

120

Emodin with PPAR? ligand-binding activity promotes adipocyte differentiation and increases glucose uptake in 3T3Ll cells  

Microsoft Academic Search

Emodin, one of the main active components in the root and rhizome of Rheum palmatum L, promoted the conversion of 3T3-L1 fibroblasts to adipocytes, as evidenced by increased glycerol-3-phosphate dehydrogenase (GPDH) activity and the expression of adipocyte aP2 mRNA, as well as accelerated triacylglycerol (TG) accumulation, which was associated with increased mRNA expression levels of both C\\/EBP? and PPAR?2. By

Ying Yang; Wenbin Shang; Libin Zhou; Boren Jiang; Hua Jin; Mingdao Chen

2007-01-01

121

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

122

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

Microsoft Academic Search

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

A. Zetterberg; Olle Larsson

1985-01-01

123

Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease.  

PubMed

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types. PMID:9227407

Pasantes-Morales, H; Sánchez Olea, R; Miranda, D; Morán, J

1997-06-01

124

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

NASA Astrophysics Data System (ADS)

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, Martin

1988-07-01

125

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

PubMed

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

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

2014-10-14

126

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

PubMed Central

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

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

2001-01-01

127

Interleukin-15 increases calcineurin expression in 3T3-L1 cells: possible involvement on in vivo adipocyte differentiation.  

PubMed

Different studies have revealed that the Ca2+-dependent serine/threonine phosphatase calcineurin is involved in the regulation of adipocyte differentiation. Calcineurin acts as a Ca2+-dependent molecular switch that negatively regulates the ability of 3T3-L1 cells to undergo adipocyte differentiation by preventing the expression of critical proadipogenic transcription factors. In this study we investigated the role of interleukin-15 (IL-15), a cytokine previously known to be involved in the control of fat accretion by adipose cells, in the differentiation of the 3T3-L1 preadipose cell line. We found that IL-15 is able to increase alpha-calcineurin mRNA content in white adipose tissue of rats chronically treated with the cytokine and also in the 3T3-L1 preadipose cell line. Moreover, IL-15 promoted a decrease in both leptin mRNA expression and lipid accumulation, as estimated by Red Oil O staining. Cotreatment with IL-15 and FK506 (a calcineurin inhibitor) resulted in no changes in lipid content compared with the non-treated group. These data suggest that IL-15 directly inhibits adipogenesis, possibly by upregulating alpha-calcineurin and preventing the induction of adipocyte differentiation. PMID:19724884

Almendro, Vanessa; Fuster, Gemma; Ametller, Elisabet; Costelli, Paola; Pilla, Federica; Busquets, Sílvia; Figueras, Maite; Argilés, Josep M; López-Soriano, Francisco J

2009-10-01

128

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

129

Degradation of erythrocyte-microinjected and scrape-loaded homologous cytosolic proteins by 3T3-L1 cells.  

PubMed Central

Homologous cytosol was introduced into 3T3-L1 cells by two different methods. Erythrocytes loaded with radiolabelled cytosolic proteins extracted from 3T3-L1 cells were fused with the aid of Sendai virus to 3T3-L1 cells, which were then seeded to confluent and non-confluent cultures. Cytosolic proteins were also introduced into cells by the technique of scrape-loading. In confluent cells, injected cytosolic proteins were recovered largely (54-93%) in a sedimentable (6 X 10(6) gav.-min) fraction from recipient cells irrespective of the method of introduction or of radiolabelling of the injected proteins [( 125I]iodination, reductive methylation with NaB3H4 and backbone labelling with L-[4,5-3H]leucine). The degradation of microinjected cytosolic proteins was in all cases inhibited by the lysosomotropic agent NH4Cl to a greater extent (32-75%) than that observed for endogenous cytosolic (less than or equal to 19%) proteins (labelled with L-[4,5-3H]leucine). In growing cells both endogenous total cell proteins and microinjected proteins were degraded at a slower rate than in confluent cell monolayers. The inhibition by NH4Cl of the degradation of both the endogenous and microinjected proteins is decreased compared with the inhibition observed in confluent monolayers. The results are discussed in terms of the cytoplasmic capacity to segregate microinjected homologous proteins before protein degradation can take place. PMID:3985941

Doherty, F J; Mayer, R J

1985-01-01

130

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

PubMed Central

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

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

2012-01-01

131

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

132

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

PubMed Central

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

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

2012-01-01

133

Enhancement of ajoene-induced apoptosis by conjugated linoleic acid in 3T3-L1 adipocytes.  

PubMed

Ajoene has been shown to induce apoptosis in 3T3-L1 adipocytes. In this report the effects on apoptosis of combinations of ajoene and trans-10, cis-12 conjugated linoleic acid (t10,c12CLA) in 3T3-L1 adipocytes were investigated. Although t10,c12CLA alone had no effect, ajoene plus t10,c12CLA reduced cell viability more than ajoene alone at 24 h (59.1 vs. 85.9% of control, respectively; p<0.05). Compared to treatment with t10,c12CLA, ajoene increased apoptosis 218% after 24 h (p<0.01), whereas ajoene plus t10,c12CLA increased apoptosis 122% over that caused by ajoene alone (p<0.01). Immunoblotting analysis also indicated that ajoene plus t10,c12CLA caused a greater increase in phosphorylation of c-Jun N-terminal kinase (JNK) and Bax expression and a greater release of mitochondrial proteins (cytochrome c, AIF) than additive responses to each compound alone. Ajoene plus t10,c12CLA also increased ROS production more than that resulting from ajoene treatment alone (264 vs 204% after 40 min, respectively; p<0.01). Furthermore, the antioxidant NAC prevented ROS generation and apoptosis by ajoene plus t10,c12CLA. Interestingly, the combination of ajoene and t10,c12CLA increased NF-kappaB activation and decreased the level of phosphorylated Akt more than each compound alone. Altogether, our observations indicate that t10,c12CLA potentiates the effect of ajoene on apoptosis in 3T3-L1 adipocytes. PMID:17318368

Yang, Jeong-Yeh; Della-Fera, Mary Anne; Hausman, Dorothy B; Baile, Clifton A

2007-06-01

134

Effect of hexavalent chromium on proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts.  

PubMed

Heavy metals contamination has become an important risk factor for public health and the environment. Chromium is a frequent industrial contaminant and is also used in orthopaedic joint replacements made from cobalt-chromium-alloy. Since hexavalent chromium (Cr(VI)) was reported as genotoxic and carcinogenic in different mammals, to further evaluate its cytotoxicity, we investigated the effect of this heavy metal in the proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts. These cells, after the addition of a mixture containing insulin, dexamethasone and methylisobutylxanthine, first proliferate, a process known as mitotic clonal expansion (MCE), and then differentiate to adipocytes. In this differentiation process a key transcription factor is induced: peroxisome proliferator-activated receptor gamma (PPAR gamma). We found that treatment of 3T3-L1 fibroblasts with potassium chromate inhibited proliferation in exponentially growing cells and MCE as well as differentiation. A decrease in PPAR gamma content, evaluated by western blot and immunofluorescence, was found in cells differentiated in the presence of chromium. On the other hand, after inhibition of differentiation with chromium, when the metal was removed, differentiation was recovered, which indicates that this may be a reversible effect. We also found an increase in the number of micronucleated cells after treatment with Cr(VI) which is associated with genotoxic effects. According to our results, Cr(VI) is able to inhibit proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts and to increase micronucleated cells, which are all indicative of alterations in cellular physiology and therefore, contributes to further elucidate the cytotoxic effects of this heavy metal. PMID:24576443

Martini, Claudia N; Brandani, Javier N; Gabrielli, Matías; Vila, María del C

2014-06-01

135

Oxidative stress impairs insulin but not platelet-derived growth factor signalling in 3T3-L1 adipocytes.  

PubMed Central

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is a common event in both insulin and platelet-derived growth factor (PDGF) signalling, but only insulin activates this enzyme in the high-speed pellet (HSP), and induces GLUT4 translocation. Recently, we have demonstrated that exposure of 3T3-L1 adipocytes to oxidative stress impairs insulin-stimulated GLUT4 translocation and glucose transport, associated with impaired PI 3-kinase translocation and activation in the HSP [Tirosh, Potashnik, Bashan and Rudich (1999) J. Biol. Chem. 274, 10595-10602]. In this study the effect of a 2 h exposure to approximately 30 microM H(2)O(2) on insulin versus PDGF-BB signalling and metabolic effects was compared. PDGF-stimulated p85-associated PI 3-kinase activity in total cell lysates, as well as co-precipitation of the PDGF receptor, were unaffected by oxidative stress. Additionally, the increase in p85 association with the plasma-membrane lawns by PDGF remained intact following oxidation, whereas the insulin effect was decreased. PDGF significantly increased protein kinase B (PKB) activity in early differentiated cells, and that of p70 S6-kinase in both early and fully differentiated 3T3-L1 adipocytes. Following oxidation the effect of PDGF on PKB and p70 S6-kinase activation remained intact, whereas significant inhibition of insulin-stimulated activation of those enzymes was observed. In accordance, in both early and fully differentiated cells, oxidative stress completely blunted insulin- but not PDGF-stimulated protein synthesis. In conclusion, oxidative stress impairs insulin, but not PDGF, signalling and metabolic actions in both early and fully differentiated 3T3-L1 adipocytes. This emphasizes compartment-specific activation of PI 3-kinase as an oxidation-sensitive step specifically leading to insulin resistance. PMID:11311139

Tirosh, A; Rudich, A; Potashnik, R; Bashan, N

2001-01-01

136

Raspberry ketone, a naturally occurring phenolic compound, inhibits adipogenic and lipogenic gene expression in 3T3-L1 adipocytes.  

PubMed

Abstract Context: Raspberry ketone (RK) is a natural phenolic compound of red raspberry. The dietary intake of RK has been reported to exert anti-obese actions and alter the lipid metabolism in vivo and human studies. Objective: To elucidate a possible mechanism for anti-obese actions of RK, the effects of RK on the adipogenic and lipogenic gene expression in 3T3-L1 adipocytes were investigated. Materials and methods: 3T3-L1 maturing pre-adipocytes were treated from day 2 to day 8 of differentiation and mature adipocytes for 24?h on day 12 with 1, 10, 20, and 50??M of RK. Triacylglycerols were assessed by spectrophotometry and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Results: Treatment of adipocytes with RK suppressed adipocyte differentiation and fat accumulation in a concentration-dependent manner. RK suppressed the expression of major genes involved in the adipogenesis pathway including peroxisome proliferator-activated receptor-? (PPAR?) and CCAAT enhancer binding protein-? (C/EBP?), which led to further down-regulation of adipocyte fatty acid-binding protein-2 (aP2). In addition, treatment with 10??M of RK also reduced mRNA levels of lipogenic genes such as acetyl-CoA carboxylase-1 (ACC1), fatty acid synthase (FASN), and stearoyl-CoA desaturase-1 (SCD1). In mature adipocytes, RK increased the transcriptional activities of genes involved in lipolysis and the oxidative pathways including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and carnitine palmitoyl transferase-1B (CPT1B). Discussion and conclusion: These findings suggest that RK holds great promise for an herbal medicine with the biological activities altering the lipid metabolism in 3T3-L1 adipocytes. PMID:25429790

Park, Kyoung Sik

2014-11-28

137

Changes in chromatin structure in NIH 3T3 cells induced by valproic acid and trichostatin A.  

PubMed

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-? (HP1-?), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05?mM) and TSA (10?ng/ml) treatments for 1?h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-? depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA. PMID:24913611

Felisbino, Marina Barreto; Gatti, Maria Silvia Viccari; Mello, Maria Luiza S

2014-11-01

138

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

139

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

140

Estimating pose statistics for robotic part feeders  

Microsoft Academic Search

In automated assembly lines, part feeders often impose a bottleneck that restricts throughput. To facilitate the design of parts and assembly lines, the authors estimate feedrates based on CAD models of parts. A previous paper (Golberg and Craig, 1995) described how to predict throughput for a vision-based robotic part feeder given the distribution of part poses when parts are randomly

Brian Mirtich; Yan Zhuang; K. Goldberg; J. Craig; R. Zanutta; B. Carlisle; J. Canny

1996-01-01

141

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

PubMed Central

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

Lanz, R; Brune, K

1981-01-01

142

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. PMID:22306825

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

2012-01-01

143

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

PubMed Central

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

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

2012-01-01

144

Ajoene exerts potent effects in 3T3-L1 adipocytes by inhibiting adipogenesis and inducing apoptosis.  

PubMed

This paper describes effects of several sulfur-containing compounds from garlic on the cell viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. In both preadipocytes and mature adipocytes, 100 and 200 microM ajoene significantly decreased cell viability and increased apoptosis. The effect on apoptosis was further confirmed with Hoechst staining. In contrast, diallyl sulfide, diallyl disulfide, diallyl trisulfide, deoxyalliin, and allyl methyl sulfide had no significant effect on cell viability or apoptosis in either preadipocytes or mature adipocytes. In maturing preadipocytes ajoene significantly decreased lipid accumulation in a dose-dependent manner and these results were further confirmed by a decrease in lipid droplet number and lipid content through Oil Red O staining. There was no significant change in lipid accumulation in maturing preadipocytes treated with other garlic derivatives. Thus, despite the same source of origin, garlic, ajoene was the only one with potent effects on cell viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. PMID:19051208

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

2009-04-01

145

Alliin, a Garlic (Allium sativum) Compound, Prevents LPS-Induced Inflammation in 3T3-L1 Adipocytes  

PubMed Central

Garlic (Allium sativum L.) has been used to alleviate a variety of health problems due to its high content of organosulfur compounds and antioxidant activity. The main active component is alliin (S-allyl cysteine sulfoxide), a potent antioxidant with cardioprotective and neuroprotective actions. In addition, it helps to decrease serum levels of glucose, insulin, triglycerides, and uric acid, as well as insulin resistance, and reduces cytokine levels. However its potential anti-inflammatory effect is unknown. We examined the effects of alliin in lipopolysaccharide- (LPS-) stimulated 3T3-L1 adipocytes by RT-PCR, Western blot, and microarrays analysis of 22,000 genes. Incubation of cells for 24?h with 100??mol/L alliin prevented the increase in the expression of proinflammatory genes, IL-6, MCP-1, and Egr-1 in 3T3-L1 adipocytes exposed to 100?ng/mL LPS for 1?h. Interestingly, the phosphorylation of ERK1/2, which is involved in LPS-induced inflammation in adipocytes, was decreased following alliin treatment. Furthermore, the gene expression profile by microarrays evidentiate an upregulation of genes involved in immune response and downregulation of genes related with cancer. The present results have shown that alliin is able to suppress the LPS inflammatory signals by generating an anti-inflammatory gene expression profile and by modifying adipocyte metabolic profile. PMID:24453416

Quintero-Fabián, Saray; Ortuño-Sahagún, Daniel; Vázquez-Carrera, Manuel; López-Roa, Rocío Ivette

2013-01-01

146

Alliin, a garlic (Allium sativum) compound, prevents LPS-induced inflammation in 3T3-L1 adipocytes.  

PubMed

Garlic (Allium sativum L.) has been used to alleviate a variety of health problems due to its high content of organosulfur compounds and antioxidant activity. The main active component is alliin (S-allyl cysteine sulfoxide), a potent antioxidant with cardioprotective and neuroprotective actions. In addition, it helps to decrease serum levels of glucose, insulin, triglycerides, and uric acid, as well as insulin resistance, and reduces cytokine levels. However its potential anti-inflammatory effect is unknown. We examined the effects of alliin in lipopolysaccharide- (LPS-) stimulated 3T3-L1 adipocytes by RT-PCR, Western blot, and microarrays analysis of 22,000 genes. Incubation of cells for 24 h with 100 ?mol/L alliin prevented the increase in the expression of proinflammatory genes, IL-6, MCP-1, and Egr-1 in 3T3-L1 adipocytes exposed to 100 ng/mL LPS for 1 h. Interestingly, the phosphorylation of ERK1/2, which is involved in LPS-induced inflammation in adipocytes, was decreased following alliin treatment. Furthermore, the gene expression profile by microarrays evidentiate an upregulation of genes involved in immune response and downregulation of genes related with cancer. The present results have shown that alliin is able to suppress the LPS inflammatory signals by generating an anti-inflammatory gene expression profile and by modifying adipocyte metabolic profile. PMID:24453416

Quintero-Fabián, Saray; Ortuño-Sahagún, Daniel; Vázquez-Carrera, Manuel; López-Roa, Rocío Ivette

2013-01-01

147

Anti-obesity and antioxidative effects of purple sweet potato extract in 3T3-L1 adipocytes in vitro.  

PubMed

The purpose of the current study was to determine the anti-obesity and anti-inflammatory effects of an extract of purple sweet potatoes (PSPs) on 3T3-L1 adipocytes. For this purpose, differentiated 3T3-L1 adipocytes were treated with a PSP extract at concentrations of 1,000, 2,000, and 3,000 ?g/mL for 24 hours. Then, we measured the changes in the sizes of the adipocytes, the secretion of leptin, and the mRNA/protein expression of lipogenic, inflammatory, and lipolytic factors after the treatment with the PSP extract. The PSP extract diminished leptin secretion, indicating that growth of fat droplets was suppressed. The extract also suppressed the expression of mRNAs of lipogenic and inflammatory factors and promoted lipolytic action. The antioxidative activity of the PSP extract was also measured using three different in vitro methods: 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity, ferric reducing ability potential assay, and chelating activity of transition metal ions. Taken together, our study shows that PSP extract has antilipogenic, anti-inflammatory, and lipolytic effects on adipocytes and has radical scavenging and reducing activity. PMID:21861722

Ju, Jae-Hyun; Yoon, Hong-Sup; Park, Hyun-Joon; Kim, Mi-Young; Shin, Hyeun-Kil; Park, Kun-Young; Yang, Jin-Oh; Sohn, Min-Shik; Do, Myoung-Sool

2011-10-01

148

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

149

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

PubMed Central

The aim of this study was to investigate whether a water extract of L. cladonioides (LC) has an anti-obesity effect in 3T3-L1 cells and obese mice. Treatment of differentiated 3T3-L1 adipocytes with LC caused a significant increase in glycerol release and reduced the protein expression of the adipogenic transcription factors, PPAR? and C/EBP?. In an animal model, obese mice were artificially induced by a high fat diet for 10 weeks. Experimental groups were treated with LC (100 mg/kg/day) by gavage for the next 10 weeks. At the end of experiment, the body weight of the LC group mice was reduced by 14.2% compared to the high fat diet (HFD) group. The treatment also decreased liver (31.0%), epididymal (18.0%) and retroperitoneal (19.3%) adipose tissue, and kidney (6.7%) weights, respectively, compared with those of the HFD group. LC prevented diet-induced increases in the serum level of TC (22.6%), TG (11.6%), and glucose (35.0%), respectively, compared with the HFD group. However, the HDL-C level was higher in the LC group (26.1%) than the HFD group. The results of this study thus suggest that LC suppressed lipid accumulation and expression of adipogenic transcription factors, and increased the amount of glycerol release. LC also indicated an anti-obese and anti-hyperlipidemic effect. PMID:22259674

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

2011-01-01

150

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

151

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

152

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

153

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

NASA Astrophysics Data System (ADS)

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

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

2010-04-01

154

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

PubMed

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

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

2014-09-01

155

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

PubMed Central

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

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

2013-01-01

156

Metformin prevents LYRM1-induced insulin resistance in 3T3-L1 adipocytes via a mitochondrial-dependent mechanism.  

PubMed

We previously proposed that LYR motif containing 1 (LYRM1)-induced mitochondrial reactive oxygen species (ROS) production contributes to obesity-related insulin resistance. Metformin inhibits ROS production and promotes mitochondrial biogenesis in specific tissues. We assessed the effects of metformin on insulin resistance in LYRM1-over-expressing 3T3-L1 adipocytes. Metformin enhanced basal and insulin-stimulated glucose uptake and GLUT4 translocation, reduced IRS-1 and Akt phosphorylation and ROS levels, and affected the expression of regulators of mitochondrial biogenesis in LYRM1-over-expressing adipocytes. Metformin may ameliorate LYRM1-induced insulin resistance and mitochondrial dysfunction in part via a direct antioxidant effect and in part by activating the adenosine monophosphate-activated protein kinase (AMPK)-PGC1/NRFs pathway. PMID:24903160

Qin, Zhen-Ying; Zhang, Min; Dai, Yong-Mei; Wang, Yu-Mei; Zhu, Guan-Zhong; Zhao, Ya-Ping; Ji, Chen-Bo; Qiu, Jie; Cao, Xin-Guo; Guo, Xi-Rong

2014-12-01

157

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

SciTech Connect

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

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

2006-09-15

158

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

NASA Astrophysics Data System (ADS)

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.

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

2014-01-01

159

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

Microsoft Academic Search

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

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

2000-01-01

160

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

Microsoft Academic Search

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

S. L Godwin; S. P Soltoff

2002-01-01

161

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

162

Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells.  

PubMed Central

AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-->Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA. PMID:10193512

Bjarnadottir, M; Wulff, B S; Sameni, M; Sloane, B F; Keppler, D; Grubb, A; Abrahamson, M

1998-01-01

163

Measuring value added characteristics in feeder cattle  

E-print Network

over seven years from regular and special feeder cattle sales at Joplin Regional Stockyards were used. The effects of explanatory variables on sale price were analyzed using ordinary least squares regression hedonic model. Type of sale, seasonality...

Mathews, Crystal Dawn

2009-05-15

164

Manually Operated Welding Wire Feeder  

NASA Technical Reports Server (NTRS)

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

Rybicki, Daniel J. (Inventor)

2001-01-01

165

Estimating Pose Statistics for Robotic Part Feeders  

Microsoft Academic Search

In automated assembly lines, part feeders often impose a bottleneck that restricts throughput. To facilitate the de sign of parts and assembly lines, we'd like to estimate feedrates based on CAD models of parts. A previous paper(8) de- scribed how to predict throughput for a vision-based roboti c part feeder given the distribution of part poses when parts are randomly

Brian Mirtich; Yan Zhuang; Ken Goldberg; John Craig; Rob Zanutta; Brian Carlisle; John Canny

166

Cytotoxicity of Zinc Oxide Nanoparticles on Antioxidant Enzyme Activities and mRNA Expression in the Cocultured C2C12 and 3T3-L1 Cells.  

PubMed

The present study was aimed to investigate the dose-dependent effect of zinc oxide (ZnO) nanoparticles on antioxidant enzyme activities and messenger RNA (mRNA) expression in the cocultured C2C12 and 3T3-L1 cells. Coculturing experiments are 3D and more reliable compared to mono-culture (2D) experiment. Even though, there are several studies on ZnO nanoparticle-mediated cytotoxicity, but there are no studies on the effect of ZnO nanoparticle on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles on the C2C12 and 3T3-L1 cell viability. At higher concentration of ZnO nanoparticles, C2C12 and 3T3-L1 cells almost die. ZnO nanoparticles increased reactive oxygen species (ROS) and lipid peroxidation and reduced glutathione (GSH) levels in a dose-dependent manner in the C2C12 and 3T3-L1 cells. In addition, ZnO nanoparticles increased antioxidant enzyme activities and their mRNA expression in the C2C12 and 3T3-L1 cells. In conclusion, the present study showed that ZnO nanoparticles increased oxidative stress, antioxidant enzyme activities, and their mRNA expression in the cocultured C2C12 and 3T3-L1 cells. PMID:25380643

Pandurangan, Muthuraman; Veerappan, Muthuviveganandavel; Kim, Doo Hwan

2015-02-01

167

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. PMID:25009600

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

2014-01-01

168

Melatonin promotes osteoblast differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions through activation of PKD/p38 pathways.  

PubMed

Osteoblastic differentiation and bone-forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3-E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time- and concentration-dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3-E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3-E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways. PMID:25250639

Son, Jang-Ho; Cho, Yeong-Cheol; Sung, Iel-Yong; Kim, In-Ryoung; Park, Bong-Soo; Kim, Yong-Deok

2014-11-01

169

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

170

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. PMID:23308295

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

2013-01-01

171

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

PubMed Central

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

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

2014-01-01

172

Chemical constituents of Triticum aestivum and their effects on adipogenic differentiation of 3T3-L1 preadipocytes.  

PubMed

In this report, we investigated the anti-obesity effect of wheat sprouts and their component compounds. Twenty compounds (1-20) were isolated from Triticum aestivum. Among them, glycolipids 1-5 were determined for the first time from T. aestivum and its sprouts. The HPLC analysis demonstrated that compounds 1-3, 5, 8, 12, and 14 were major peak in the HPLC chromatogram of the active fraction. The effects of the compounds on lipid accumulation were assessed at concentrations ranging from 1.0 to 100 ?M. At concentration of 10.0 ?M, compounds 1-7, 10-15, and 17-19 significantly decreased lipid accumulation in 3T3-L1 preadipocytes. Glycolipids 1, 2, and phenolic 17 significantly reduced lipid accumulation in the differentiated adipocytes in a concentration-dependent manner. Quantitative analysis based on measurement of the optical density of Oil Red O indicated that, at 100 ?M, compounds 1, 2, and 17 reduced lipid accumulation by 41, 37, and 48 %, respectively, compared with the positive control. PMID:25241774

Luyen, Bui Thi Thuy; Thao, Nguyen Phuong; Tai, Bui Huu; Lim, Ji Young; Ki, Hyeon Hui; Kim, Dae Ki; Lee, Young Mi; Kim, Young Ho

2014-09-23

173

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

174

C-terminal bombesin sequence requirements for binding and effects on protein synthesis in Swiss 3T3 cells.  

PubMed Central

1. Synthetic peptides corresponding to the five, seven, nine and eleven C-terminal amino acids of the tetradecapeptide bombesin as well as bombesin itself and gastrin-releasing peptide have been evaluated in Swiss 3T3 cells in order to define the minimal peptide length needed for biological responsiveness. 2. Gastrin-releasing peptide, bombesin, the undecapeptide and nonapeptide had nearly equipotent abilities to compete for binding of labelled gastrin-releasing peptide to the cell receptors and showed half-maximal competition at 5-10 nM. The heptapeptide and pentapeptide were ineffective. 3. Cross-linking experiments demonstrated specific binding of gastrin-releasing peptide to a 100 kDa receptor subunit. 4. Total cell protein synthesis was stimulated equally by the nonapeptide and longer peptides with a half-maximal effect at 0.5 nM, while a more than 1000-fold higher concentration of the heptapeptide was required to produce a similar response. Comparable results were found when insulin was also present. 5. Neither an inhibition of protein breakdown nor a stimulation of DNA labelling could be demonstrated by bombesin or gastrin-releasing peptide. 6. We conclude that a C-terminal peptide ligand comprising more than seven but no more than nine amino acids is required to achieve high-affinity binding and receptor-mediated responses via the bombesin receptor. Images Fig. 2. PMID:3426545

Gargosky, S E; Wallace, J C; Upton, F M; Ballard, F J

1987-01-01

175

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

PubMed Central

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

176

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

177

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

NASA Technical Reports Server (NTRS)

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

Fitzgerald, J.; Hughes-Fulford, M.

1999-01-01

178

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

PubMed Central

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

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

2014-01-01

179

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. PMID:22292054

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

2012-01-01

180

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

PubMed

The phytotherapeutic protein stem bromelain (SBM) is used as an anti-obesity alternative medicine. We show at the cellular level that SBM irreversibly inhibits 3T3-L1 adipocyte differentiation by reducing adipogenic gene expression and induces apoptosis and lipolysis in mature adipocytes. At the molecular level, SBM suppressed adipogenesis by downregulating C/EBP? and PPAR? independent of C/EBP? gene expression. Moreover, mRNA levels of adipocyte fatty acid-binding protein (ap2), fatty acid synthase (FAS), lipoprotein lipase (LPL), CD36, and acetyl-CoA carboxylase (ACC) were also downregulated by SBM. Additionally, SBM reduced adiponectin expression and secretion. SBM's ability to repress PPAR? expression seems to stem from its ability to inhibit Akt and augment the TNF? pathway. The Akt-TSC2-mTORC1 pathway has recently been described for PPAR? expression in adipocytes. In our experiments, TNF? upregulation compromised cell viability of mature adipocytes (via apoptosis) and induced lipolysis. Lipolytic response was evident by downregulation of anti-lipolytic genes perilipin, phosphodiestersae-3B (PDE3B), and GTP binding protein G(i)?(1), as well as sustained expression of hormone sensitive lipase (HSL). These data indicate that SBM, together with all-trans retinoic-acid (atRA), may be a potent modulator of obesity by repressing the PPAR?-regulated adipogenesis pathway at all stages and by augmenting TNF?-induced lipolysis and apoptosis in mature adipocytes. PMID:22292054

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

2012-01-01

181

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

SciTech Connect

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

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

2009-12-18

182

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

PubMed

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

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

2013-09-01

183

Role of protease inhibitors and acylation stimulating protein in the adipogenesis in 3T3-L1 cells  

PubMed Central

Treatment of AIDS (HIV) and hepatitis C virus needs protease inhibitors (PI) to prevent viral replication. Uses of PI in therapy are usually associated with a decrease in body weight and dyslipidemia. Acylation stimulating protein (ASP) is a protein synthesized in adipocytes to increase triglycerides biosynthesis, for that the relation of PI and ASP to adipogenesis is tested in this work. ASP expression was increased during 3T3-L1 differentiation and reached a peak at day 8 with cell maturation. Addition of PI during adipocytes differentiation dose dependently and significantly (p < 0.5) inhibited the degree of triglycerides (TG) accumulation. Moreover, presence of ASP (450 ng/mL) in media significantly (p < 0.5) stimulated the degree of TG accumulation and there was additive stimulation for ASP when added with insulin (10 µg/mL). Finally, when ASP in different doses (Low, 16.7; Medium, 45 and High, 450 ng/mL) incubated with a dose of ×150 PI, ASP partially inhibited the PI-inhibited adipogenesis and TG accumulation. The results in this study show that PI inhibit lipids accumulation and confirm role of ASP in TG biosynthesis and adipogenesis. PMID:19687619

El-Senosi, Yakut Abdel-Fattah; Salem, Maysara Mahmoud; Hamid, Omniya Mahmoud Abdel; Kazuhiro, Kimura

2009-01-01

184

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

PubMed

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

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

2014-01-01

185

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

186

Chlamydia induces anchorage independence in 3T3 cells and detrimental cytological defects in an infection model.  

PubMed

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

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

2013-01-01

187

Regulation of bone-related genes expression by bone-like apatite in MC3T3-E1 cells.  

PubMed

Bone-like apatite on HA/TCP ceramics sintered at 1,100 degrees C (HT1) and 1,200 degrees C (HT2) could be obtained via immersing substrates into simulated body fluid (SBF) for 3 days. When MC3T3-E1 preosteoblastic cells cultured on the surface of the bone-like apatite for 3 days, SEM observations revealed cell membrane features with secreted crystals very similar to in vivo bone formation during intramembranous ossification with a direct bone apposition on the ceramics. According to semi-quantitative RT-PCR method, mRNA expressions of osteocalcin (marker of late-stage differentiation) and type 1 collagen were increased in cultures with HT1S and HT2S when compared to HT1 and HT2 after cultured for 6 days. The results indicated that bone-like apatite had the ability to support the growth of osteoblast-like cells in vitro and to promote osteoblast differentiation by stimulating the expression of major phenotypic markers. Taken together, our findings will be helpful in understanding the mechanism of osteoinductivity of calcium phosphate ceramics and in constructing more appropriate biomimetic substrate. PMID:17597361

Tan, Y F; Hong, S F; Wang, X L; Lu, J; Wang, H; Zhang, X D

2007-11-01

188

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

189

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

PubMed

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

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

2014-01-01

190

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

191

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

192

Enhanced MC3T3-E1 preosteoblast response and bone formation on the addition of nano-needle and nano-porous features to microtopographical titanium surfaces.  

PubMed

Micro/nanotopographical modifications on titanium surfaces constitute a new process to increase osteoblast response to enhance bone formation. In this study, we utilized alkali heat treatment at high (SB-AH1) and low temperatures (SB-AH2) to nano-modify sandblasted titanium with microtopographical surfaces. Then, we evaluated the surface properties, biocompatibility and osteogenic capability of SB-AH1 and SB-AH2 in vitro and in vivo, and compared these with conventional sandblast-acid etching (SLA) and Ti control surfaces. SB-AH1 and SB-AH2 surfaces exhibited micro/nanotopographical modifications of nano-needle structures and nano-porous network layers, respectively, compared with the sole microtopographical surface of macro and micro pits on the SLA surface and the relatively smooth surface on the Ti control. SB-AH1 and SB-AH2 showed different roughness and elemental components, but similar wettability. MC3T3-E1 preosteoblasts anchored closely on the nanostructures of SB-AH1 and SB-AH2 surfaces, and these two surfaces more significantly enhanced cell proliferation and alkaline phosphatase (ALP) activity than others, while the SB-AH2 surface exhibited better cell proliferation and higher ALP activity than SB-AH1. All four groups of titanium domes with self-tapping screws were implanted in rabbit calvarial bone models, and these indicated that SB-AH1 and SB-AH2 surfaces achieved better peri-implant bone formation and implant stability, while the SB-AH2 surface achieved the best percentage of bone-implant contact (BIC%). Our study demonstrated that the micro/nanotopographical surface generated by sandblasting and alkali heat treatment significantly enhanced preosteoblast proliferation, ALP activity and bone formation in vitro and in vivo, and nano-porous network topography may further induce better preosteoblast proliferation, ALP activity and BIC%. PMID:24945708

Zhuang, X-M; Zhou, B; Ouyang, J-L; Sun, H-P; Wu, Y-L; Liu, Q; Deng, F-L

2014-08-01

193

Ginsenoside Rc Promotes Anti-Adipogenic Activity on 3T3-L1 Adipocytes by Down-Regulating C/EBP? and PPAR?.  

PubMed

Panax ginseng and its major components, the ginsenosides, are widely used in oriental medicine for the prevention of various disorders. In the present study, the inhibitory activity of ginsenoside Rc on adipogenesis was investigated using the 3T3-L1 cell line. The results obtained showed that Rc reduced the proliferation and viability of 3T3-L1 preadipocytes in a dose-dependent manner. Treatment with Rc decreased the number of adipocytes and reduced lipid accumulation in maturing 3T3-L1 preadipocytes, demonstrating an inhibitory effect on lipogenesis. Moreover, it was found that Rc directly induced lipolysis in adipocytes and down-regulated the expression of major transcription factors of the adipogenesis pathway, such as PPAR? and C/EBP?. These findings indicate that Rc is capable of suppressing adipogenesis and therefore they seem to be natural bioactive factors effective in adipose tissue mass modulation. PMID:25594343

Yang, Ji-Won; Kim, Sung Soo

2014-01-01

194

Optimal compensation of nonuniformly loaded radial feeders with applications  

SciTech Connect

This paper presents a generalized mathematical model for assessing the loss reduction in nonuniformly loaded radial feeders by capacitor shunt compensation. The model allows for the simultaneous presence of linearly increasing feeder load density in addition to a concentrated load at the feeder end. Any number of compensating capacitors can be included in the analysis. The model is then applied to study the effect of the number of feeders on the achievable optimal reduction in the feeder copper loss. The paper also gives the optimal size and locations of these capacitors, for any number of radial feeders emanating from a distribution substation to serve a certain total load.

Saied, M.M. (Electrical and Computer Engineering Dept., Kuwait Univ., P.O. Box 5969, 13060 Safat (KW))

1990-01-01

195

Power budget analysis of dual/single feeder fiber WDMPON  

NASA Astrophysics Data System (ADS)

This paper investigates how to reduce the cost of wavelength division multiplexing passive optical network (WDMPON) by comparing the transmission performance of bidirectional single feeder fiber and dual feeder fiber. Comparison is performed on the basis of power budgeting and cost of both arrangements. Simulation results using Optisystem show that the performance of a single feeder fiber is almost equivalent to that of a dual feeder fiber. Therefore, the single feeder fiber WDM-PON can efficiently replace the dual feeder fiber WDM-PON with the minimum deterioration in system performance and reduction in cost.

Imtiaz, Waqas A.; Khan, Yousaf; Qamar, Affaq; Khan, Jehanzeb; Khan, Noaman Ahmed

2014-03-01

196

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

197

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

198

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

PubMed Central

Early passages of the NIH 3T3 mouse cell line undergo spontaneous neoplastic transformation leading to the development of transformed foci if grown to confluence in 2% (vol/vol) calf serum (CS) and left there for more than a week. Transfer of the postconfluent cultures results in the appearance of large numbers of transformed foci; many of them are larger and denser than those in the original culture. If the cells are continually kept at low population densities by frequent passages in 10% CS, they lose the capacity to undergo spontaneous transformation. If however the low-density passages are made in 2% CS or in 10% (vol/vol) fetal bovine serum, both of which support lower growth rates and saturation densities than does 10% CS, they gain the capacities to grow to high saturation densities and produce more foci when grown to confluence in 2% CS. These increases are proportional to the population densities used in the frequent passages, although the densities are all kept well below confluence. We conclude that the combined constraints of submaximal serum plus those of the limited cell contacts of the low cell densities used here elicit an adaptive response that endows the entire population with increased growth capacity. The increased growth capacity of the heterogeneous population in turn increases the capacity of a fraction of the population to initiate distinctive transformed foci. Similar studies have indicated that the capacity of cells to produce tumors and metastases in mice and rats is enhanced by prior maintenance at high density in culture. We propose the concept of progressive state selection to account for the general increase in the growth capacity of cells that is elicited by moderate constraints on their growth and metabolism. PMID:1502159

Yao, A; Rubin, H

1992-01-01

199

Progesterone inhibits glucose uptake by affecting diverse steps of insulin signaling in 3T3-L1 adipocytes.  

PubMed

Maternal insulin resistance is essential for efficient provision of glucose to the fetus. Although elevation of placental hormones is known to relate to the development of insulin resistance, the precise underlying mechanism of maternal insulin resistance is unknown. Therefore, we examined the molecular mechanisms of progesterone causing insulin resistance in 3T3-L1 adipocytes. Progesterone at 10(-4) M, but not 10(-5) M, reduced the amount of IRS-1. As a result, insulin-induced phosphorylation of IRS-1, the association of IRS-1 with p85alpha, and subsequent phosphorylation of Akt1 and -2 was decreased moderately by 10(-4) M progesterone. Subsequently, insulin-induced translocation of GLUT4 to the plasma membrane evaluated by immunostaining on the plasma membrane sheet by confocal laser microscope was also decreased by 10(-4) M progesterone. In contrast, 2-[(3)H]deoxyglucose (2DG) uptake was markedly inhibited by both 10(-5) and 10(-4) M progesterone in a dose-dependent manner. Surprisingly, 2DG uptake elicited by adenovirus-mediated expression of constitutive-active mutant of PI 3-kinase (myr-p110) and Akt (myr-Akt) was suppressed by progesterone. Interestingly, insulin-induced tyrosine phosphorylation of Cbl and activation of TC10 were inhibited by progesterone at 10(-5) M. These results indicate that progesterone is implicated in insulin resistance during pregnancy by inhibiting the PI 3-kinase pathway at the step of 1) IRS-1 expression and 2) distal to Akt and 3) by suppressing the PI 3-kinase-independent pathway of TC10 activation by affecting Cbl phosphorylation. PMID:20071559

Wada, Tsutomu; Hori, Satoko; Sugiyama, Maine; Fujisawa, Eriko; Nakano, Tetsuro; Tsuneki, Hiroshi; Nagira, Kiyofumi; Saito, Shigeru; Sasaoka, Toshiyasu

2010-04-01

200

Butyrate and other short-chain fatty acids increase the rate of lipolysis in 3T3-L1 adipocytes.  

PubMed

We determined the effect of butyrate and other short-chain fatty acids (SCFA) on rates of lipolysis in 3T3-L1 adipocytes. Prolonged treatment with butyrate (5 mM) increased the rate of lipolysis approximately 2-3-fold. Aminobutyric acid and acetate had little or no effect on lipolysis, however propionate stimulated lipolysis, suggesting that butyrate and propionate act through their shared activity as histone deacetylase (HDAC) inhibitors. Consistent with this, the HDAC inhibitor trichostatin A (1 µM) also stimulated lipolysis to a similar extent as did butyrate. Western blot data suggested that neither mitogen-activated protein kinase (MAPK) activation nor perilipin down-regulation are necessary for SCFA-induced lipolysis. Stimulation of lipolysis with butyrate and trichostatin A was glucose-dependent. Changes in AMP-activated protein kinase (AMPK) phosphorylation mediated by glucose were independent of changes in rates of lipolysis. The glycolytic inhibitor iodoacetate prevented both butyrate- and tumor necrosis factor-alpha-(TNF-?) mediated increases in rates of lipolysis indicating glucose metabolism is required. However, unlike TNF-?- , butyrate-stimulated lipolysis was not associated with increased lactate release or inhibited by activation of pyruvate dehydrogenase (PDH) with dichloroacetate. These data demonstrate an important relationship between lipolytic activity and reported HDAC inhibitory activity of butyrate, other short-chain fatty acids and trichostatin A. Given that HDAC inhibitors are presently being evaluated for the treatment of diabetes and other disorders, more work will be essential to determine if these effects on lipolysis are due to inhibition of HDAC. PMID:25320679

Rumberger, John M; Arch, Jonathan R S; Green, Allan

2014-01-01

201

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. PMID:23444368

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

2013-01-01

202

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. PMID:24278099

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

2013-01-01

203

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

204

Butyrate and other short-chain fatty acids increase the rate of lipolysis in 3T3-L1 adipocytes  

PubMed Central

We determined the effect of butyrate and other short-chain fatty acids (SCFA) on rates of lipolysis in 3T3-L1 adipocytes. Prolonged treatment with butyrate (5 mM) increased the rate of lipolysis approximately 2–3-fold. Aminobutyric acid and acetate had little or no effect on lipolysis, however propionate stimulated lipolysis, suggesting that butyrate and propionate act through their shared activity as histone deacetylase (HDAC) inhibitors. Consistent with this, the HDAC inhibitor trichostatin A (1 µM) also stimulated lipolysis to a similar extent as did butyrate. Western blot data suggested that neither mitogen-activated protein kinase (MAPK) activation nor perilipin down-regulation are necessary for SCFA-induced lipolysis. Stimulation of lipolysis with butyrate and trichostatin A was glucose-dependent. Changes in AMP-activated protein kinase (AMPK) phosphorylation mediated by glucose were independent of changes in rates of lipolysis. The glycolytic inhibitor iodoacetate prevented both butyrate- and tumor necrosis factor-alpha-(TNF-?) mediated increases in rates of lipolysis indicating glucose metabolism is required. However, unlike TNF-?– , butyrate-stimulated lipolysis was not associated with increased lactate release or inhibited by activation of pyruvate dehydrogenase (PDH) with dichloroacetate. These data demonstrate an important relationship between lipolytic activity and reported HDAC inhibitory activity of butyrate, other short-chain fatty acids and trichostatin A. Given that HDAC inhibitors are presently being evaluated for the treatment of diabetes and other disorders, more work will be essential to determine if these effects on lipolysis are due to inhibition of HDAC. PMID:25320679

Rumberger, John M.; Arch, Jonathan R.S.

2014-01-01

205

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

PubMed Central

Background Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting. Results We identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes. Conclusions We conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis. PMID:22436005

2012-01-01

206

Phytic acid and myo-inositol support adipocyte differentiation and improve insulin sensitivity in 3T3-L1 cells.  

PubMed

Phytic acid, also known as myo-inositol hexaphosphate, has been shown to lower blood glucose levels and to improve insulin sensitivity in rodents. We investigated the effects of phytic acid and myo-inositol on differentiation, insulin-stimulated glucose uptake, and lipolysis of adipocytes to test the hypothesis that the antidiabetic properties of phytic acid and myo-inositol are mediated directly through adipocytes. 3T3-L1 cells were treated with 10, 50, or 200 ?mol/L of phytic acid or myo-inositol. Oil Red O staining and an intracellular triacylglycerol assay were used to determine lipid accumulation during adipocyte differentiation. Immunoblotting and real-time polymerase chain reaction (PCR) were performed to evaluate expression of transcription factors, a target protein, and insulin signaling molecules. Phytic acid and myo-inositol exposures increased lipid accumulation in a dose-dependent manner (P < .01). The expression of key transcription factors associated with adipocyte differentiation, such as peroxisome proliferator-activated receptor ? (PPAR?) and sterol regulatory element-binding protein 1c, and the expression of fatty acid synthase increased upon treatments with phytic acid and myo-inositol (P < .05). Insulin-stimulated glucose uptake in mature adipocytes increased with phytic acid and myo-inositol treatments (P < .01). In addition, mRNA levels of insulin receptor substrate 1 (IRS1), mRNA levels of glucose transporter 4, and phosphorylation of tyrosine in IRS1 increased upon phytic acid and myo-inositol treatments. In fully differentiated adipocytes, phytic acid and myo-inositol reduced basal lipolysis dose dependently (P < .01). These results suggest that phytic acid and myo-inositol increase insulin sensitivity in adipocytes by increasing lipid storage capacity, improving glucose uptake, and inhibiting lipolysis. PMID:25174657

Kim, Jin Nam; Han, Sung Nim; Kim, Hye-Kyeong

2014-08-01

207

Oroxylin A, a constituent of Oroxylum indicum inhibits adipogenesis and induces apoptosis in 3T3-L1 cells.  

PubMed

Oroxylin A (OA) is a flavonoid found in Oroxylum indicum, a medicinal plant with multiple biological activities. This study was taken up to investigate the effect of OA, on adipogenesis, lipolysis and apoptosis in 3T3 L1 cells. Pre-adipocytes were treated with 10-40?M OA on various days of adipogenesis treatment schedule. Mature adipocytes were treated with OA for lipolysis and apoptosis studies. In maturing pre-adipocytes, 10?M OA suppressed intracellular lipid accumulation by 42.19% which was confirmed by lipidTox imaging of cells. In addition, OA decreased the nuclear translocation of PPAR? and mRNA expression of its downstream genes (FAS and LPL) along with adiponectin secretion. In mature adipocytes, 40?M of OA decreased cell viability by 30% of control. Annexin V/PI staining showed induction of apoptosis which was further confirmed by enhanced levels of pro-apoptotic proteins Bax, cyt c, AIF and chromatin condensation. OA enhanced TNF-? secretion, lipolysis and decreased Akt phosphorylation in mature adipocytes. Findings suggest that OA possibly exerts its anti-obesity effect by affecting adipocyte life cycle at critical points of differentiation and maturity. When we compared the potency of OA with non-methoxylated flavonoids morin, naringenin and kaempferol on adipocyte life cycle OA was far more potent. Thus, study clearly indicates a new role for oroxylin A as regulator of adipocyte life cycle. In addition, study also suggested a specific role of methoxylated group in exerting lipolysis and cytotoxic effects in mature adipocytes. PMID:25442284

Singh, Jyotsna; Kakkar, Poonam

2014-10-15

208

Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling.  

PubMed

Autophagy is essential for successful white adipocyte differentiation but the data regarding the timing and relevance of autophagy action during different phases of adipogenesis are limited. We subjected 3T3-L1 preadipocytes to a standard differentiation protocol and inhibited the autophagy within time-limited periods (days 0-2; 2-4; 4-6; 6-8) by asparagine or 3-methyladenine. In the normal course of events, both autophagy flux and the mRNA expression of autophagy related genes (Atg5, Atg12, Atg16, beclin 1) is most intensive at the beginning of differentiation (days 0-4) and then declines. The initiation of differentiation is associated with a 50% reduction of the mitochondrial copy number on day 2 followed by rapid mitochondrial biogenesis. Preadipocytes and differentiated adipocytes differ in the mRNA expression of genes involved in electron transport (Nufsd1, Sdhb, Uqcrc1); ATP synthesis (ATP5b); fatty acid metabolism (CPT1b, Acadl); mitochondrial transporters (Hspa9, Slc25A1) and the TCA cycle (Pcx, Mdh2) as well as citrate synthase activity. Autophagy inhibition during the first two days of differentiation blocked both phenotype changes (lipid accumulation) and the gene expression pattern, while having no or only a marginal effect over any other time period. Similarly, autophagy inhibition between days 0-2 inhibited mitotic clonal expansion as well as mitochondrial network remodeling. In conclusion, we found that autophagy is essential and most active during an initial stage of adipocyte differentiation but it is dispensable during its later stages. We propose that the degradation of preadipocyte cytoplasmic structures, predominantly mitochondria, is an important function of autophagy during this phase and its absence prevents remodeling of the mitochondrial gene expression pattern and mitochondrial network organization. PMID:25041706

Skop, Vojtech; Cahova, Monika; Dankova, Helena; Papackova, Zuzana; Palenickova, Eliska; Svoboda, Petr; Zidkova, Jarmila; Kazdova, Ludmila

2014-06-01

209

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

PubMed

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

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

2013-04-01

210

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

PubMed

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

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

2015-03-01

211

Coal gasification: New challenge for the Beaumont rotary feeder  

NASA Technical Reports Server (NTRS)

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

Stelian, J.

1977-01-01

212

Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells  

SciTech Connect

G/sub 0//G/sub 1/ traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G/sub 0//G/sub 1/. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. The authors have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10 fold more (/sup 3/H)-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca/sub 3/ (PO/sub 4/)/sub 2/ and was not due to cell-cycle traverse. Stimulation of (/sup 3/H)-GlcN incorporation by PDGF was time dependent, and increased incorporation of (/sup 3/H)-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.

Harrington, M.A.; Wharton, W.; Pledger, W.J.

1987-01-01

213

Proteomics of Oxidative Stress Using Inducible CYP2E1 Expressing HepG2 Cells and 3T3-L1 Adipocytes as Model Systems  

E-print Network

The overall goal of this research was to investigate oxidative stress related changes to the proteomes of 3T3-L1 adipocytes and an inducible CYP2E1 expressing HepG2 cells. Enhanced oxidative stress in hypertrophic adipocytes is associated...

Newton, Billy Walker

2012-07-16

214

NIH3T3 cells overexpressing CD98 heavy chain resist early G1 arrest and apoptosis induced by serum starvation.  

PubMed

CD98 is a heterodimeric glycoprotein of 125-kDa, which consists of a 90-kDa heavy chain (hc) subunit and 35-kDa to 55-kDa light chain (lc) subunits. It is strongly expressed on the surface of proliferating normal cells and almost all tumor cells. To investigate the participation of CD98 in cellular proliferation and malignant transformation, we analyzed cell-cycle progression of NIH3T3 clones transfected with cDNA of human CD98hc. Although NIH3T3 and control transfectant cells grown to the subconfluent state were arrested in the G0/G1 phase by serum starvation, considerable portions of CD98hc-transfected cells resided at S and G2/M phases. Under serum-starved and confluent conditions, significant fractions (20-25%) of NIH3T3 and control transfectant cells contained less than 2n content DNA, indicating occurrence of apoptosis, whereas no apoptotic cells were detected in CD98hc-transfectant cells. Under serum-starved conditions, a marked increase in the levels of cyclin D1 and cyclin E and a decrease in p16 were observed in CD98hc-transfectant cells. The reverse was true for NIH3T3 and control transfectant cells. Our results suggest that resistance to G1 arrest and apoptosis by CD98 overexpression are associated with high G1-cyclins and low p16 levels. PMID:22497681

Hara, Kaori; Ueda, Shiho; Ohno, Yoshiya; Tanaka, Toshiyuki; Yagi, Hideki; Okazaki, Shogo; Kawahara, Rieko; Masayuki, Takechi; Enomoto, Takemi; Hashimoto, Yoshiyuki; Masuko, Kazue; Masuko, Takashi

2012-08-01

215

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

PubMed

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

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

2015-01-01

216

Soyasaponins Aa and Ab Exert an Anti-Obesity Effect in 3T3-L1 Adipocytes Through Downregulation of PPAR?.  

PubMed

Saponins are a diverse group of biologically functional products in plants. Soyasaponins are usually glycosylated, which give rise to a wide diversity of structures and functions. In this study, we investigated the effects and molecular mechanism of soyasaponins Aa and Ab in regulating adipocyte differentiation and expression of adipogenic marker genes in 3T3-L1 adipocytes. Soyasaponins Aa and Ab dose-dependently inhibited the accumulation of lipids and the expression of adiponectin, adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c, adipocyte fatty acid-binding protein 2, fatty acid synthase, and resistin in 3T3-L1 adipocytes. In addition, soyasaponins Aa and Ab suppressed the transcriptional activity of peroxisome proliferator-activated receptor ? (PPAR?) in HEK 293T cells. Furthermore, we confirmed that the expression of PPAR? and of CCAAT-enhancer-binding protein ? (C/EBP?) was suppressed at both the mRNA and protein levels in 3T3-L1 adipocytes by treatment with soyasaponins Aa and Ab. Taken together, these findings indicate that soyasaponin Aa and Ab markedly inhibit adipocyte differentiation and expression of various adipogenic marker genes through the downregulation of the adipogenesis-related transcription factors PPAR? and C/EBP? in 3T3-L1 adipocytes. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25366162

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

2015-02-01

217

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

PubMed Central

SUMMARY Survivin is an inhibitor of apoptosis (IAP) family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone 3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes, and identify intermolecular hydrogen bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and that this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket. PMID:22244766

Du, Jiamu; Kelly, Alexander E.; Funabiki, Hironori; Patel, Dinshaw J.

2012-01-01

218

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. PMID:21350939

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

2011-01-01

219

Integrated Smart Feeder \\/ Shuttle Bus Service  

Microsoft Academic Search

This paper presents the design of an integrated smart feeder\\/shuttle system. The design of such a system was motivated by the need to provide easy access to main haul transit services. Park and ride lots in many train stations can no longer accommodate automobiles brought to the stations. Some train riders have switched their mode of transportation from public transit

Avishai Ceder; Youngbin Yim

2003-01-01

220

Using Bird Feeders for Wintertime Ecological Instruction.  

ERIC Educational Resources Information Center

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

Gannon, John E.; Weber, Peter G.

1985-01-01

221

Group Regulation of Urban 4KV Feeders  

Microsoft Academic Search

The best way, theoretically, to maintain ideal voltage conditions on a given feeder is to equip each of its phases with a regulator. However, other factors in addition to voltage regulation must be considered in a practical co-ordinated design. The engineering approach toward accomplishing a purpose with the minimum physical facilities must be adhered to both in improving an old

C. L. Grim; H. B. Peck

1951-01-01

222

Estimating Throughput for a Flexible Part Feeder  

Microsoft Academic Search

To rapidly feed industrial parts on an assembly line, Carlisle et. al. [4] proposed a flexible part feeding system that drops parts on a flat conveyor belt, determines their pose (position and orientation) with a vision system and uses a high-speed scara robot to move them to a pallet in a desired pose. Such a feeder can be rapidly configured

Kenneth Y. Goldberg; John Craig; Brian Carlisle; Rob Zanutta

1995-01-01

223

Trap Design for Vibratory Bowl Feeders  

Microsoft Academic Search

The vibratory bowl feeder is the oldest and still most common ap- proach to the automated feeding (orienting) of industrial parts. In this paper we consider a class of vibratory bowl lters that can be described by removing polygonal sections from the track; we refer to this class of lters as traps. For an n-sided polygonal part and an m-sided

Robert-paul Berretty; Kenneth Y. Goldberg; Lawrence Cheung; Mark H. Overmars; Gordon Smith; A. Frank Van Der Stappen

1999-01-01

224

Trap Design for Vibratory Bowl Feeders  

Microsoft Academic Search

The vibratory bowl feeder is the oldest and still most common ap- proach to the automated feeding (orienting) of industrial parts. In this paper, the authors consider a class of vibratory bowl filters that can be described by removing polygonal sections from the track; this class of filters is referred to as traps. For an n-sided polygonal part and an

Robert-paul Berretty; Kenneth Y. Goldberg; Mark H. Overmars; A. Frank Van Der Stappen

2001-01-01

225

Feeder and Stocker Health and Management Practices  

E-print Network

of disease loss. Goals for herd health management in a stocker setting should be centered aroundFeeder and Stocker Health and Management Practices W. Dee Whittier, Extension Specialist Specialist and Professor, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech Disease

Liskiewicz, Maciej

226

46 CFR 58.25-65 - Feeder circuits.  

Code of Federal Regulations, 2011 CFR

...58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear...at least two feeder circuits, which must be separated... (d) Each feeder circuit must have a current-carrying...current rating of the electric steering-gear...

2011-10-01

227

46 CFR 58.25-65 - Feeder circuits.  

Code of Federal Regulations, 2013 CFR

...58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear...at least two feeder circuits, which must be separated... (d) Each feeder circuit must have a current-carrying...current rating of the electric steering-gear...

2013-10-01

228

46 CFR 58.25-65 - Feeder circuits.  

Code of Federal Regulations, 2012 CFR

...58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear...at least two feeder circuits, which must be separated... (d) Each feeder circuit must have a current-carrying...current rating of the electric steering-gear...

2012-10-01

229

46 CFR 58.25-65 - Feeder circuits.  

Code of Federal Regulations, 2010 CFR

...58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear...at least two feeder circuits, which must be separated... (d) Each feeder circuit must have a current-carrying...current rating of the electric steering-gear...

2010-10-01

230

Trap Design for Vibratory Bowl Feeders Robert-Paul Berretty  

E-print Network

Trap Design for Vibratory Bowl Feeders Robert-Paul Berretty Ken Goldberg §¶ Mark H. Overmars A. Frank van der Stappen Abstract The vibratory bowl feeder is the oldest and still most common approach are exponential in the number of trap parameters, many indus- trial part feeders use few-parameter traps

Utrecht, Universiteit

231

7 CFR 58.625 - Fruit or syrup feeders.  

Code of Federal Regulations, 2010 CFR

...Agriculture 3 2010-01-01 2010-01-01 false Fruit or syrup feeders. 58.625 Section 58.625...Service 1 Equipment and Utensils § 58.625 Fruit or syrup feeders. Fruit or syrup feeders inject flavoring material into...

2010-01-01

232

7 CFR 58.625 - Fruit or syrup feeders.  

Code of Federal Regulations, 2012 CFR

...Agriculture 3 2012-01-01 2012-01-01 false Fruit or syrup feeders. 58.625 Section 58.625...Service 1 Equipment and Utensils § 58.625 Fruit or syrup feeders. Fruit or syrup feeders inject flavoring material into...

2012-01-01

233

7 CFR 58.625 - Fruit or syrup feeders.  

Code of Federal Regulations, 2013 CFR

...Agriculture 3 2013-01-01 2013-01-01 false Fruit or syrup feeders. 58.625 Section 58.625...Service 1 Equipment and Utensils § 58.625 Fruit or syrup feeders. Fruit or syrup feeders inject flavoring material into...

2013-01-01

234

7 CFR 58.625 - Fruit or syrup feeders.  

Code of Federal Regulations, 2011 CFR

...Agriculture 3 2011-01-01 2011-01-01 false Fruit or syrup feeders. 58.625 Section 58.625...Service 1 Equipment and Utensils § 58.625 Fruit or syrup feeders. Fruit or syrup feeders inject flavoring material into...

2011-01-01

235

7 CFR 58.625 - Fruit or syrup feeders.  

...Agriculture 3 2014-01-01 2014-01-01 false Fruit or syrup feeders. 58.625 Section 58.625...Service 1 Equipment and Utensils § 58.625 Fruit or syrup feeders. Fruit or syrup feeders inject flavoring material into...

2014-01-01

236

Photocopy of drawing, "Sluiceway at Combined Locks, Glens Falls Feeder" ...  

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

Photocopy of drawing, "Sluiceway at Combined Locks, Glens Falls Feeder" (from Champlain canal structure book) 6-45, New York State Archives and Manuscripts, Albany, New York), c. 1858 - Glens Falls Feeder, Sluice, Along south side of Glens Falls Feeder between locks 10 & 20, Hudson Falls, Washington County, NY

237

Phorbol ester stimulates choline uptake in Swiss 3T3 fibroblasts following introduction of the gene encoding protein kinase C alpha.  

PubMed Central

Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation into phosphatidylcholine (PtdCho) in a time- and concentration-dependent manner in wild-type NIH 3T3 fibroblasts. The accumulation of labelled choline induced by PMA was paralled by an increase in choline mass. The results implicate protein kinase C (PKC) in the regulation of choline uptake. In order to address the PKC-subtype specificity of this response, a study was undertaken in Swiss 3T3 fibroblast cells, which normally express very low levels of PKC alpha. A retroviral expression system was used to introduce the genes for PKC alpha and neomycin resistance (used for selection) into the cells. Two resulting lines expressed PKC alpha at levels that were 20-fold higher than those found in the control (neomycin-resistant) line, or in the wild-type cells. In control Swiss 3T3 fibroblasts, 1 microM PMA elevated choline levels by only 30%, whereas, in Swiss 3T3 cell lines that stably over-expressed PKC alpha, PMA caused a 5-fold enhancement in [14C]choline accumulation. This concentration of PMA significantly increased [14C]PtdCho levels in both control and PKC alpha-over-expressing lines, although the effect in the latter was significantly greater. The effects of PMA were inhibited by the PKC antagonist sphingosine. These results implicate PKC alpha in the regulation of choline accumulation and phospholipid synthesis in fibroblasts. Although additional PKC subtypes appear to participate in the control of PtdCho synthesis in these cells, PMA-stimulated choline uptake in Swiss 3T3 fibroblasts is almost entirely dependent on the presence of PKC alpha. Images Figure 1 PMID:7832781

Slack, B E; Breu, J; Livneh, E; Eldar, H; Wurtman, R J

1995-01-01

238

Efficient induction of focus formation in a subclone of NIH3T3 cells by c-myc and its inhibition by serum and by growth factors.  

PubMed

In both experimental and spontaneous tumors, c-myc expression is often enhanced following its amplification or its rearrangement adjacent to a strong promotor/enhancer. However, c-myc by itself does not induce foci efficiently in fibroblast cultures. The effect of high levels of c-myc expression from a retroviral construct was investigated in several rodent fibroblast cell lines grown in medium containing 10% fetal calf serum or in serum-free PC-1 medium. c-myc-infected NIH3T3 clone 7 cells exhibited efficient quantitative focus formation when grown in PC-1 medium, whereas foci were not detected when grown in serum-supplemented medium. NIH3T3 clone 7 was the only cell line found to be sensitive to c-myc; other clones of NIH3T3 or other rodent fibroblast cell lines proved to be resistant to c-myc focus formation. At least two major types of morphologically distinct c-myc-induced foci were observed; the first was similar to ras-transformed foci induced in NIH3T3 and other fibroblast cell lines, and the second type was composed of adipocyte-like cells similar to NIH3T3 L1 cells. The c-myc infected cells cloned from these two types of foci expressed high levels of retrovirus-derived c-myc RNA and exhibited elevated levels of immunoreactive myc protein, as detected by immunofluorescent staining with an anti-myc polyclonal antibody. c-myc-transformed clones displayed only a limited ability to grow in soft-agar in the presence of serum and were not tumorigenic in nude mice. Focus formation by c-myc was quantitatively inhibited by the addition of interferon alpha + beta (INF alpha, beta), tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta 1 (TGF beta 1) to the serum-free PC-1 medium, and, in correlation, NIH3T3 clone 7 cells produced the lowest level of endogenous TGF beta of the various cell lines tested. PMID:2193293

Blondel, B; Talbot, N; Merlo, G R; Wychowski, C; Yokozaki, H; Valverius, E M; Salomon, D S; Bassin, R H

1990-06-01

239

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

240

Maximum Photovoltaic Penetration Levels on Typical Distribution Feeders: Preprint  

SciTech Connect

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

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

2012-07-01

241

Ex vivo bio-compatibility of honey-alginate fibrous matrix for HaCaT and 3T3 with prime molecular expressions.  

PubMed

Honey's inherent compositional diversity, bio-compatibility and time tested therapeutic efficacy, especially in tissue repair as a topical agent, attract researchers towards harnessing its biomaterial potential particularly in developing matrix for tissue engineering applications. Hence, this study fabricates fibrous mat from optimum honey-alginate formulation and alginate solution using wet spinning technology. The physical and morphological properties of the scaffolds are assessed and finally their comparative biological performances are evaluated through in vitro studies on adherence, viability and prime molecular expression of HaCaT and 3T3 cells. The honey-alginate scaffold demonstrates better performance than that of alginate in terms of cellular adherence, viability and proper expression of cell-cell adhesion molecule (E-cadherin) and prime molecules of extra cellular matrix (Collagen I and III) by HaCaT and 3T3 respectively. PMID:22042457

Barui, Ananya; Khare, Ritesh; Dhara, Santanu; Banerjee, Provas; Chatterjee, Jyotirmoy

2014-12-01

242

Pentacyclic Triterpenoids from Spikes of Prunella vulgaris L. Inhibit Glycogen Phosphorylase and Improve Insulin Sensitivity in 3T3-L1 Adipocytes.  

PubMed

Phytochemical investigation of methanol extract from the spikes of Prunella vulgaris L. led to the isolation of two new pentacyclic triterpenoid glycosides Vulgasides I (1) and II (2) along with 13 known compounds (3-15). Their structures were established on the basis of nuclear magnetic resonance (1D and 2D) and mass spectroscopic data analysis. All the isolated compounds were screened for glycogen phosphorylase inhibitory activity and also evaluated for their effect on insulin sensitivity in 3T3-L1 adipocytes. Two new compounds (1, 2) did not demonstrate the glycogen phosphorylase inhibitory activity, but other compounds (3-11) exhibited varying degrees of glycogen phosphorylase inhibitory activity with IC50 values in the range from 30.69 to 68.85??M. Compounds 3, 6, 7, 11, and 13 demonstrated markedly increased insulin-mediated glucose consumption in 3T3-L1 adipocytes. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25278372

Yu, Qian; Qi, Jin; Wang, Lu; Liu, Shou-Jin; Yu, Bo-Yang

2014-10-01

243

Pneumatic solids feeder for coal gasification reactor  

SciTech Connect

This invention is comprised of a pneumatic feeder system for a coal gasification reactor which includes one or more feeder tubes entering the reactor above the level of the particle bed inside the reactor. The tubes are inclined downward at their outer ends so that coal particles introduced into the tubes through an aperture at the top of the tubes slides downward away from the reactor and does not fall directly into the reactor. Pressurized gas introduced into, or resulting from ignition of recycled combustible gas in a chamber adjacent to the tube ends, propels the coal from the tube into the reactor volume and onto the particle bed. Leveling of the top of the bed is carried out by a bladed rotor mounted on the reactor stirring shaft. Coal is introduced into the tubes from containers above the tubes by means of rotary valves placed across supply conduits. This system avoids placement of feeder hardware in the plenum above the particle bed and keeps the coal from being excessively heated prior to reaching the particle bed.

Notestein, J.E.; Halow, J.S.

1991-12-31

244

Differentially expressed genes and signalling pathways are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-? estradiol  

PubMed Central

Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation. In this study, complementary DNA (cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells' response to 17-? estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha (?-MEM) cell culture supplemented with 17-? estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with 10?8 mol?L?1 17-? estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase (ALP) activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5?403 differentially expressed genes, of which 1?996 genes were upregulated and 3?407 genes were downregulated, 1?553 different functional classifications were identified by gene ontology (GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta (TGF-?)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to ?-MEM supplemented with 17-? estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies. PMID:24556956

Shang, Zhen-Zhen; Li, Xin; Sun, Hui-Qiang; Xiao, Guo-Ning; Wang, Cun-Wei; Gong, Qi

2014-01-01

245

Ginsenoside F2 possesses anti-obesity activity via binding with PPAR? and inhibiting adipocyte differentiation in the 3T3-L1 cell line.  

PubMed

Abstract Panax ginseng Meyer has been shown to be effective in mitigating various diseases. Protopanaxadiols (PPD) and protopanaxatriols (PPT), which are the main constituents of ginseng, have been shown to impact obesity. Therefore, we selected several important ginsenosides to perform our docking study and determine if they had binding affinity with the peroxisome proliferator activated receptor gamma (PPAR?), which is a major transcription factor in adipocytes. Among them, only a few ginsenosides demonstrated binding affinity with PPAR?. Other than ginsenoside F2 rest of them were previously reported by the researchers in experimental study in case of obesity cell line 3T3-L1 adipocyte. In few recent studies, it was reported that F2 has protective effects on malignant brain tumors as well as anti-cancer activity in breast cancer. Therefore, we felt it was important to focus on F2 when considering obesity. Our study focused on this ginsenoside and analyzed its impact on 3T3-L1 adipocytes. Following the molecular interaction studies, further experimental studies were carried out and demonstrated that ginsenoside F2 when treated with different doses reduces the level of lipid accumulated by the 3T3-L1 cell line during adipogenesis. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR results showed reduction in PPAR? and perilipin gene expression levels compared to that of differentiated adipocytes without any treatment. So considering the binding with a major adipocyte transcription factor and the performed experiments, we suggest that ginsenoside F2 may reduce obesity via the inhibition of adipogenesis in the 3T3-L1 cell line. PMID:24666293

Siraj, Fayeza Md; SathishKumar, Natarajan; Kim, Yeon Ju; Kim, Se Young; Yang, Deok Chun

2015-02-01

246

Increased cell proliferation of mouse fibroblast NIH-3T3 in vitro induced by excretory/secretory product(s) from Opisthorchis viverrini.  

PubMed

Infection by Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. However, the mechanism by which the parasite is involved in carcinogenesis is not clear. In addition to the direct damage of the bile duct epithelium via direct contact with O. viverrini, the excretory/secretory (ES) product(s) released from the parasites may play important roles in this process. We therefore investigated the responses of a fibroblast cell line, NIH-3T3, to ES product(s) released from O. viverrini by using a non-contact co-culture technique. In this culture system, the parasites in the upper chamber had no direct contact with the NIH-3T3 cells in the lower chamber of the culture plate. The results indicated a marked increase in NIH-3T3 cell proliferation in the non-contact co-culture condition with either 0% or 10% calf serum in the medium compared with that without parasites. ES product(s) increased cell proliferation by stimulating the expression of phosphorylated retinoblastoma (pRB) and cyclin D1, the key proteins in driving cells through the G1/S transition point of the cell cycle. This led to the induction of cells going into the S-phase of the cell cycle. ES product(s) also changed the morphology of NIH-3T3 cells to a refractive and narrow shape, which allowed the cells to proliferate in the limited culture area. For the first time, we have been able to demonstrate increased cell proliferation induced by the ES product(s) from O. viverrini; this finding may clarify how O. viverrini ES product(s) affect human bile duct epithelium during cholangiocarcinogenesis. PMID:15521634

Thuwajit, C; Thuwajit, P; Kaewkes, S; Sripa, B; Uchida, K; Miwa, M; Wongkham, S

2004-10-01

247

Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes  

PubMed Central

The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor ? (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes. PMID:25161868

Kajimoto, Kazuaki; Minami, Yoshitaka; Harashima, Hideyoshi

2014-01-01

248

Type-2 cannabinoid receptor regulates proliferation, apoptosis, differentiation, and OPG/RANKL ratio of MC3T3-E1 cells exposed to Titanium particles.  

PubMed

The type-2 cannabinoid receptor (CB2) is expressed in osteoblasts and plays a role in bone metabolism through regulation on bone mass and bone turnover, but the functional importance of CB2 in osteoblasts under Titanium (Ti) stimulation is incompletely understood. This study aimed to investigate the CB2 expression in osteoblasts under Ti stimulation and the effects of CB2 activation on proliferation, apoptosis, differentiation, mineralization, OPG, and RANKL expression of MC3T3-E1 cells exposed to Ti particles. MC3T3-E1 cells were incubated in the presence of Ti particles with or without CB2-specific agonist HU-308 and antagonist SR144528. Ti particles treatment obviously induced the CB2 expression in MC3T3-E1 cells, and reduced the cell survival in a dose- and time-dependent manner (p < 0.05). Addition of HU-308 could dose-dependently alleviate the Ti-induced decrease of cell survival (p < 0.05). The flow cytometry assay showed that comparing with the control group, the apoptosis rate and caspase-3 activity in the Ti group were significantly elevated (p < 0.05), which could be alleviated by HU-308. Moreover, HU-308 effectively attenuated the decrease of cell mineralization capability, alkaline phosphates (ALP) and osteocalcin activity, and increase of OPG/RANKL ratio induced by Ti particles treatment (p < 0.05). These effects were partially counteracted by combined treatment of CB2 antagonist SR144528 (p < 0.05). In conclusion, CB2 activation has a favorable inhibitory effect on Ti-induced reactions in MC3T3-E1 cell through modulating proliferation, apoptosis, differentiation, and RANKL expression. These findings suggest that activation of CB2 might be an effective therapeutic strategy to promote bone formation and reduce bone dissolution. PMID:25292314

Qiu, Shang; Zhao, Fengchao; Tang, Xianye; Pei, Fang; Dong, Hongyan; Zhu, Liang; Guo, Kaijin

2015-01-01

249

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

PubMed Central

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

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

2014-01-01

250

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

PubMed Central

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

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

2015-01-01

251

Defective regulation of mitogen-activated protein kinase activity in a 3T3 cell variant mitogenically nonresponsive to tetradecanoyl phorbol acetate.  

PubMed Central

Mitogen-activated protein (MAP) kinase is a serine/threonine-specific protein kinase which is activated in response to various mitogenic agonists (e.g., epidermal growth factor, insulin, and the tumor promoter tetradecanoyl phorbol acetate [TPA]) and requires both threonine and tyrosine phosphorylation for activity. This enzyme has recently been shown to be identical or closely related to pp42, a protein which becomes tyrosine phosphorylated in response to mitogenic stimulation. Neither the kinases which regulate MAP kinase/pp42 nor the in vivo substrates for this enzyme are known. Because MAP MAP kinase is activated and phosphorylated in response both to agents which stimulate tyrosine kinase receptors and to agents which stimulate protein kinase C, a serine/threonine kinase, we have examined the regulation and phosphorylation of this enzyme in 3T3-TNR9 cells, a variant cell line partially defective in protein kinase C-mediated signalling. In this communication, we show that in the 3T3-TNR9 variant cell line, TPA does not cause the characteristically rapid phosphorylation of pp42 or the activation and phosphorylation of MAP kinase. This defective response is not due to the absence of the MAP kinase/pp42 protein itself because both tyrosine phosphorylation of MAP kinase/pp42 and its enzymatic activation could be induced by platelet-derived growth factor in the 3T3-TNR9 cells. Thus, the defect in these variant cells apparently resides in some aspect of the regulation of MAP kinase phosphorylation. Since the 3T3-TNR9 cells are also defective with respect to the TPA-induced increase in ribosomal protein S6 kinase, these in vivo results reinforce the earlier in vitro finding that MAP kinase can regulate S6 kinase activity. These findings suggest a key role for MAP kinase in a kinase cascade cascade involved in the control of cell proliferation. Images PMID:1990261

L'Allemain, G; Sturgill, T W; Weber, M J

1991-01-01

252

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. PMID:24963319

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

253

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. PMID:24302962

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

254

Quercetin reversed lipopolysaccharide-induced inhibition of osteoblast differentiation through the mitogen?activated protein kinase pathway in MC3T3-E1 cells.  

PubMed

Quercetin, a flavonoid found in onions and other vegetables, has potential inhibitory effects on bone resorption in vivo and in vitro. In our previous study it was identified that quercetin triggered the apoptosis of lipopolysaccharide (LPS)?induced osteoclasts and inhibited bone resorption. Currently, little information is available detailing the effect of quercetin on osteoblast differentiation and bone formation in bacteria?induced inflammatory diseases. The present study aimed to investigate the effect of quercetin on osteoblast differentiation in MC3T3?E1 osteoblasts stimulated with LPS. LPS significantly downregulated the mRNA expression of osteoblast?related genes in the MC3T3?E1 cells. By contrast, quercetin significantly restored the LPS?suppressed mRNA expression of osteoblast?related genes in a dose?dependent manner. Quercetin also restored the protein expression of Osterix in MC3T3?E1 cells suppressed by LPS. Furthermore, quercetin selectively triggered the activation of the mitogen?activated protein kinase (MAPK) pathway by enhancing the expression of extracellular signal-regulated kinase and reducing the expression of c?Jun N?terminal kinase. These data suggest that quercetin reversed the inhibition of osteoblast differentiation induced by LPS through MAPK signaling. These findings suggest that quercetin may be of potential use as a therapeutic agent to restore osteoblast function in bacteria?induced bone diseases. PMID:25323558

Wang, Xin-Chun; Zhao, Nzhi-Jun; Guo, Chun; Chen, Jing-Tao; Song, Jin-Ling; Gao, Li

2014-12-01

255

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. PMID:24967352

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

2014-01-01

256

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

257

Activation of AMPK participates hydrogen sulfide-induced cyto-protective effect against dexamethasone in osteoblastic MC3T3-E1 cells.  

PubMed

Long-time glucocorticoids (GCs) usage causes osteoporosis. In the present study, we explored the potential role of hydrogen sulfide (H2S) against dexamethasone (Dex)-induced osteoblast cell damage, and focused on the underlying mechanisms. We showed that two H2S-producing enzymes, cystathionine ?-synthase (CBS) and cystathionine ?-lyase (CSE), were significantly downregulated in human osteonecrosis tissues as well as in Dex-treated osteoblastic MC3T3-E1 cells. H2S donor NaHS as well as the CBS activator S-adenosyl-l-methionine (SAM) inhibited Dex-induced viability reduction, death and apoptosis in MC3T3-E1 cells. NaHS activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling, which participated its cyto-protective activity. AMPK inhibition by its inhibitor (compound C) or reduction by targeted-shRNA suppressed its pro-survival activity against Dex in MC3T3-E1 cells. Further, we found that NaHS inhibited Dex-mediated reactive oxygen species (ROS) production and ATP depletion. Such effects by NaHS were again inhibited by compound C and AMPK?1-shRNA. In summary, we show that H2S inhibits Dex-induced osteoblast damage through activation of AMPK signaling. H2S signaling might be further investigated as a novel target for anti-osteoporosis treatment. PMID:25445596

Yang, Ming; Huang, Yue; Chen, Jia; Chen, Yi-Lei; Ma, Jian-Jun; Shi, Pei-Hua

2014-10-14

258

Regulation of the levels of Smad1 and Smad5 in MC3T3-E1 cells by Icariine in vitro.  

PubMed

The purpose of this study was to investigate the role of Icariine on the expression of Smadl and Smad5 mRNA and protein levels in MC3T3-E1 cells in vitro. MC3T3-E1 cells were cultured in the presence of different concentrations of Icariine (0, 10, 40 and 80 ng/ml). Smad1 and Smad5 mRNA levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and the expression of proteins was determined by western blotting, immunohistochemistry staining and immunofluorescence. Smad1 and Smad5 mRNA levels continuously increased in 10, 40 and 80 ng/ml of Icariine with time and the differences indicated statistical significance. Western blot analysis demonstrated that the Smad1 and Smad5 protein levels in the 10, 40 and 80 ng/ml groups were higher compared with the 0 ng/ml group at 24, 48 and 72 h, and the difference was statistically significant. Immunohistochemistry staining and immunofluorescence showed that the expression of the Smad1 and Smad5 proteins was higher in the cytoplasm and nuclei in the 10, 40 and 80 ng/ml groups compared with the 0 ng/ml group. Icariine has a direct stimulatory function on the differentiation of MC3T3-E1 osteoblastic cells in vitro, which may be mediated by increasing the production of Smad1 and Smad5 in osteoblasts. PMID:24297369

Zhou, Huifang; Wang, Shuo; Xue, Yuan; Shi, Nianke

2014-02-01

259

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. PMID:23766875

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

2013-01-01

260

Gadolinium promoted proliferation in mouse embryo fibroblast NIH3T3 cells through Rac and PI3K/Akt signaling pathways.  

PubMed

Nephrogenic systemic fibrosis (NSF) is a fibrosing disorder disease developed in patients with underlying renal insufficiency following exposure to gadolinium-based contrast agents (GBCAs). Previous studies have demonstrated that GdCl3 can promote NIH3T3 fibroblast cell proliferation, which provide a new clue to the role of GBCAs in the development of NSF. In the present study, we further clarify the molecular mechanism of Gd-promoted proliferation. The results showed that intervention with the Rac inhibitor NSC23766 abrogated Gd-promoted proliferation. The levels of active Rac1 significantly increased in Gd-treated cells detected by pull-down assays. In addition, the phosphorylation of Akt was significantly elevated in the treatment group, which was blocked by NSC23766. NSC23766 also reduced the migration of NIH3T3 cells enhanced by Gd. Moreover, the F-actin cytoskeleton was strengthened and the mitotic cell numbers was significantly increased after exposure to Gd. These results suggest that Rac and PI3K/Akt signaling pathways, as well as integrin-mediated signal pathway may play important roles in Gd-induced cell proliferation. In addition, under serum-free condition, Gd could decrease ROS accumulation and increase NIH3T3 cell survival. PMID:25037060

Shen, Liming; Yang, Aochu; Yao, Pengwei; Sun, Xiaohong; Chen, Cheng; Mo, Cuiping; Shi, Lei; Chen, Youjiao; Liu, Qiong

2014-08-01

261

Adlay seed extract (Coix lachryma-jobi L.) decreased adipocyte differentiation and increased glucose uptake in 3T3-L1 cells.  

PubMed

The aim of the present study was to investigate effects of the ethyl acetate fraction of an ethanol extract of Coix lachryma-jobi (ECLJ) on glucose uptake and adipocyte differentiation in 3T3-L1 cells. ECLJ phosphorylated AMP-activated protein kinase (AMPK) and its downstream substrate acetyl-coenzymeA carboxylase in 3T3-L1 cells in a time- and dose-dependent manner. Moreover, we discovered that compound C inhibits ECLJ-stimulated ACC phosphorylation. In addition, ECLJ exhibited a dose-dependent stimulation of glucose uptake in 3T3-L1 cells, and this increase was obviously attenuated by compound C. ECLJ also caused a decrease in the expression levels of adipogenesis factors such as fatty acid synthase, sterol-regulatory-element-binding protein-1c, peroxisome proliferator-activated receptor ?, and CAATT/enhancer binding protein ? in a dose-dependent manner. Differentiation was examined by Oil red O staining activity after ECLJ treatment for 6 days. ECLJ decreased mean droplet size. These results suggest a possible role for AMPK in the process of adipose differentiation and that ECLJ targeted for adipocyte functions could be effective in improving the symptoms of metabolic syndrome. PMID:21091246

Ha, Do Thi; Nam Trung, Trinh; Bich Thu, Nguyen; Van On, Tran; Hai Nam, Nguyen; Van Men, Chu; Thi Phuong, Tran; Bae, KiHwan

2010-12-01

262

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. PMID:24711359

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

2014-01-01

263

Blueberry Peel Extracts Inhibit Adipogenesis in 3T3-L1 Cells and Reduce High-Fat Diet-Induced Obesity  

PubMed Central

This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBP?, as well as the C/EBP? and PPAR? genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3?, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPAR? and C/EBP? and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBP?, C/EBP?, and PPAR? and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity. PMID:23936120

Jang, Sun-Hee; Lee, Soo-Jung; Ko, Yeoung-Gyu; Kim, Gon-Sup; Cho, Jae-Hyeon

2013-01-01

264

The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets.  

PubMed

Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1?. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening. PMID:22445253

Nagamoto, Yasuhito; Tashiro, Katsuhisa; Takayama, Kazuo; Ohashi, Kazuo; Kawabata, Kenji; Sakurai, Fuminori; Tachibana, Masashi; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

2012-06-01

265

The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells  

PubMed Central

Objectives The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period. PMID:25110642

Jeong, Youngdan; Yang, Wonkyung; Ko, Hyunjung

2014-01-01

266

Effects of different fatty acids and dietary lipids on adiponectin gene expression in 3T3-L1 cells and C57BL/6J mice adipose tissue.  

PubMed

Obesity is positively correlated to dietary lipid intake, and the type of lipid may play a causal role in the development of obesity-related pathologies. A major protein secreted by adipose tissue is adiponectin, which has antiatherogenic and antidiabetic properties. The aim of this study was to evaluate the effects of four different high-fat diets (enriched with soybean oil, fish oil, coconut oil, or lard) on adiponectin gene expression and secretion by the white adipose tissue (WAT) of mice fed on a selected diet for either 2 (acute treatment) or 60 days (chronic treatment). Additionally, 3T3-L1 adipocytes were treated for 48 h with six different fatty acids: palmitic, linoleic, eicosapentaenoic (EPA), docosahexaenoic (DHA), lauric, or oleic acid. Serum adiponectin concentration was reduced in the soybean-, coconut-, and lard-enriched diets in both groups. Adiponectin gene expression was lower in retroperitoneal WAT after acute treatment with all diets. The same reduction in levels of adiponectin gene expression was observed in epididymal adipose tissue of animals chronically fed soybean and coconut diets and in 3T3-L1 cells treated with palmitic, linoleic, EPA, and DHA acids. These results indicate that the intake of certain fatty acids may affect serum adiponectin levels in mice and adiponectin gene expression in mouse WAT and 3T3-L1 adipocytes. The effects appear to be time dependent and depot specific. It is postulated that the downregulation of adiponectin expression by dietary enrichment with soybean oil or coconut oil may contribute to the development of insulin resistance and atherosclerosis. PMID:17717684

Bueno, Allain Amador; Oyama, Lila Missae; de Oliveira, Cristiane; Pisani, Luciana Pelegrini; Ribeiro, Eliane Beraldi; Silveira, Vera Lucia Flor; Oller do Nascimento, Cláudia Maria

2008-01-01

267

Receptors for NPY and PACAP differ in expression and activity during adipogenesis in the murine 3T3-L1 fibroblast cell line  

PubMed Central

Background and purpose: Neuropeptides are involved in the regulation of food intake in the central nervous system, but they might also act on peripheral fat tissue via neuropeptide receptors. Experimental approach: We investigated the receptor expression and activity of pituitary adenylate cyclase-activating polypeptide (PACAP) and of neuropeptide Y at the mRNA and protein levels in the 3T3-L1 fibroblast line during differentiation into adipocytes. Intracellular calcium concentration was measured by calcium imaging. Key results: The PACAP receptors PAC1 and VPAC2 as well as the neuropeptide Y1 receptor were expressed at the mRNA level in fibroblasts, pre-adipocytes and adipocytes. The mRNA profile of the PAC1 receptor isoforms showed the HOP sequence, whereas the HIP-isoform was present in subconfluent 3T3-L1 fibroblasts only. At the protein level, the mature 3T3-L1 adipocytes produced the PAC1 and Y1 receptors; only the PAC1 receptor showed carbohydrate residues. Both neuropeptides induced an increase of intracellular calcium in mature adipocytes, which was absent in the precursor cells. These changes in calcium were mediated by Y1 and PAC1 receptors as demonstrated by the effects of specific receptor agonists and antagonists. Conclusions and implications: As the PAC1-HOP receptor variant seems to be responsible for PACAP-mediated calcium influx in many cell types, the HOP sequence might also mediate the increase in intracellular calcium in adipocytes. Because a high intracellular calcium level is associated with lipogenesis, peptidergic innervation of adipose tissue might be involved in stress-induced obesity. British Journal of Pharmacology (2009) 157, 620–632; doi:10.1111/j.1476-5381.2009.00164.x; published online 27 April 2009 PMID:19422400

Gericke, Martin T; Kosacka, Joanna; Koch, Daniela; Nowicki, Marcin; Schröder, Thomas; Ricken, Albert M; Nieber, Karen; Spanel-Borowski, Katharina

2009-01-01

268

Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Omega-3 PUFA has a direct anti-oxidant effect in adipocytes. Black-Right-Pointing-Pointer EPA and DHA induce HO-1 expression in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer Omega-3 PUFA and its end-product, 4-HHE, activates the Nrf-2/HO-1 pathway. Black-Right-Pointing-Pointer Omega-3 PUFA protects against oxidative stress-induced cytotoxicity. -- Abstract: Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid ({omega}3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of {omega}3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with {omega}3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). {omega}3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with {omega}3-PUFA prevented H{sub 2}O{sub 2}-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.

Kusunoki, Chisato, E-mail: yosizaki@belle.shiga-med.ac.jp [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan); Yang, Liu; Yoshizaki, Takeshi; Nakagawa, Fumiyuki; Ishikado, Atsushi; Kondo, Motoyuki; Morino, Katsutaro; Sekine, Osamu; Ugi, Satoshi [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan); Nishio, Yoshihiko [Division of Diabetes, Metabolism and Endocrinology, Department of Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)] [Division of Diabetes, Metabolism and Endocrinology, Department of Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kashiwagi, Atsunori; Maegawa, Hiroshi [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)

2013-01-04

269

p300-Dependent Acetylation of Activating Transcription Factor 5 Enhances C/EBP? Transactivation of C/EBP? during 3T3-L1 Differentiation  

PubMed Central

Adipogenesis is a multistep process by which 3T3-L1 preadipocytes differentiate into mature adipocytes through mitotic clonal expansion (MCE) and terminal differentiation. The CCAAT/enhancer-binding protein ? (C/EBP?) is an important transcription factor that takes part in both of these processes. C/EBP? not only transactivates C/EBP? and the peroxisome proliferator-activated receptor ? (PPAR?), which cause 3T3-L1 preadipocytes to enter terminal adipocyte differentiation, but also is required to activate cell cycle genes necessary for MCE. The identification of potential cofactors of C/EBP? will help to explain how C/EBP? undertakes these specialized roles during the different stages of adipogenesis. In this study, we found that activating transcription factor 5 (ATF5) can bind to the promoter of C/EBP? via its direct interaction with C/EBP? (which is mediated via the p300-dependent acetylation of ATF5), leading to enhanced C/EBP? transactivation of C/EBP?. We also show that p300 is important for the interaction of ATF5 with C/EBP? as well as for the binding activity of this complex on the C/EBP? promoter. Consistent with these findings, overexpression of ATF5 and an acetylated ATF5 mimic both promoted 3T3-L1 adipocyte differentiation, whereas short interfering RNA-mediated ATF5 downregulation inhibited this process. Furthermore, we show that the elevated expression of ATF5 is correlated with an obese phenotype in both mice and humans. In summary, we have identified ATF5 as a new cofactor of C/EBP? and examined how C/EBP? and ATF5 (acetylated by a p300-dependent mechanism) regulate the transcription of C/EBP?. PMID:24216764

Zhao, Yue; Zhang, Ya-Dong; Zhang, You-You; Qian, Shu-Wen; Zhang, Zhi-Chun; Li, Shu-Fen; Guo, Liang; Liu, Yuan; Wen, Bo; Lei, Qun-Ying; Tang, Qi-Qun

2014-01-01

270

Anti-transforming nature of ascorbic acid and its derivatives examined by two-stage cell transformation using BALB\\/c 3T3 cells  

Microsoft Academic Search

The anti-transforming effects of sodium ascorbate and its stable derivatives were examined in the two-stage transformation assay. When BALB\\/c 3T3 cells were treated with 0.2 ?g\\/ml 20-methylcholanthrene as an initiator, and 100 ng\\/ml 12-O-tetradecanoylphorbol-13-acetate as a promoter, the addition at the promotion stage of l-ascorbic acid-2-phosphate ester magnesium (APM) was most marked in the inhibition of transformation. The inhibitory effects

Toshiyuki Tsuchiya; Eiko Kato-Masatsuji; Toshi Tsuzuki; Makoto Umeda

2000-01-01

271

The study of oleanolic acid on the estrodiol production and the fat production of mouse preadipocyte 3T3-L1 in vitro.  

PubMed

The women during the menopause period have an increased tendency for the obesity, which represents the more fat production than during the premenopausal period. Although this is not beneficial overall, it could provide a compensatory source for the estrogen production for the menopausal women. So it would be meaningful to find an agent that could inhibit the fat production while does not disturb the total estrogen production by fat tissues. In the present study, the effect of oleanolic acid (OA) on the fat production and the total estrogen production of the differentiating mouse preadipocyte 3T3-L1 as well as the mechanisms behind those effects were preliminarily investigated. The cell line 3T3-L1 was chosen as the model cell because it is usually used for the research about the obesity. During the induced differentiation of 3T3-L1 cells, cells were intervened continuously with OA. The fat production was determined with the oil red staining assay and the total estrogen production was measured with the ELISA assay. Finally, the expression patterns for important genes of the fat production and the estrogen production were studied, respectively with the real-time fluorescence quantitative PCR (qPCR). The results showed that for the differentiating 3T3-L1 cells, OA could significantly inhibit the fat production and did not disturb the total estrogen production significantly. In the mechanism studies, OA was found to significantly down-regulate ACC, the key gene for fat synthesis, which could explain the inhibitory effect of OA on the fat production; OA was also found to significantly up-regulate CYP11A1, CYP17, CYP19, the key genes for the estrogen synthesis and significantly down-regulate CYP1A1, the key gene for the estrogen decomposition, which preliminarily explained the lack of the effect of OA on the total estrogen production. In conclusion, OA was found able to inhibit the fat production while maintaining the total estrogen level and the mechanisms for the above findings were preliminarily clarified, which suggests that OA may be useful to treat the menopausal obesity. PMID:25027016

Wan, Qian; Lu, Hua; Liu, Xia; Yie, Shangmian; Xiang, Junbei; Yao, Zouying

2015-01-01

272

Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase Aâ  

Microsoft Academic Search

In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin Eâ (PGEâ) synthesis. The ECââ values for stimulation of PGEâ synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-(..gamma..-thio)triphosphate stimulated PGEâ synthesis and InsP formation, and guanosine-5'-(..beta..-thio)diphosphate inhibited both PGEâ synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGEâ synthesis nor

R. M. Burch; J. Axelrod

1987-01-01

273

The influence of EPA and DHA on markers of inflammation in 3T3-L1 cells at different stages of cellular maturation  

PubMed Central

Background EPA and DHA have been reported to have anti-obesity and anti-inflammatory properties. Recent studies revealed that these positive actions of n-3 PUFA at least partially are connected with their influence on metabolism and secretory functions of the adipose tissue. However, their impact on old adipocytes is still poorly understood. Therefore the aim of the present study was to evaluate the influence of EPA and DHA on markers of inflammation in 3T3-L1 cells at different stages of cellular maturation. Methods Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 ?M EPA, or 50 ?M DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits. Results The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted. Conclusions In summary, age is an important determinant of fat accumulation in adipocytes and affects adipokines secretion by these cells. Moreover, the impact of investigated fatty acids: EPA and DHA on fat cells varies depending on the stage of maturation, and seems to be stronger in young cells than in mature and old ones. Docosahexaenoic acid exerts an anti-inflammatory action; however, on the basis of the obtained data it was not possible to determine whether eicosapentaenoic acid shows anti- or pro-inflammatory properties. PMID:24387137

2014-01-01

274

Down-regulation of cyclic-nucleotide phosphodiesterase 3B in 3T3-L1 adipocytes induced by tumour necrosis factor alpha and cAMP.  

PubMed Central

We have used murine 3T3-L1 cells, which differentiate in culture and acquire morphological and biochemical features of mature adipocytes, as a model for studying the expression of cyclic-nucleotide phosphodiesterase (PDE) 3B activity, protein and mRNA during differentiation and during long-term treatment of the cells with tumour necrosis factor alpha (TNF-alpha), a cytokine associated with insulin resistance, and a cAMP analogue, N(6),2'-O-dibutyryl cAMP (dbcAMP). PDE3B activity, protein and mRNA could be detected 4 days after the initiation of differentiation of 3T3-L1 preadipocytes. Treatment of 3T3-L1 adipocytes with 10 ng/ml TNF-alpha for 24 h produced a maximal (50%) decrease in PDE3B activity, protein and mRNA, which was well correlated with both activation of protein kinase A (PKA) and stimulation of lipolysis, presumably reflecting an increase in intracellular cAMP concentration. To investigate the effect of cAMP on PDE3B we treated 3T3-L1 adipocytes with dbcAMP. After 4 h with 0.5 mM dbcAMP, PDE3B activity was decreased by 80%, which was also correlated with a decrease in PDE3B protein and mRNA. This effect was abolished in the presence of N-[2-(bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide] (H-89), a specific PKA inhibitor. We conclude that the lipolytic effect of TNF-alpha involves the down-regulation of PDE3B, which is associated with increased activation of PKA, presumably owing to increased levels of cAMP. In addition, the PKA activation induced by dbcAMP resulted in the down-regulation of PDE3B. These results, which suggest that PDE3B is a novel target for long-term regulation by TNF-alpha and cAMP, could contribute to the understanding of the mechanisms of insulin resistance. PMID:10677351

Rahn Landström, T; Mei, J; Karlsson, M; Manganiello, V; Degerman, E

2000-01-01

275

50. BOAT GOING THROUGH FEEDER LOCK FROM LAKE HOPATCONG. A ...  

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

50. BOAT GOING THROUGH FEEDER LOCK FROM LAKE HOPATCONG. A BYPASS FLUME (LEFT OF LOCK) ALLOWED A CONTINUOUS FLOW OF WATER INTO THE FEEDER WHILE LOCK WAS IN USE TO MAINTAIN THE LEVEL OF THE MORRIS CANAL. - Morris Canal, Phillipsburg, Warren County, NJ

276

A hybrid genetic algorithm for component sequencing and feeder arrangement  

Microsoft Academic Search

This paper presents a hybrid genetic algorithm to optimize the sequence of component placements on a printed circuit board and the arrangement of component types to feeders simultaneously for a pick-and-place machine with multiple stationary feeders, a fixed board table and a movable placement head. The objective of the problem is to minimize the total traveling distance, or the traveling

William Ho; Ping Ji

2004-01-01

277

Tuning robotic part feeder parameters to maximize throughput  

Microsoft Academic Search

We study a programmable robotic part feeder that relies on a sequence of three conveyor belts to singulate and re-circulate parts. In industrial practice, belt speeds are set in an ad hoc fashion. Experience with real feeders reveals that throughput can suffer owing to: starvation where no parts are visible to the camera; and saturation, where too many parts are

Dadi Gudmundsson; Ken Goldberg

1999-01-01

278

Tuning robotic part feeder parameters to maximize throughput  

Microsoft Academic Search

We study a programmable robotic part feeder that relies on a sequence of three conveyor belts to separate and re-circulate parts. In industrial practice, belt speeds are set in an ad-hoc fashion. Experience with real feeders reveals that throughput can suffer due to (1) starvation where no parts are visible to the camera and (2) saturation, where too many parts

D. Gudmundsson; Ken Goldberg

1997-01-01

279

Fuzzy PI control design for an industrial weigh belt feeder  

Microsoft Academic Search

An industrial weigh belt feeder is used to transport solid materials into a manufacturing process at a constant feedrate. It exhibits nonlinear behavior because of motor friction, saturation, and quantization noise in the sensors, which makes standard autotuning methods difficult to implement. The paper proposes and experimentally demonstrates two types of fuzzy logic controllers for an industrial weigh belt feeder.

Yanan Zhao

2003-01-01

280

Tests of a UâOâ tubular feeder  

Microsoft Academic Search

This paper is devoted to tests of a tubular feeder for the purpose of assessing the prospects for its utilization for batching uranium containing materials. Batching operations of friable materials are an important component part of the technological processes of uranium production. In chemical technology, belt, screw conveyor, disk and vibration feeders are usually used for bulk batching. The effect

V. A. Zuev; A. A. Kryuchkov; Y. A. Repkin; S. F. Romanov; A. I. Tseilkovskaya

1986-01-01

281

On the design of guillotine traps for vibratory bowl feeders  

Microsoft Academic Search

The vibratory bowl feeder remains the most common approach to the automated feeding (orienting) of industrial parts. We study the algorithmic design of a trap in the bowl feeder track that filters out all but one orientation of a given polygonal part. We propose a new class of traps that we call guillotine traps, which remove a portion of the

Onno C. Goemans; Anthony Levandowski; Ken Goldberg; A. Frank Van Der Stappen

2005-01-01

282

On the Design of Guillotine Traps for Vibratory Bowl Feeders  

Microsoft Academic Search

ó The vibratory bowl feeder remains the most com- mon approach to the automated feeding (orienting) of industrial parts. We study the algorithmic design of a trap in the bowl feeder track that lters out all but one orientation of a given polygonal part. We propose a new class of traps that we call guillotine traps, which remove a portion

Onno C. Goemans; Anthony Levandowskiz; Ken Goldbergz; A. Frank van der Stappen

283

Automated fault location and diagnosis on electric power distribution feeders  

Microsoft Academic Search

This paper presents new techniques for locating and diagnosing faults on electric power distribution feeders. The proposed fault location and diagnosis scheme is capable of accurately identifying the location of a fault upon its occurrence, based on the integration of information available from disturbance recording devices with knowledge contained in a distribution feeder database. The developed fault location and diagnosis

Jun Zhu; D. L. Lubkeman; A. A. Girgis

1997-01-01

284

Fucoidan from the sporophyll of Undaria pinnatifida suppresses adipocyte differentiation by inhibition of inflammation-related cytokines in 3T3-L1 cells.  

PubMed

Obesity is a metabolic disorder, associated with cardiovascular disease and type 2 diabetes mellitus. Recent studies suggest that seaweed extracts are a significant source of bioactive compounds that are similar to dietary phytochemicals. Fucoidan, which is extracted from brown seaweeds, has a number of physiological functions. However, it is still unclear whether fucoidan would be beneficial in adipogenesis. In this study, we hypothesized that fucoidan extracted from the sporophyll of U pinnatifida exerts anti-obesity effects via inhibition of inflammatory-related cytokines. Thus, to test our hypothesis, we determined the obesity-specific therapeutic action of fucoidan in 3T3-L1 adipocytes. Herein, we showed that proliferator-activated receptor ?, CCAAR/enhancer-binding protein ?, and adipocyte protein 2 were significantly suppressed in the presence of fucoidan, which decreased expression of the inflammation-related genes during adipogenesis in 3T3-L1 adipocytes. Moreover, fucoidan also reduced the accumulation of lipids and reactive oxygen species production in adipocytes. In conclusion, these results demonstrate that fucoidan from the sporophyll of U pinnatifida suppresses adipogenesis through the inhibition of major markers and inflammation-related cytokines in adipocytes. Hence, these findings indicate that fucoidan may afford some potential to control or reduce obesity. PMID:22749180

Kim, Kui-Jin; Lee, Boo-Yong

2012-06-01

285

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells  

NASA Technical Reports Server (NTRS)

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

2002-01-01

286

Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors.  

PubMed Central

Extracellular ATP in concentrations of 5-50 microM displayed very little mitogenic activity by itself but it caused synergistic stimulation of [3H]thymidine incorporation in the presence of phorbol 12-tetradecanoate 13-acetate, epidermal growth factor, platelet-derived growth factor, insulin, adenosine, or 5'-(N-ethyl)carboxamidoadenosine. Cultures of Swiss 3T3, Swiss 3T6, A431, DDT1-MF2, and HFF cells were used. The percent of cell nuclei labeled with [3H]thymidine and cell number were also increased. ADP was equally mitogenic, while UTP and ITP were much less active. The effect of ATP was not due to hydrolysis by ectoenzymes to form adenosine, a known growth factor. Thus, the nonhydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate was mitogenic. In addition, it was found that ATP showed synergism in 3T6 and 3T3 cells when present for only the first hour of an incorporation assay, during which time no significant hydrolysis occurred. Furthermore, prolonged preincubation of cells with ATP reduced the mitogenic response to ATP but not to adenosine; preincubation with adenosine or N6-(R-phenylisopropyl)adenosine had the reverse effect. Finally, the effect of adenosine, but not of ATP, was inhibited by aminophylline. We conclude that extracellular ATP is a mitogen that interacts with P2 purinoceptors on the plasma membrane. PMID:2813367

Huang, N; Wang, D J; Heppel, L A

1989-01-01

287

Effect of Ganoderma lucidum extract on adipocyte differentiation and adiponectin gene expression in the murine pre-adipocyte cell line, 3T3-L1.  

PubMed

Ganoderma lucidum (G. lucidum), a traditional Chinese medicine, has been used for the treatment of various diseases including cancer and atherosclerosis. In this study, the positive effect of G. lucidum on metabolic syndrome was investigated in more detail by the use of 3T3-L1 pre-adipocyte cells. Treatment of 3T3-L1 cells with G. lucidum extract (GE) significantly promoted adipocyte differentiation and adiponectin production in a dose-dependent manner, as assessed by Oil-Red O staining, quantitative RT-PCR and ELISA. Treatment with GW9662, an inhibitor for peroxisome proliferator-activated receptor-gamma (PPARgamma), significantly attenuated GE-dependent adipocyte differentiation and adiponectin gene expression, suggesting the involvement of PPARgamma. Moreover, a reporter gene assay using GAL4-PPAR fusion proteins revealed that GE enhances GAL4-PPARgamma and GAL4-PPARalpha activities. These results indicate the presence of natural compounds possessing PPARgamma and PPARalpha activating properties in G. lucidum. PMID:20632304

Shimojo, Yosuke; Kosaka, Kunio; Shirasawa, Takuji

2011-02-01

288

Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR-103 in MC3T3-E1 osteoblasts  

PubMed Central

L-type voltage-sensitive calcium channels (LTCCs), particularly Cav1.2 LTCCs, play fundamental roles in cellular responses to mechanical stimuli in osteoblasts. Numerous studies have shown that mechanical loading promotes bone formation, whereas the removal of this stimulus under microgravity conditions results in a reduction in bone mass. However, whether microgravity exerts an influence on LTCCs in osteoblasts and whether this influence is a possible mechanism underlying the observed bone loss remain unclear. In the present study, we demonstrated that simulated microgravity substantially inhibited LTCC currents and suppressed Cav1.2 at the protein level in MC3T3-E1 osteoblast-like cells. In addition, reduced Cav1.2 protein levels decreased LTCC currents in MC3T3-E1 cells. Moreover, simulated microgravity increased miR-103 expression. Cav1.2 expression and LTCC current densities both significantly increased in cells that were transfected with a miR-103 inhibitor under mechanical unloading conditions. These results suggest that simulated microgravity substantially inhibits LTCC currents in osteoblasts by suppressing Cav1.2 expression. Furthermore, the down-regulation of Cav1.2 expression and the inhibition of LTCCs caused by mechanical unloading in osteoblasts are partially due to miR-103 up-regulation. Our study provides a novel mechanism for microgravity-induced detrimental effects on osteoblasts, offering a new avenue to further investigate the bone loss induced by microgravity. PMID:25627864

Sun, Zhongyang; Cao, Xinsheng; Zhang, Zhuo; Hu, Zebing; Zhang, Lianchang; Wang, Han; Zhou, Hua; Li, Dongtao; Zhang, Shu; Xie, Manjiang

2015-01-01

289

Amelioration of Mitochondrial Dysfunction-Induced Insulin Resistance in Differentiated 3T3-L1 Adipocytes via Inhibition of NF-?B Pathways  

PubMed Central

A growing body of evidence suggests that activation of nuclear factor kappa B (NF-?B) signaling pathways is among the inflammatory mechanism involved in the development of insulin resistance and chronic low-grade inflammation in adipose tissues derived from obese animal and human subjects. Nevertheless, little is known about the roles of NF-?B pathways in regulating mitochondrial function of the adipose tissues. In the present study, we sought to investigate the direct effects of celastrol (potent NF-?B inhibitor) upon mitochondrial dysfunction-induced insulin resistance in 3T3-L1 adipocytes. Celastrol ameliorates mitochondrial dysfunction by altering mitochondrial fusion and fission in adipocytes. The levels of oxidative DNA damage, protein carbonylation and lipid peroxidation were down-regulated. Further, the morphology and quantification of intracellular lipid droplets revealed the decrease of intracellular lipid accumulation with reduced lipolysis. Moreover, massive production of the pro-inflammatory mediators tumor necrosis factor-? (TNF-?) and interleukin-1? (IL-1?) were markedly depleted. Insulin-stimulated glucose uptake activity was restored with the enhancement of insulin signaling pathways. This study signified that the treatments modulated towards knockdown of NF-?B transcription factor may counteract these metabolic insults exacerbated in our model of synergy between mitochondrial dysfunction and inflammation. These results demonstrate for the first time that NF-?B inhibition modulates mitochondrial dysfunction induced insulin resistance in 3T3-L1 adipocytes. PMID:25474091

Hafizi Abu Bakar, Mohamad; Sarmidi, Mohamad Roji; Kai, Cheng Kian; Huri, Hasniza Zaman; Yaakob, Harisun

2014-01-01

290

Effect of Sutherlandia frutescens on the lipid metabolism in an insulin resistant rat model and 3T3-L1 adipocytes.  

PubMed

High fat diet induced insulin resistance correlates with dyslipidaemia and ectopic fat deposits in skeletal muscle and liver. The effects of Sutherlandia frutescens, an antidiabetic medicinal plant, on lipid metabolism were evaluated in an insulin resistant (IR) rat model and in 3?T3-preadipocytes. Wistar rats received normal diet (ND) or high fat diet (HFD). After the onset of IR in the HFD group, the rats were subdivided into two subgroups, which either continued with HFD or were treated with 50?mg S. frutescens/kg BW/day and HFD (HFD?+?SF). After 4?weeks, the HFD?+?SF rats had a significantly lower body weight than the HFD rats (p?3?T3-L1 cells were used as a model. Treatment with S. frutescens led to a decrease in triglyceride accumulation, whilst glucose consumption and lactate production were increased (p?

MacKenzie, Janine; Koekemoer, Trevor C; Roux, Saartjie; van de Venter, Maryna; Dealtry, Gill B

2012-12-01

291

Cell competition in mouse NIH3T3 embryonic fibroblasts is controlled by the activity of Tead family proteins and Myc.  

PubMed

Cell competition is a short-range communication originally observed in Drosophila. Relatively little is known about cell competition in mammals or in non-epithelial cells. Hippo signaling and its downstream transcription factors of the Tead family, control cell proliferation and apoptosis. Here, we established an in vitro model system that shows cell competition in mouse NIH3T3 embryo fibroblast cells. Co-culture of Tead-activity-manipulated cells with normal (wild-type) cells caused cell competition. Cells with reduced Tead activity became losers, whereas cells with increased Tead activity became super-competitors. Tead directly regulated Myc RNA expression, and cells with increased Myc expression also became super-competitors. At low cell density, cell proliferation required both Tead activity and Myc. At high cell density, however, reduction of either Tead activity or Myc was compensated for by an increase in the other, and this increase was sufficient to confer 'winner' activity. Collectively, NIH3T3 cells have cell competition mechanisms similar to those regulated by Yki and Myc in Drosophila. Establishment of this in vitro model system should be useful for analyses of the mechanisms of cell competition in mammals and in fibroblasts. PMID:25588835

Mamada, Hiroshi; Sato, Takashi; Ota, Mitsunori; Sasaki, Hiroshi

2015-02-15

292

Targeting the Autophagy Pathway Using Ectopic Expression of Beclin 1 in Combination with Rapamycin in Drug-Resistant v-Ha-ras-Transformed NIH 3T3 Cells  

PubMed Central

The effectiveness of an apoptosis-targeting therapy may be limited in tumor cells with defects in apoptosis. Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target (mTOR), inhibition of which results in autophagic cell death. In our study using multidrug-resistant v-Ha-ras-transformed NIH3T3 (Ras-NIH 3T3/Mdr) cells, we demonstrated that rapamycin-induced cell death may result from 2 different mechanisms. At high rapamycin concentrations (? 100 nM), cell death may occur via an autophagy-dependent pathway, whereas at lower concentrations (?10 nM), cell death may occur after G1-phase cell cycle arrest. This effect was accompanied by upregulation of p21Cip1 and p27Kip1 expression via an autophagy-independent pathway. We also tested whether inhibition of mTOR with low concentrations of rapamycin and ectopic Beclin-1 expression would further sensitize multidrug resistance (MDR)-positive cancer cells by upregulating autophagy. Rapamycin at low concentrations might be insufficient to initiate autophagosome formation in autophagy but Beclin-1 overexpression triggered additional processes downstream of mTOR during G1 cell cycle arrest by rapamycin. Our findings suggest that these combination strategies targeting autophagic cell death may yield significant benefits for cancer patients, because lowering rapamycin concentration for cancer treatment minimizes its side effects in patients undergoing chemotherapy. PMID:21350938

Eum, Ki-Hwan; Lee, Michael

2011-01-01

293

Melatonin rescues 3T3-L1 adipocytes from FFA-induced insulin resistance by inhibiting phosphorylation of IRS-1 on Ser307.  

PubMed

Melatonin is biosynthesized in the pineal gland and secreted into the bloodstream. Evidences indicate a role of melatonin in the regulation of glucose metabolism. The objective of this study was to investigate the effect of melatonin on insulin sensitivity in insulin resistant adipocytes. Following a preincubation with melatonin or vehicle for 30 min, insulin resistant cells of 3T3-L1 adipocytes were induced by palmitic acids (300 ?M, 6 h). Our results showed that palmitic acids inhibited both the basal and insulin-stimulated uptake of [(3)H]-2-Deoxyglucose, down-regulated the levels of IRS-1 and GLUT-4. However, compared to the vehicle group, melatonin pre-treatment increased significantly the uptake of [(3)H]-2-Deoxyglucose as well as the level of GLUT-4, and decreased phosphorylated IRS-1 (Ser307) although total IRS-1 did not change significantly. These data suggest that palmitic acids impair insulin signal via down-regulating the expressions of IRS-1 and GLUT-4; whereas melatonin can ameliorate insulin sensitivity by inhibiting Ser307 phosphorylation in IRS-1 and increasing GLUT-4 expressions in insulin resistant 3T3-L1 adipocytes. We conclude that melatonin regulates the insulin sensitivity and glucose homeostasis via inhibiting Ser-phosphorylation and improving function of IRS-1. PMID:24846082

She, Meihua; Hou, Hongjie; Wang, Zongbao; Zhang, Chi; Laudon, Moshe; Yin, Weidong

2014-08-01

294

The neck of caveolae is a distinct plasma membrane subdomain that concentrates insulin receptors in 3T3-L1 adipocytes.  

PubMed

Insulin receptors (IRs) segregate on plasma membrane microvilli, but in cells devoid of microvilli, such as adipocytes, the localization of IRs is a matter of controversy. In the present study, we examined the distribution of IRs in the plasma membrane of 3T3-L1 adipocytes. Quantitative electron microscopy indicates that IRs are predominantly associated with the neck, but not the bulb, of caveolae. Caveola necks represent distinct microdomains of the plasma membrane. Indeed, as shown by freeze-fracture analysis, intramembrane particles are concentrated as necklaces around the craters of caveolae. In addition, subcellular fractionation suggests that the neck and the bulb of caveolae present a different resistance to detergent solubility. Finally, cytoskeletal components, including actin, are highly enriched in the membrane area underlying the neck part of caveolae. IRs coimmunoprecipitate with cytoskeletal components, and disruption of the actin cytoskeleton alters IRs expression, localization, and signaling, thus supporting the notion that caveola necks are involved in intracellular signaling by IRs. Together, these results suggest that cytoskeletal proteins anchor IRs to microdomains in the caveola necks of 3T3-L1 adipocytes. By homology with IR localization in other cell types, we suggest that the necks of caveolae may represent the counterpart of microvillar domains in cells poor in microvilli such as adipocytes and that they play an important role as signaling platforms. PMID:17227843

Foti, Michelangelo; Porcheron, Geneviève; Fournier, Margot; Maeder, Christine; Carpentier, Jean-Louis

2007-01-23

295

Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR-103 in MC3T3-E1 osteoblasts.  

PubMed

L-type voltage-sensitive calcium channels (LTCCs), particularly Cav1.2 LTCCs, play fundamental roles in cellular responses to mechanical stimuli in osteoblasts. Numerous studies have shown that mechanical loading promotes bone formation, whereas the removal of this stimulus under microgravity conditions results in a reduction in bone mass. However, whether microgravity exerts an influence on LTCCs in osteoblasts and whether this influence is a possible mechanism underlying the observed bone loss remain unclear. In the present study, we demonstrated that simulated microgravity substantially inhibited LTCC currents and suppressed Cav1.2 at the protein level in MC3T3-E1 osteoblast-like cells. In addition, reduced Cav1.2 protein levels decreased LTCC currents in MC3T3-E1 cells. Moreover, simulated microgravity increased miR-103 expression. Cav1.2 expression and LTCC current densities both significantly increased in cells that were transfected with a miR-103 inhibitor under mechanical unloading conditions. These results suggest that simulated microgravity substantially inhibits LTCC currents in osteoblasts by suppressing Cav1.2 expression. Furthermore, the down-regulation of Cav1.2 expression and the inhibition of LTCCs caused by mechanical unloading in osteoblasts are partially due to miR-103 up-regulation. Our study provides a novel mechanism for microgravity-induced detrimental effects on osteoblasts, offering a new avenue to further investigate the bone loss induced by microgravity. PMID:25627864

Sun, Zhongyang; Cao, Xinsheng; Zhang, Zhuo; Hu, Zebing; Zhang, Lianchang; Wang, Han; Zhou, Hua; Li, Dongtao; Zhang, Shu; Xie, Manjiang

2015-01-01

296

Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells  

PubMed Central

This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC–MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells.

Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

2014-01-01

297

Effects of Panicum miliaceum L. extract on adipogenic transcription factors and fatty acid accumulation in 3T3-L1 adipocytes.  

PubMed

The dietary intake of whole grains is known to reduce the incidence of chronic diseases such as obesity, diabetes, cardiovascular disease, and cancer. To investigate whether there are anti-adipogenic activities in various Korean cereals, we assessed water extracts of nine cereals. The results showed that treatment of 3T3-L1 adipocytes with Sorghum bicolor L. Moench, Setaria italica Beauvois, or Panicum miliaceum L. extract significantly inhibited adipocyte differentiation, as determined by measuring oil red-O staining, triglyceride accumulation, and glycerol 3-phosphate dehydrogenase activity. Among the nine cereals, P. miliaceum L. showed the highest anti-adipogenic activity. The effects of P. miliaceum L. on mRNA expression of peroxisome proliferator-activated receptor-?, sterol regulatory element-binding protein 1, and the CCAAT/enhancer binding protein-? were evaluated, revealing that the extract significantly decreased the expression of these genes in a dose-dependent manner. Moreover, P. miliaceum L. extract changed the ratio of monounsaturated fatty acids to saturated fatty acids in adipocytes, which is related to biological activity and cell characteristics. These results suggest that some cereals efficiently suppress adipogenesis in 3T3-L1 adipocytes. In particular, the effect of P. miliaceum L. on adipocyte differentiation is associated with the downregulation of adipogenic genes and fatty acid accumulation in adipocytes. PMID:21779521

Park, Mi-Young; Seo, Dong-Won; Lee, Jin-Young; Sung, Mi-Kyung; Lee, Young-Min; Jang, Hwan-Hee; Choi, Hae-Yeon; Kim, Jae-Hyn; Park, Dong-Sik

2011-06-01

298

Effects of Panicum miliaceum L. extract on adipogenic transcription factors and fatty acid accumulation in 3T3-L1 adipocytes  

PubMed Central

The dietary intake of whole grains is known to reduce the incidence of chronic diseases such as obesity, diabetes, cardiovascular disease, and cancer. To investigate whether there are anti-adipogenic activities in various Korean cereals, we assessed water extracts of nine cereals. The results showed that treatment of 3T3-L1 adipocytes with Sorghum bicolor L. Moench, Setaria italica Beauvois, or Panicum miliaceum L. extract significantly inhibited adipocyte differentiation, as determined by measuring oil red-O staining, triglyceride accumulation, and glycerol 3-phosphate dehydrogenase activity. Among the nine cereals, P. miliaceum L. showed the highest anti-adipogenic activity. The effects of P. miliaceum L. on mRNA expression of peroxisome proliferator-activated receptor-?, sterol regulatory element-binding protein 1, and the CCAAT/enhancer binding protein-? were evaluated, revealing that the extract significantly decreased the expression of these genes in a dose-dependent manner. Moreover, P. miliaceum L. extract changed the ratio of monounsaturated fatty acids to saturated fatty acids in adipocytes, which is related to biological activity and cell characteristics. These results suggest that some cereals efficiently suppress adipogenesis in 3T3-L1 adipocytes. In particular, the effect of P. miliaceum L. on adipocyte differentiation is associated with the downregulation of adipogenic genes and fatty acid accumulation in adipocytes. PMID:21779521

Park, Mi-Young; Seo, Dong-Won; Lee, Jin-Young; Sung, Mi-Kyung; Lee, Young-Min; Jang, Hwan-Hee; Choi, Hae-Yeon; Kim, Jae-Hyn

2011-01-01

299

Hop and Acacia Phytochemicals Decreased Lipotoxicity in 3T3-L1 Adipocytes, db/db Mice, and Individuals with Metabolic Syndrome  

PubMed Central

The plant-based compounds rho-iso-alpha acids (RIAA) from Humulus lupulus (hops) and proanthocyanidins (PAC) from Acacia nilotica have been shown to modulate insulin signaling in vitro. We investigated their effects on triglyceride (TG) deposition in 3T3-L1 adipocytes, glucose and insulin in obese mouse models, and metabolic syndrome markers in adults with metabolic syndrome. The combination of RIAA and PAC synergistically increased TG content and adiponectin secretion in 3T3-L1 adipocytes under hyperinsulinemic conditions and reduced glucose or insulin in obese mice. In a clinical trial, tablets containing 100?mg RIAA and 500?mg PAC or placebo were administered to metabolic syndrome subjects (3 tablets/day, n = 35; 6 tablets/day, n = 34; or placebo, n = 35) for 12 weeks. Compared to placebo, subjects taking 3 tablets daily showed greater reductions in TG, TG : HDL, fasting insulin, and HOMA scores. The combination of RIAA : PAC at 1 : 5 (wt : wt) favorably modulates dysregulated lipids in insulin resistance and metabolic syndrome. PMID:20721358

Minich, Deanna M.; Lerman, Robert H.; Darland, Gary; Babish, John G.; Pacioretty, Linda M.; Bland, Jeffrey S.; Tripp, Matthew L.

2010-01-01

300

Mechanism of inhibition of growth of 3T3-L1 fibroblasts and their differentiation to adipocytes by dehydroepiandrosterone and related steroids: role of glucose-6-phosphate dehydrogenase.  

PubMed Central

Dehydroepiandrosterone (DHEA) and certain structural analogues block the differentiation of 3T3-L1 mouse embryo fibroblasts to adipocytes. These steroids also are potent uncompetitive inhibitors of mammalian glucose-6-phosphate dehydrogenases (G6PDs). We provide direct evidence that treatment of the 3T3-L1 cells with DHEA and its analogues results in intracellular inhibition of G6PD, which is associated with the block of differentiation: (i) Levels of 6-phosphogluconate and other products of the pentose phosphate pathway are decreased; (ii) the magnitude of these decreases depends on the potency of steroids as inhibitors of G6PD and on concentration and duration of exposure, and it is accompanied by a proportionate block of differentiation; (iii) in cells exposed to 16 alpha-bromoepiandrosterone (a more potent inhibitor of G6PD than DHEA) at concentrations that block differentiation, introduction of exogenous 6-phosphogluconate in liposomes raises the levels of 6-phosphogluconate and other products of the pentose phosphate pathway and partially relieves the steroid block of cell growth and differentiation. Images PMID:2524835

Shantz, L M; Talalay, P; Gordon, G B

1989-01-01

301

Distinct Roles of the Phosphatidate Phosphatases Lipin 1 and 2 during Adipogenesis and Lipid Droplet Biogenesis in 3T3-L1 Cells*  

PubMed Central

Lipins are evolutionarily conserved Mg2+-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis. PMID:24133206

Sembongi, Hiroshi; Miranda, Merce; Han, Gil-Soo; Fakas, Stylianos; Grimsey, Neil; Vendrell, Joan; Carman, George M.; Siniossoglou, Symeon

2013-01-01

302

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.  

PubMed Central

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides. Images Fig. 5. Fig. 6. PMID:2844145

Brown, K D; Laurie, M S; Littlewood, C J; Blakeley, D M; Corps, A N

1988-01-01

303

Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: An ultrastructural, compositional and comparative analysis with mouse bone.  

PubMed

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone. PMID:25460184

Addison, W N; Nelea, V; Chicatun, F; Chien, Y-C; Tran-Khanh, N; Buschmann, M D; Nazhat, S N; Kaartinen, M T; Vali, H; Tecklenburg, M M; Franceschi, R T; McKee, M D

2015-02-01

304

Adipogenic effects of piperlonguminine in 3T3-L1 cells and plasma concentrations of several amide constituents from Piper chaba extracts after treatment of mice.  

PubMed

In our previous study, piperlonguminine from the fruit of Piper chaba was reported to promote adipogenesis in 3T3-L1 cells like the peroxisome proliferator-activated receptor-? (PPAR?) agonist, troglitazone. In the present study, the mode of action of piperlonguminine in cells was examined. Piperlonguminine increased mRNA levels of adiponectin, glucose transporter 4, and fatty acid-binding protein (aP2). It also increased mRNA levels of PPAR?2 but, unlike troglitazone, piperlonguminine did not activate PPAR? directly in a nuclear receptor cofactor assay. Analyses of plasma from mice treated with piperlonguminine, piperine, and retrofractamide A, and an extract of the fruit, showed that concentrations of piperlonguminine were higher than those of piperine and retrofractamide A, and that the "area-under-the-curve" of piperine increased following in vivo administration of the extract. PMID:23584920

Yamaguchi, Itadaki; Matsuda, Hisashi; Zhang, Hailong; Hamao, Makoto; Yamashita, Chihiro; Kogami, Yuichiro; Kon'I, Haruka; Murata, Megumi; Nakamura, Seikou; Yoshikawa, Masayuki

2014-01-01

305

ER stress-inducible ATF3 suppresses BMP2-induced ALP expression and activation in MC3T3-E1 cells.  

PubMed

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts. PMID:24315873

Park, Jae-kyung; Jang, Hoon; Hwang, SeongSoo; Kim, Eun-Jung; Kim, Dong-Ern; Oh, Keon-Bong; Kwon, Dae-Jin; Koh, Jeong-Tae; Kimura, Kumi; Inoue, Hiroshi; Jang, Won-Gu; Lee, Jeong-Woong

2014-01-01

306

Reduction of adipogenesis and lipid accumulation by Taraxacum officinale (Dandelion) extracts in 3T3L1 adipocytes: an in vitro study.  

PubMed

In this in vitro study, we have investigated the ability of Taraxacum officinale (dandelion) to inhibit adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes. HPLC analysis of the three plant extracts used in this study-leaf and root extracts and a commercial root powder-identified caffeic and chlorogenic acids as the main phenolic constituents. Oil Red O staining and triglyceride levels analysis showed decreased lipid and triglyceride accumulation, respectively. Cytotoxicity was assessed with the MTT assay showing non-toxic effect among the concentrations tested. DNA microarray analysis showed that the extracts regulated the expression of a number of genes and long non-coding RNAs that play a major role in the control of adipogenesis. Taken together, our results indicate that the dandelion extracts used in this study may play a significant role during adipogenesis and lipid metabolism, and thus, supporting their therapeutic interest as potential candidates for the treatment of obesity. PMID:23956107

González-Castejón, Marta; García-Carrasco, Belén; Fernández-Dacosta, Raquel; Dávalos, Alberto; Rodriguez-Casado, Arantxa

2014-05-01

307

Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes  

SciTech Connect

Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-{alpha} and PPAR{gamma}. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPAR{gamma} antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPAR{gamma} activations. The results indicate that auraptene activates PPAR{gamma} in adipocytes to control adipocytekines such as adiponectin and MCP-1 and suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.

Kuroyanagi, Kayo; Kang, Min-Sook; Goto, Tsuyoshi; Hirai, Shizuka; Ohyama, Kana; Kusudo, Tatsuya [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Yu, Rina [Department of Food Science and Nutrition, University of Ulsan, Ulsan 680-749 (Korea, Republic of); Yano, Masamichi [Department of Citriculture, National Institute of Fruit Tree Science, Ministry of Agriculture, Forestry and Fisheries, Shimizu, Shizuoka 424-0292 (Japan); Sasaki, Takao [ARKRAY Inc., Kyoto 601-8045 (Japan); Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)], E-mail: fat@kais.kyoto-u.ac.jp

2008-02-01

308

Both type I and II IFN induce insulin resistance by inducing different isoforms of SOCS expression in 3T3-L1 adipocytes.  

PubMed

Although elevation of the blood glucose level is a causal adverse effect of treatment with interferon (IFN), the precise underlying molecular mechanism is largely unknown. We examined the effects of type I and type II IFN (IFN-? and IFN-?) on insulin-induced metabolic signaling leading to glucose uptake in 3T3-L1 adipocytes. IFN-? suppressed insulin-induced tyrosine phosphorylation of IRS-1 without affecting its expression, whereas IFN-? reduced both the protein level and tyrosine phosphorylation. Although both IFNs stimulated phosphorylation of STAT1 (at Tyr(701)) and STAT3 (at Tyr(705)) after treatment for 30 min, subsequent properties of induction of the SOCS isoform were different. IFN-? preferentially induced SOCS1 rather than SOCS3, whereas IFN-? strongly induced SOCS3 expression alone. In addition, adenovirus-mediated overexpression of either SOCS1 or SOCS3 inhibited insulin-induced tyrosine phosphorylation of IRS-1, whereas the reduction of IRS-1 protein was observed only in SOCS3-expressed cells. Notably, IFN-?-induced SOCS1 expression and suppression of insulin-induced tyrosine phosphorylation of IRS-1 were attenuated by siRNA-mediated knockdown of STAT1. In contrast, adenovirus-mediated expression of a dominant-negative STAT3 (F-STAT3) attenuated IFN-?-induced SOCS3 expression, reduction of IRS-1 protein, and suppression of insulin-induced glucose uptake but did not have any effect on the IFN-?-mediated SOCS1 expression and inhibition of insulin-induced glucose uptake. Interestingly, pretreatment of IFN-? with IL-6 synergistically suppressed insulin signaling, even when IL-6 alone had no significant effect. These results indicate that type I and type II IFN induce insulin resistance by inducing distinct SOCS isoforms, and IL-6 synergistically augments IFN-?-induced insulin resistance by potentiating STAT3-mediated SOCS3 induction in 3T3-L1 adipocytes. PMID:21386060

Wada, Tsutomu; Hoshino, Masashi; Kimura, Yukari; Ojima, Minoru; Nakano, Tetsuro; Koya, Daisuke; Tsuneki, Hiroshi; Sasaoka, Toshiyasu

2011-06-01

309

A homeopathic remedy from arnica, marigold, St. John’s wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts  

PubMed Central

Background Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. Methods We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712–2), its succussed hydroalcoholic solvent (0712–1) and unsuccussed solvent (0712–3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined “wound field”. All assays were performed in three independent controlled experiments. Results None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712–2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712–1) at 1:100 dilutions (p??0.05). Preparation (0712–2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p?3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis. PMID:22809174

2012-01-01

310

miR-135a-5p inhibits 3T3-L1 adipogenesis through activation of canonical Wnt/?-catenin signaling.  

PubMed

MicroRNAs are endogenous, conserved, and non-coding small RNAs that function as post-transcriptional regulators of fat development and adipogenesis. Adipogenic marker genes, such as CCAAT/enhancer binding protein ? (Cebpa), peroxisome proliferator-activated receptor ? (Pparg), adipocyte fatty acid binding protein (Ap2), and fatty acid synthase (Fas), are regarded as the essential transcriptional regulators of preadipocyte differentiation and lipid storage in mature adipocytes. Canonical Wnt/?-catenin signaling is recognized as a negative molecular switch during adipogenesis. In the present work we found that miR-135a-5p is markedly downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-135a-5p impairs the expressions of adipogenic marker genes as well as lipid droplet accumulation and triglyceride content, indicating the importance of miR-135a-5p for adipogenic differentiation and adipogenesis. Further studies show that miR-135a-5p directly targets adenomatous polyposis coli (Apc), contributes to the translocation of ?-catenin from cytoplasm to nucleus, and then activates the expressions of cyclin D1 (Ccnd1) and Cmyc, indicating the induction of canonical Wnt/?-catenin signaling. In addition, inhibition of APC with siRNA exhibits the same effects as overexpression of miR-135a-5p. Our findings demonstrate that miR-135a-5p suppresses 3T3-L1 preadipocyte differentiation and adipogenesis through the activation of canonical Wnt/?-catenin signaling by directly targeting Apc. Taken together, these results offer profound insights into the adipogenesis mechanism and the development of adipose tissue. PMID:24850830

Chen, Chen; Peng, Yongdong; Peng, Yinglin; Peng, Jian; Jiang, Siwen

2014-06-01

311

Isolation of cellular genes differentially expressed in mouse NIH 3T3 cells and a simian virus 40-transformed derivative: growth-specific expression of VL30 genes.  

PubMed Central

We constructed and screened a cDNA library made from simian virus 40 (SV40)-transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression. Images PMID:3016508

Singh, K; Saragosti, S; Botchan, M

1985-01-01

312

Transformer 2? homolog (Drosophila) (TRA2B) regulates protein kinase C ?I (PKC?I) splice variant expression during 3T3L1 preadipocyte cell cycle.  

PubMed

Obesity is characterized by adipocyte hyperplasia and hypertrophy. We previously showed that PKC? expression is dysregulated in obesity (Carter, G., Apostolatos, A., Patel, R., Mathur, A., Cooper, D., Murr, M., and Patel, N. A. (2013) ISRN Obes. 2013, 161345). Using 3T3L1 preadipocytes, we studied adipogenesis in vitro and showed that expression of PKC? splice variants, PKC?I and PKC?II, have different expression patterns during adipogenesis (Patel, R., Apostolatos, A., Carter, G., Ajmo, J., Gali, M., Cooper, D. R., You, M., Bisht, K. S., and Patel, N. A. (2013) J. Biol. Chem. 288, 26834-26846). Here, we evaluated the role of PKC?I splice variant during adipogenesis. Our results indicate that PKC?I expression level is high in preadipocytes and decreasing PKC?I accelerated terminal differentiation. Our results indicate that PKC?I is required for mitotic clonal expansion of preadipocytes. We next evaluated the splice factor regulating the expression of PKC?I during 3T3L1 adipogenesis. Our results show TRA2B increased PKC?I expression. To investigate the molecular mechanism, we cloned a heterologous splicing PKC? minigene and showed that inclusion of PKC? exon 9 is increased by TRA2B. Using mutagenesis and a RNA-immunoprecipitation assay, we evaluated the binding of Tra2? on PKC?I exon 9 and show that its association is required for PKC?I splicing. These results provide a better understanding of the role of PKC?I in adipogenesis. Determination of this molecular mechanism of alternative splicing presents a novel therapeutic target in the management of obesity and its co-morbidities. PMID:25261467

Patel, Rekha S; Carter, Gay; Cooper, Denise R; Apostolatos, Hercules; Patel, Niketa A

2014-11-14

313

Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3  

PubMed Central

Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Results Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Conclusion Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health. PMID:24119256

2013-01-01

314

The cytotoxicity of the autonomous parvovirus minute virus of mice nonstructural proteins in FR3T3 rat cells depends on oncogene expression.  

PubMed Central

The nonstructural (NS) proteins of the autonomous parvovirus minute virus of mice are involved in viral DNA replication and in the regulation of homologous and heterologous promoters. Moreover, NS products have proved to be cytotoxic, especially for transformed cells. We show here that intracellular accumulation of NS products is not sufficient to kill rat fibroblasts from the established cell line FR3T3, which is phenotypically normal in several respects. FRNS cell lines were obtained by stable transfection of FR3T3 cells by a vector carrying the NS genes under the control of the hormone-inducible long terminal repeat promoter of the mouse mammary tumor virus. In the presence of dexamethasone, the NS proteins were synthesized without associated cell death. Transformation of FRNS cells with the c-Ha-ras oncogene or polyomavirus oncogenes had little effect on their capacity for NS induction, as measured at both concentration and transactivating activity levels, yet the transformants were now dying within a few days in the presence of the inducer. The same results were obtained with cells stably transfected by a vector expressing the NS1 product alone, suggesting that in this system there is no cooperation between NS1 and NS2 for maximal cytopathic effect. Cell mortality after NS protein induction was quantitatively related to the yield of oncogene expression, while NS-1 was not limiting in this respect. Our results show that the NS1 protein is not lethal unless cellular factors that may depend on oncogene expression trigger its cytotoxicity. Images PMID:8083981

Mousset, S; Ouadrhiri, Y; Caillet-Fauquet, P; Rommelaere, J

1994-01-01

315

Correlations between radiation-induced double strand breaks, cell division delay, and cyclin-dependent signaling in x-irradiated NIH3T3 fibroblasts  

NASA Astrophysics Data System (ADS)

Molecular responses to radiation-induced DNA double strand breaks (DSB) are mediated by the phosphorylation of the histone variant H2AX which forms identifiable gamma-H2AX foci at the site of the DSB. This event is thought to be linked with the down-regulation of signaling proteins contributing to the checkpoints regulating cell cycle progression and, vis-a-vis , the induction of cell division delay. However, it is unclear whether this division delay is directly related to the number of DSB (gamma-H2AX foci) sustained by an irradiated cell and, if so, whether this number drives cells into cell cycle delay or apoptosis. For this reason, studies were conducted in the immortalized NIH/3T3 fibroblast cell in order to establish correlations between the temporal appearance of the gamma-H2AX foci (a DSB) and the expression of the cell cycle regulatory proteins, cyclin E, A, B1, and their cyclin kinase inhibitor, p21. Cell cycle kinetics and flow cytometry were used to establish radiation-induced division delay over a dose range of 1--6 Gy where a mitotic delay of 2.65 min/cGy was established. Correlations between the expression of cyclin E, A, B1, p21, and the generation of DSB were established in NIH/3T3 cells exposed to 2 or 4 Gy x-irradiation. The data suggest that the G1/S and S phase delay (cyclin E and cyclin A protein levels) are dependent on the dose of radiation while the G2/M (cyclin B1 protein levels) delay is dependent on the quantity of DSB sustained by the irradiated cell.

Cariveau, Mickael J.

2005-07-01

316

Enhanced osteogenic fate and function of MC3T3-E1 cells on nanoengineered polystyrene surfaces with nanopillar and nanopore arrays.  

PubMed

During in vitro culture, cell fate and function, including cell adhesion, morphology, proliferation and differentiation, are affected by surface characteristics, such as geometry, wettability, hardness, chemistry and charge. This study replicated two different types of nanoengineered polystyrene surfaces (NPS) containing nanopillar (NPS-Pi) or nanopore (NPS-Po) arrays by hot embossing and investigated their topographical effects on cell behavior using osteoblast-like MC3T3-E1 cells. To mass-replicate NPS, rigid metal nano-stamps were manufactured by nickel electroforming onto two different nano-templates: (1) a nanopore-arrayed anodic aluminum oxide nano-template using two-step electrochemical oxidation and (2) a nanopillar-arrayed polymer using hot embossing process. The physical and mechanical properties of the NPS, including geometry, wettability, hardness and elastic modulus, were evaluated with the help of field emission-scanning electron microscopy, a contact angle meter, and a nanoindenter. The nanotopography maintained the bulk property, while drastically changing the surface properties. In vitro the NPS had significant effects on MC3T3-E1 cell morphology, attachment, proliferation and osteogenic differentiation compared to a flat substrate due to the altered physical and mechanical surface properties of the nanoengineered surface. Interestingly, the NPS-Po was more effective at enhancing cell proliferation and osteogenesis differentiation. One potential explanation for these results may be that the subcellular binding sites induced by the nanostructures changed the cell morphology and promoted contractile cytoskeletons, thereby enhancing osteogenic differentiation. This, which allows for the cost-effective replication of NPS and the control of cell behavior, has various applications with respect to biomedical and cell surface interaction studies, in addition to enhanced osteogenic cell fate and function. PMID:23548407

Cha, Kyoung Je; Hong, Jung Min; Cho, Dong-Woo; Kim, Dong Sung

2013-06-01

317

Mechanisms for proteinase-activated receptor 1-triggered prostaglandin E2 generation in mouse osteoblastic MC3T3-E1 cells.  

PubMed

Abstract We analyzed signaling mechanisms for prostaglandin E2 (PGE2) production following activation of proteinase-activated receptor-1 (PAR1), a thrombin receptor, in preosteoblastic MC3T3-E1 cells. PAR1 stimulation caused PGE2 release, an effect suppressed by inhibitors of COX-1, COX-2, iPLA2, cPLA2, MAP kinases (MAPKs), Src, EGF receptor (EGFR) tyrosine kinase (EGFR-TK) and matrix metalloproteinase (MMP), but not by an intracellular Ca2+ chelator or inhibitors of PI3 kinase, protein kinase C (PKC) and NF-?B. PAR1 activation induced phosphorylation of MAPKs and upregulation of COX-2. The phosphorylation of p38 MAPK was suppressed by inhibitors of Src and EGFR-TK. The COX-2 upregulation was dependent on ERK, p38, EGFR-TK, Src, and COX-2 itself. PAR1 activation also induced MEK-dependent phosphorylation of cAMP response element binding protein (CREB). All inhibitors of EP1, EP2, EP3 and EP4 receptors suppressed the PAR1-triggered PGE2 release. Exogenously applied PGE2 facilitated PAR1-triggered COX-2 upregulation, but it alone had no effect. Together, the PAR1-mediated PGE2 production in MC3T3-E1 cells appears to involve iPLA2 and cPLA2 for arachidonic acid release, and the MEK/ERK/CREB and Src/MMP/EGFR/p38 pathways for COX-2 upregulation, which is facilitated by endogenous PGE2 formed by COX-2. These signaling mechanisms might underlie the role of the thrombin/PAR1/PGE2 system in the early stage of the bone healing. PMID:25205726

Maeda, Yuma; Sekiguchi, Fumiko; Yamanaka, Rumi; Sugimoto, Ryo; Yamasoba, Daichi; Tomita, Shiori; Nishikawa, Hiroyuki; Kawabata, Atsufumi

2015-02-01

318

Kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/?-catenin signaling pathways in MC3T3-E1 cells.  

PubMed

Kirenol has been reported to possess anti-oxidant, anti-inflammatory, anti-allergic, anti-adipogenic, and anti-arthritic activities; however, its effect on osteoblast differentiation has not yet been reported. The aim of the present study was to evaluate the effect of kirenol on osteoblast differentiation through activation of the bone morphogenetic protein (BMP) and Wnt/?-catenin signaling pathways in MC3T3-E1 cells. Kirenol markedly promoted alkaline phosphatase (ALP) activity and mineralization. Kirenol not only increased the expression of osteoblast differentiation markers, such as ALP, type I collagen (ColA1), and osteopontin (OPN), but also increased the expression of osteoprotegerin/receptor activator of nuclear factor kappa B ligand (OPG/RANKL) ratio. The effects of kirenol on osteoblast differentiation were accompanied by stimulating the expression of the BMP and Wnt/?-catenin signaling pathways, including BMP2, runt-related transcription factor 2 (Runx2), osterix (Osx), low density lipoprotein receptor related protein 5 (LRP5), disheveled 2 (DVL2), ?-catenin, cyclin D1 (CCND1), and phosphorylated glycogen synthase kinase 3? (GSK3?). In addition, kirenol up-regulated the expression of ?-catenin, CCND1, ALP, and ColA1 which were down-regulated by siRNA knockdown of ?-catenin. Overall, these results demonstrate that kirenol is capable of promoting osteoblast differentiation in MC3T3-E1 cells through activation of the BMP and Wnt/?-catenin signaling pathways, suggesting that it is a potential candidate target for treating or preventing osteoporosis. PMID:25062891

Kim, Mi-Bo; Song, Youngwoo; Hwang, Jae-Kwan

2014-10-01

319

Coexpression of luxA and luxB genes of Vibrio fischeri in NIH3T3 mammalian cells and evaluation of its bioluminescence activities.  

PubMed

Expression of bacterial luciferase enzyme (lux) in eukaryotic cells would provide a new bioreporter system for in vivo imaging and diagnostics technology. In spite of this, until now only a few efforts have been made to express bacterial luciferase enzyme in eukaryotic cells. We attempted to synthesize an expression construct of luxA and luxB genes from Vibrio fischeri. The luxA and luxB genes were cloned into the MCS of pTZ57R via the 5' kpnI, BamHI and BamHI, EcoRI restriction sites to generate pTZ57R/luxA and pTZ57R/luxB respectively, then newly synthesized constructs were cleaved with the same enzymes and respectively cloned into the pcDNA3.1(+) (Hyg) and pcDNA3.1(+) (Neo) expression vectors to create pcDNA3.1(+) (Hyg)/luxA and pcDNA3.1(+) (neo)/luxB. Recombinant constructs were cotransfected to the NIH3T3 cell line. Gene expression was confirmed by reverse transcription-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; in addition, bioluminescence characteristics of transfected NIH3T3 cell lines were evaluated by decanal supplement. In conclusion, in the current research, separate vector systems were constructed, which are composed of bacterial luciferase genes (luxA and luxB) that accordingly have not already been reported. These results hold promise toward the potential development of an autonomous light-generating lux reporter system in eukaryotic cells. PMID:23616465

Tehrani, Golnaz Asaadi; Mirzaahmadi, Sina; Bandehpour, Mojgan; Kazemi, Bahram

2014-02-01

320

PPAR? agonist fenofibrate attenuates TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway  

SciTech Connect

The ligand-activated transcription factor peroxisome proliferator-activated receptor-? (PPAR?) participates in the regulation of cellular inflammation. More recent studies indicated that sirtuin1 (SIRT1), a NAD{sup +}-dependent deacetylase, regulates the inflammatory response in adipocytes. However, whether the role of PPAR? in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPAR? agonist fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 and underlying mechanisms in 3T3-L1 adipocytes. We found that fenofibrate inhibited CD40 expression and up-regulated SIRT1 expression in tumor necrosis factor-? (TNF-?)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPAR? antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-?-induced CD40 expression in adipocytes. Importantly, NF-?B inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression in adipocytes. Further study found that fenofibrate decreased the expression of acetylated-NF-?B p65 (Ac-NF-?B p65) in TNF-?-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-?-stimulated adipocytes. Taken together, these findings indicate that PPAR? agonist fenofibrate inhibits TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. -- Highlights: • Fenofibrate up-regulates SIRT1 expression in TNF-?-stimulated adipocytes. • Fenofibrate inhibits CD40 expression through SIRT1 in adipocytes. • The effects of fenofibrate on CD40 and SIRT1 expressions are dependent on PPAR?. • Fenofibrate inhibits CD40 expression via SIRT1-dependent deacetylation of NF-?B. • Fenofibrate increases SIRT1 expression through PPAR? and AMPK in adipocytes.

Wang, Weirong [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Lin, Qinqin [Physical Education College, Yanshan University, Qinhuangdao, Hebei 066004 (China); Lin, Rong, E-mail: linrong63@yahoo.com.cn [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Zhang, Jiye [Faculty of Pharmacy, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Ren, Feng; Zhang, Jianfeng; Ji, Meixi; Li, Yanxiang [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China)

2013-06-10

321

46 CFR 113.25-8 - Distribution of general emergency alarm system feeders and branch circuits.  

Code of Federal Regulations, 2012 CFR

...emergency alarm system feeders and branch circuits. 113.25-8 Section 113.25-8...emergency alarm system feeders and branch circuits. (a) Each system must have a...zone feeder is necessary; then a branch circuit distribution panel or feeder...

2012-10-01

322

46 CFR 113.25-8 - Distribution of general emergency alarm system feeders and branch circuits.  

...emergency alarm system feeders and branch circuits. 113.25-8 Section 113.25-8...emergency alarm system feeders and branch circuits. (a) Each system must have a...zone feeder is necessary; then a branch circuit distribution panel or feeder...

2014-10-01

323

46 CFR 113.25-8 - Distribution of general emergency alarm system feeders and branch circuits.  

Code of Federal Regulations, 2013 CFR

...emergency alarm system feeders and branch circuits. 113.25-8 Section 113.25-8...emergency alarm system feeders and branch circuits. (a) Each system must have a...zone feeder is necessary; then a branch circuit distribution panel or feeder...

2013-10-01

324

46 CFR 113.25-8 - Distribution of general emergency alarm system feeders and branch circuits.  

Code of Federal Regulations, 2011 CFR

...emergency alarm system feeders and branch circuits. 113.25-8 Section 113.25-8...emergency alarm system feeders and branch circuits. (a) Each system must have a...zone feeder is necessary; then a branch circuit distribution panel or feeder...

2011-10-01

325

46 CFR 111.75-1 - Lighting feeders.  

Code of Federal Regulations, 2010 CFR

...CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 Lighting...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

2010-10-01

326

46 CFR 111.75-1 - Lighting feeders.  

Code of Federal Regulations, 2013 CFR

...CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 Lighting...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

2013-10-01

327

46 CFR 111.75-1 - Lighting feeders.  

Code of Federal Regulations, 2012 CFR

...CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 Lighting...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

2012-10-01

328

46 CFR 111.75-1 - Lighting feeders.  

Code of Federal Regulations, 2011 CFR

...CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 Lighting...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

2011-10-01

329

46 CFR 111.75-1 - Lighting feeders.  

...CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 Lighting...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

2014-10-01

330

46 CFR 112.43-15 - Emergency lighting feeders.  

Code of Federal Regulations, 2010 CFR

... 2010-10-01 2010-10-01 false Emergency lighting feeders. 112.43-15 Section...SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15...

2010-10-01

331

Analysis of Feeder Bus Network Design and Scheduling Problems  

PubMed Central

A growing concern for public transit is its inability to shift passenger's mode from private to public transport. In order to overcome this problem, a more developed feeder bus network and matched schedules will play important roles. The present paper aims to review some of the studies performed on Feeder Bus Network Design and Scheduling Problem (FNDSP) based on three distinctive parts of the FNDSP setup, namely, problem description, problem characteristics, and solution approaches. The problems consist of different subproblems including data preparation, feeder bus network design, route generation, and feeder bus scheduling. Subsequently, descriptive analysis and classification of previous works are presented to highlight the main characteristics and solution methods. Finally, some of the issues and trends for future research are identified. This paper is targeted at dealing with the FNDSP to exhibit strategic and tactical goals and also contributes to the unification of the field which might be a useful complement to the few existing reviews. PMID:24526890

Almasi, Mohammad Hadi; Karim, Mohamed Rehan

2014-01-01

332

11. MOVABLE BED SEDIMENTATION MODELS. AUTOMATIC SEDIMENT FEEDER DESIGNED AND ...  

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

11. MOVABLE BED SEDIMENTATION MODELS. AUTOMATIC SEDIMENT FEEDER DESIGNED AND BUILT BY WES. - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

333

39. CLOSE UP DETAIL OF THE FEEDER AND STAMP CONNECTION. ...  

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

39. CLOSE UP DETAIL OF THE FEEDER AND STAMP CONNECTION. THE STAMP AN MORTAR BOX ARE ON THE LEFT AND THE FEEDER WITH ITS FEEDER DISK IS ON THE RIGHT. NOTE THE COLLAR ON THE CENTER STAMP STEM (UPPER LEFT CORNER OF THE IMAGE) THAT ACTIVATES THE LEVER IN THE CENTER OF THE PHOTO. THE COLLAR IS POSITIONED SUCH THAT WHEN THE LEVEL OF THE MATERIAL REACHES A LOW POINT IN THE MORTAR BOX IT PUSHES DOWN ON THE LEVER WHICH IN TURN ACTIVATES THE AUTOMATIC FEEDER DRIVE MECHANISM WHICH THEM DELIVERS ORE INTO THE BACKSIDE OF THE MORTAR BOX. - Standard Gold Mill, East of Bodie Creek, Northeast of Bodie, Bodie, Mono County, CA

334

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.  

PubMed Central

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7568234

Isola, L M; Zhou, S L; Kiang, C L; Stump, D D; Bradbury, M W; Berk, P D

1995-01-01

335

Functional Proteomic Analysis of Long-term Growth Factor Stimulation and Receptor Tyrosine Kinase Coactivation in Swiss 3T3 Fibroblasts*  

PubMed Central

In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after 35S- and 33P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated. PDGF stimulation produced the highest overall rate of protein synthesis at any given time and induced the most sustained phospho-signaling. Simultaneous activation with two or three of the growth factors revealed both synergistic and antagonistic effects on protein synthesis and expression levels with PDGF showing dominance over both IGF-1 and EGF in generating distinct proteome compositions. Using signaling pathway inhibitors, PI3K was identified as an early site for signal diversification, with sustained activity of the PI3K/AKT pathway critical for regulating late protein synthesis and phosphorylation of target proteins and required for maintaining the PDGF-dependent motile phenotype. Several proteins were identified with novel PI3K/Akt-dependent synthesis and phosphorylations including eEF2, PRS7, RACK-1, acidic calponin, NAP1L1, Hsp73, and fascin. The data also reveal induction/suppression of key F-actin and actomyosin regulators and chaperonins that enable PDGFR to direct the assembly of a motile cytoskeleton, despite simultaneous antagonistic signaling activities. Together, the study demonstrates that long-term exposure to different growth factors results in receptor tyrosine kinase-specific regulation of relatively small subproteomes, and implies that the strength and longevity of receptor tyrosine kinase-specific signals are critical in defining the composition and functional activity of the resulting proteome. PMID:22956732

Nagano, Kohji; Akpan, Akunna; Warnasuriya, Gayathri; Corless, Steven; Totty, Nick; Yang, Alice; Stein, Robert; Zvelebil, Marketa; Stensballe, Allan; Burlingame, Al; Waterfield, Michael; Cramer, Rainer; Timms, John F.; Naaby-Hansen, Søren

2012-01-01

336

Autocrine/paracrine function of globular adiponectin: inhibition of lipid metabolism and inflammatory response in 3T3-L1 adipocytes.  

PubMed

Adiponectin is an adipose-derived hormone, with beneficial effects on insulin sensitivity and inflammation. The aim of this study was to clarify the autocrine/paracrine effects of globular adiponectin (gAd) administrated during differentiation on the function of the mature adipocytes. Experiments were performed on 3T3-L1 preadipocytes treated with gAd (10nM), starting at an early stage of differentiation. gAd treatment during differentiation was without effect on mRNA expression of adiponectin and AdipoR2, but increased AdipoR1 expression. PPARgamma, perillipin and FABP4 mRNA expressions were lower in gAd-treated adipocytes, accompanied by a reduction in lipid accumulation. While mRNA expression of HSL was not affected by gAd compared to untreated adipocytes, both ATGL and FAS were reduced, indicating that gAd regulates both lipolysis and lipogenesis. PPAR?, ACOX2 and UCPs mRNA expressions were not affected by gAd, indicating that the reduction in lipid content is not attributed to an increase in fatty-acid oxidation. In accord with the lower PPAR? expression, there was reduced Glut4 mRNA expression; however, insulin-induced PKB phosphorylation was enhanced by gAd, and glucose uptake was not altered. To investigate the effect of gAd on LPS-induced inflammatory response, cells were treated with gAd either during differentiation or 24 hours before induction of inflammation in differentiated adipocytes. LPS induced inflammatory response, as indicated by increase in IL6 and MCP-1 mRNA expression. gAd given through differentiation was much more effective in abrogating LPS-dependent cytokines production than gAd given to the mature adipocyte. In conclusion, elevated gAd at differentiation of 3T3-L1 cells leads to reduced lipid content, reduced lipid metabolism and high resistance to inflammation. This article is protected by copyright. All rights reserved. PMID:25491932

Lazra, Y; Falach, A; Frenkel, L; Rozenberg, K; Sampson, Sr; Rosenzweig, T

2014-12-10

337

Ghrelin augments the expressions and secretions of proinflammatory adipokines, VEGF120 and MCP-1, in differentiated 3T3-L1 adipocytes.  

PubMed

Ghrelin is a physiological-active peptide with growth hormone-releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin-direct-effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3-L1 adipocytes stimulated with ghrelin in vitro . The stimulation with 50?nmol/L ghrelin for 24?h resulted in the significant 1.9-fold increase on vascular endothelial growth factor-120 (VEGF(120)) releases (p?3T3-L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor-? (TNF-?), interleukin-1? (IL-1?), IL-6, IL-10 and adiponectin secretions. In addition, Akt phosphorylation on Ser473 and c-Jun NH2 -terminal protein kinase (JNK) phosphorylation on Thr183/Tyr185 were markedly enhanced 1.4-fold (p?

Kitahara, Atsuko; Takahashi, Kazuto; Moriya, Rie; Onuma, Hirohisa; Handa, Keiko; Sumitani, Yoshikazu; Tanaka, Toshiaki; Katsuta, Hidenori; Nishida, Susumu; Sakurai, Takuya; Inukai, Kouichi; Ohno, Hideki; Ishida, Hitoshi

2015-01-01

338

Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes  

SciTech Connect

In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

Chung, Le Thi Kim, E-mail: ngocanh@nutr.med.tokushima-u.ac.jp [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Hosaka, Toshio [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan)] [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Teshigawara, Kiyoshi [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan); Sakai, Tohru [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan)] [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Nakaya, Yutaka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Funaki, Makoto, E-mail: m-funaki@clin.med.tokushima-u.ac.jp [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)

2010-01-01

339

Constitutively active Ras negatively regulates Erk MAP kinase through induction of MAP kinase phosphatase 3 (MKP3) in NIH3T3 cells.  

PubMed

The Ras/Raf/MEK/Erk signaling pathway is important for regulation of cell growth, proliferation, differentiation, survival, and apoptosis in response to a variety of extracellular stimuli. Lack of Erk MAPK activation is observed in several cancer cells despite active activation of Ras. However, little is known about the modulation of Erk1/2 activity by active Ras. Here, we show that overexpression of active H-Ras (H-RasG12R) in NIH3T3 fibroblasts impaired FGF2-induced Erk1/2 phosphorylation, as compared to wild-type cells. Northern blot analysis revealed that prolonged expression of active Ras increased MAP kinase phosphatase 3 (MKP3) mRNA expression, a negative regulator of Erk MAPK. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway abrogated active Ras-induced up-regulation of MKP3 expression, leading to the rescue of Erk1/2 phosphorylation. Our results demonstrated that the Ras/Raf/MEK/Erk signaling cascade is negatively regulated by the PI3K/Akt dependent transcriptional activation of the MKP3 gene. [BMB Reports 2014; 47(12): 685-690]. PMID:24602610

Park, Young Jae; Lee, Jong Min; Shin, Soon Young; Kim, Young Ho

2014-12-01

340

Anti-Obesity Effects of Boussingaulti gracilis Miers var. pseudobaselloides Bailey via Activation of AMP-Activated Protein Kinase in 3T3-L1 Cells  

PubMed Central

Abstract In a previous study, we demonstrated the anti-obesity and hypolipidemic effects of Boussingaulti gracilis Miers var. pseudobaselloides Bailey in high-fat diet-induced obese rats. The present study investigated the molecular mechanisms by which B. gracilis Miers var. pseudobaselloides Bailey ethanol extract (BGE) conferred antidifferentiation and anti-adipogenic effects in the 3T3-L1 preadipocyte differentiation model. BGE treatment significantly and dose-dependently suppressed lipid accumulation and down-regulated the expression of major transcription factors involved in adipogenesis, such as peroxisome proliferator-activated receptor-?, CCAAT/enhancer binding protein ?, sterol regulatory element-binding proteins, and their target genes. It is important that treatment with BGE increased phosphorylation of AMP-activated protein kinase (AMPK), which is one of the rate-limiting enzymes in the fatty acid synthesis pathway, and its direct downstream protein, acetyl-coenzyme A carboxylase. These results suggest that BGE may exert anti-adipogenic effects through regulation of AMPK activity and expression of genes involved in lipogenesis. PMID:22871035

Kim, Hana

2012-01-01

341

Changes in the phospholipid fatty acid composition of the lipid droplet during the differentiation of 3T3-L1 adipocytes.  

PubMed

Lipid droplets (LDs) are independent organelles in adipocytes that are composed of a lipid ester core surrounded by a phospholipid monolayer. The fatty acid composition of the phospholipid monolayer should determine the metabolism and dynamics of LDs. In this study, we examined the fatty acid composition of phospholipid monolayer in LDs during the differentiation of 3T3-L1 adipocytes. The levels of saturated fatty acids (SFAs), such as 16:0 and 18:0, in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of LDs decreased during differentiation. In contrast, the levels of monounsaturated fatty acids (MUFAs) such as 16:1n-7 and 18:1n-9 in PC and PE of LDs and the level of polyunsaturated fatty acids (PUFAs) such as 20:3n-6, 20:4n-6 and 22:6n-3 in PE of LDs increased during differentiation. These results suggest that the phospholipid monolayer in mature LDs is more fluid than that in nascent LDs. The fatty acid compositions of the LD monolayer were different from those of the microsome bilayer in the early stage of differentiation, but similar to those of the microsome bilayer in the late stage of differentiation. These data provide evidence that biophysical properties of the phospholipid monolayer in LDs change during adipocyte differentiation. PMID:23760554

Arisawa, Kotoko; Ichi, Ikuyo; Yasukawa, Yukiko; Sone, Yasuko; Fujiwara, Yoko

2013-09-01

342

Induction of phospho-Thr-172 AMPK in 3T3-L1 adipocytes exposed to cold or treated with anisomycin, mithramycin A, and ionic compounds.  

PubMed

Cold exposure induces cellular responses, including subcellular molecule expression and transport responses, similar to those stimulated by insulin in 3T3-L1 (L1) adipocytes. The transport response is induced in L1 adipocytes treated with translation inhibitors. We examined the level of phospho-Thr-172 AMPK (an active form of AMPK, a known energy-state sensor) in L1 adipocytes exposed to different temperatures of 4-37 degrees C or stressors, including chemical inhibitors and activators. The phospho-AMPK level increased in cold-exposed cells and their subcellular fractions and decreased after rewarming and serum depletion. The phospho-molecule was also induced by anisomycin, which induces protein kinase activation and translation inhibition; mithramycin A, an inhibitor of transcription factor binding; and ionic compounds, which stimulate molecular signaling and alter several gene expression. These results indicate that temperature responses are mimicked by metabolic stressors through phospho-molecule alteration. Our results provide possible clues for clarifying the mechanisms underlying cold responses in L1 adipocytes. PMID:20919451

Ohsaka, Yasuhito; Nishino, Hoyoku; Nomura, Yasuyuki

2010-01-01

343

(-)-Hydroxycitric acid attenuates endoplasmic reticulum stress-mediated alterations in 3T3-L1 adipocytes by protecting mitochondria and downregulating inflammatory markers.  

PubMed

Endoplasmic reticulum (ER) stress is an emerging potential therapeutic target for metabolic syndrome due to its role in synthesis, secretion, and folding of proteins. It leads to an increased production of reactive oxygen species (ROS) which, along with mitochondrial dysfunction and reduced antioxidant defense, causes chronic cell injury. The present investigation aims to observe the alterations in adipocytes due to ER stress and the protective effect of hydroxycitric acid (HCA), a bioactive from Garcinia species, to develop the same as a nutraceutical. ER stress was induced in mature 3T3-L1 adipocytes by treating them with tunicamycin (2?g/ml) for 18 h. Alterations in cell viability, innate antioxidant system (superoxide dismutase, glutathione peroxidase, and glutathione reductase), mitochondria (membrane potential, biogenesis, and transition pore opening), and inflammatory cytokines (tumor necrosis factor, monocyte chemoattractant protein, interferon-?, interleukin (IL)-10, IL-6, and IL-1?) during ER stress, and co-treatment with HCA were analyzed. Endocrine function of adipocytes was also assessed by measuring adiponectin and leptin secretion levels. HCA protected the cells from ER stress by improving the antioxidant status and mitochondrial functions. The results validate nutraceutical properties of the edible bioactive, commonly used for culinary purpose. A more detailed study on the mechanism of action of HCA is required for developing it as a therapeutic agent for metabolic syndrome. PMID:25175938

Nisha, V M; Priyanka, A; Anusree, S S; Raghu, K G

2014-11-01

344

Sheets of Vertically Aligned BaTiO3 Nanotubes Reduce Cell Proliferation but Not Viability of NIH-3T3 Cells  

PubMed Central

All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Nanotechnology has the potential to address this problem by virtue of the ability of some nanomaterials to modulate interactions with cells, thereby inducing specific biological responses to implanted foreign materials. To this effect in the present study, we have characterised the growth of fibroblasts on nano-structured sheets constituted by BaTiO3, a material extensively used in biomedical applications. We found that sheets of vertically aligned BaTiO3 nanotubes inhibit cell cycle progression - without impairing cell viability - of NIH-3T3 fibroblast cells. We postulate that the 3D organization of the material surface acts by increasing the availability of adhesion sites, promoting cell attachment and inhibition of cell proliferation. This finding could be of relevance for biomedical applications designed to prevent or minimize fibrous encasement by uncontrolled proliferation of fibroblastic cells with loss of material-tissue interface underpinning long-term function of implants. PMID:25506693

Giannini, Marianna; Giannaccini, Martina; Sibillano, Teresa; Giannini, Cinzia; Liu, Dun; Wang, Zhigang; Baù, Andrea; Dente, Luciana; Cuschieri, Alfred; Raffa, Vittoria

2014-01-01

345

Genistein inhibits CCAAT/enhancer-binding protein beta (C/EBPbeta) activity and 3T3-L1 adipogenesis by increasing C/EBP homologous protein expression.  

PubMed Central

The tyrosine kinase inhibitor genistein inhibits 3T3-L1 adipogenesis when present during the first 72 h of differentiation. In this report, we investigated the underlying mechanisms involved in the anti-adipogenic effects of genistein. We found that genistein blocked the DNA binding and transcriptional activity of CCAAT/enhancer-binding protein beta (C/EBPbeta) during differentiation by promoting the expression of C/EBP homologous protein, a dominant-negative member of the C/EBP family. Loss of C/EBPbeta activity was manifested as a loss of differentiation-induced C/EBPalpha and peroxisome-proliferator-activated receptor gamma protein expression and a dramatic reduction in lipid accumulation. Further, we documented for the first time that C/EBPbeta was tyrosine-phosphorylated in vivo during differentiation and in vitro by activated epidermal growth factor receptor. Genistein inhibited both of these events. Collectively, these results indicate that genistein blocks adipogenesis and C/EBPbeta activity by increasing the level of C/EBP homologous protein and possibly by inhibiting the tyrosine phosphorylation of C/EBPbeta. PMID:12095417

Harmon, Anne W; Patel, Yashomati M; Harp, Joyce B

2002-01-01

346

In vitro biomimetic construction of hydroxyapatite-porcine acellular dermal matrix composite scaffold for MC3T3-E1 preosteoblast culture.  

PubMed

The application of porous hydroxyapatite-collagen (HAp-Collagen) as a bone tissue engineering scaffold is hindered by two main problems: its high cost and low initial strength. As a native 3-dimenssional collagen framework, purified porcine acellular dermal matrix (PADM) has been successfully used as a skin tissue engineering scaffold. Here we report its application as a matrix for the preparation of HAp to produce a bone tissue scaffold through a biomimetic chemical process. The HAp-PADM scaffold has two-level pore structure, with large channels (?100??m in diameter) inherited from the purified PADM microstructure and small pores (<100?nm in diameter) formed by self-assembled HAp on the channel surfaces. The obtained HAp-PADM scaffold (S15D) has a compressive elastic modulus as high as 600?kPa. The presence of HAp in sample S15D reduces the degradation rate of PADM in collagenase solution at 37°C. After 7 day culture of MC3T3-E1 pre-osteroblasts, MTT data show no statistically significant difference on pure PADM framework and HAp-PADM scaffold (p?>?0.05). Because of its high strength and nontoxicity, its simple preparation method, and designable and tailorable properties, the HAp-PADM scaffold is expected to have great potential applications in medical treatment of bone defects. PMID:20964580

Zhao, Hongshi; Wang, Guancong; Hu, Shunpeng; Cui, Jingjie; Ren, Na; Liu, Duo; Liu, Hong; Cao, Chengbo; Wang, Jiyang; Wang, Zhonglin

2011-03-01

347

New vinegar produced by tomato suppresses adipocyte differentiation and fat accumulation in 3T3-L1 cells and obese rat model.  

PubMed

There is an increasing surplus of tomatoes that are abandoned due to their failure to meet customer standards. Therefore, to allow both value additions and the effective reuse of surplus tomatoes, we developed tomato vinegar (TV) containing phytochemicals and evaluated its anti-obesity effects in vitro and in vivo. TV inhibited adipocyte differentiation of 3T3-L1 preadipocyte and lipid accumulation during differentiation. TV supplementation in rats fed a high-fat diet (HFD) markedly decreased visceral fat weights without changing the food and calories intakes. TV significantly decreased hepatic triglyceride and cholesterol levels compared to the HFD group. Furthermore, TV lowered plasma LDL-cholesterol level and antherogenic index compared to the HFD group, whereas elevated HDL-cholesterol to total cholesterol ratio. These results show that TV prevented obesity by suppressing visceral fat and lipid accumulation in adipocyte and obese rats, and suggest that TV can be used as an anti-obesity therapeutic agent or functional food. PMID:23871083

Lee, Ju-Hye; Cho, Hyun-Dong; Jeong, Ji-Hye; Lee, Mi-Kyung; Jeong, Yong-Ki; Shim, Ki-Hwan; Seo, Kwon-Il

2013-12-01

348

Comparative proteome analysis of 3T3-L1 adipocyte differentiation using iTRAQ-coupled 2D LC-MS/MS.  

PubMed

Adipose tissue is critical in obesity and type II diabetes. Blocking of adipocyte differentiation is one of the anti-obesity strategies targeting on strong rise in fat storage and secretion of adipokine(s). However, the molecular basis of adipocyte differentiation and its regulation remains obscure. Therefore, we exposed 3T3-L1 cell line to appropriate hormonal inducers as adipocyte differentiation model. Using iTRAQ-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, we nearly quantitated 1,000 protein species and found 106 significantly altered proteins during adipocyte differentiation. The great majority of differentially expressed proteins were related to metabolism enzymes, structural molecules, and proteins involved in signal transduction. In addition to previously reported differentially expressed molecules, more than 20 altered proteins previously unknown to be involved with adipogenic process were firstly revealed (e.g., HEXB, DPP7, PTTG1IP, PRDX5, EPDR1, SPNB2, STEAP3, TPP1, etc.). The partially differential proteins were verified by Western blot and/or real-time PCR analysis. Furthermore, the association of PCX and VDAC2, two altered proteins, with adipocyte conversion was analyzed using siRNA method, and the results showed that they could contribute considerably to adipogenesis. In conclusion, our data provide valuable information for further understanding of adipogenesis. PMID:21678470

Ye, Feng; Zhang, Huoming; Yang, Yi-Xuan; Hu, Huai-Dong; Sze, Siu Kwan; Meng, Wei; Qian, Jingru; Ren, Hong; Yang, Bao-Lin; Luo, Ming-Ying; Wu, Xiaoqiong; Zhu, Wu; Cai, Wei-Jun; Tong, Jian-Bin

2011-10-01

349

Pycnogenol® inhibits lipid accumulation in 3T3-L1 adipocytes with the modulation of reactive oxygen species (ROS) production associated with antioxidant enzyme responses.  

PubMed

Pycnogenol® is a group of flavonoids with antioxidant effects. Adipogenesis is the process of adipocyte differentiation. It causes the increase of lipids as well as ROS (reactive oxygen species). Lipid accumulation and ROS production were determined in 3?T3-L1 adipocyte, and the effect of Pycnogenol® was evaluated. Lipid accumulation was elevated in adipocyte treated with hydrogen peroxide, one of the ROS. Pycnogenol® showed an inhibitory effect on the lipid accumulation and ROS production during the adipogenesis. We also investigated the molecular events associated with ROS production and lipid accumulation. Our results showed that Pycnogenol® inhibited the mRNA expression of pro-oxidant enzymes, such as NOX4 (NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase 4), and the NADPH-producing G6PDH (glucose-6-phosphate dehydrogenase) enzyme. In addition, Pycnogenol® suppressed the mRNA abundance of adipogenic transcription factors, PPAR-? (peroxisome proliferator-activated receptor ?) and C/EBP-? (CCAAT/enhancer binding protein ?), and their target gene, aP2 (adipocyte protein 2) responsible for fatty acid transportation. On the other hand, Pycnogenol® increased the abundance of antioxidant proteins such as Cu/Zn-SOD (copper-zinc superoxide dismutase), Mn-SOD (manganese superoxide dismutase), GPx (glutathione peroxidase) and GR (glutathione reductase). Our results suggest that Pycnogenol® inhibits lipid accumulation and ROS production by regulating adipogenic gene expression and pro-/antioxidant enzyme responses in adipocytes. PMID:21796705

Lee, Ok-Hwan; Seo, Min-Jung; Choi, Hyeon-Son; Lee, Boo-Yong

2012-03-01

350

Inactivation of lipoprotein lipase in 3T3-L1 adipocytes by angiopoietin-like protein 4 requires that both proteins have reached the cell surface.  

PubMed

Lipoprotein lipase (LPL) and angiopoietin-like protein 4 (Angptl4) were studied in 3T3-L1 adipocytes. Transfections of the adipocytes with Angptl4 esiRNA caused reduction of the expression of Angptl4 to about one fourth of that in cells treated with vehicle only. This resulted in higher levels of LPL activity both on cell surfaces (heparin-releasable) and in the medium, while LPL activity within the cells remained unaffected. This demonstrated that even though both proteins are made in the same cell, Angptl4 does not inactivate LPL during intracellular transport. Most of the Angptl4 protein was present as covalent dimers and tetramers on cell surfaces, while within the cells there were only monomers. LPL gradually lost activity when incubated in medium, but there was no marked difference between conditioned medium from normal cells (rich in Angptl4) and medium after knockdown of Angptl4. Hence Angptl4 did not markedly accelerate inactivation of LPL in the medium. Experiments with combinations of different cells and media indicated that inactivation of LPL occurred on the surfaces of cells producing Angptl4. PMID:24220340

Makoveichuk, Elena; Vorrsjö, Evelina; Olivecrona, Thomas; Olivecrona, Gunilla

2013-11-29

351

Influence of heating and cyclic tension on the induction of heat shock proteins and bone-related proteins by MC3T3-E1 cells.  

PubMed

Stress conditioning (e.g., thermal, shear, and tensile stress) of bone cells has been shown to enhance healing. However, prior studies have not investigated whether combined stress could synergistically promote bone regeneration. This study explored the impact of combined thermal and tensile stress on the induction of heat shock proteins (HSPs) and bone-related proteins by a murine preosteoblast cell line (MC3T3-E1). Cells were exposed to thermal stress using a water bath (44°C for 4 or 8 minutes) with postheating incubation (37°C for 4 hours) followed by exposure to cyclic strain (equibiaxial 3%, 0.2?Hz, cycle of 10-second tensile stress followed by 10-second rest). Combined thermal stress and tensile stress induced mRNA expression of HSP27 (1.41 relative fold induction (RFI) compared to sham-treated control), HSP70 (5.55?RFI), and osteopontin (1.44?RFI) but suppressed matrix metalloproteinase-9 (0.6?RFI) compared to the control. Combined thermal and tensile stress increased vascular endothelial growth factor (VEGF) secretion into the culture supernatant (1.54-fold increase compared to the control). Therefore, combined thermal and mechanical stress preconditioning can enhance HSP induction and influence protein expression important for bone tissue healing. PMID:25013774

Chung, Eunna; Sampson, Alana Cherrell; Rylander, Marissa Nichole

2014-01-01

352

Influence of Heating and Cyclic Tension on the Induction of Heat Shock Proteins and Bone-Related Proteins by MC3T3-E1 Cells  

PubMed Central

Stress conditioning (e.g., thermal, shear, and tensile stress) of bone cells has been shown to enhance healing. However, prior studies have not investigated whether combined stress could synergistically promote bone regeneration. This study explored the impact of combined thermal and tensile stress on the induction of heat shock proteins (HSPs) and bone-related proteins by a murine preosteoblast cell line (MC3T3-E1). Cells were exposed to thermal stress using a water bath (44°C for 4 or 8 minutes) with postheating incubation (37°C for 4 hours) followed by exposure to cyclic strain (equibiaxial 3%, 0.2?Hz, cycle of 10-second tensile stress followed by 10-second rest). Combined thermal stress and tensile stress induced mRNA expression of HSP27 (1.41 relative fold induction (RFI) compared to sham-treated control), HSP70 (5.55?RFI), and osteopontin (1.44?RFI) but suppressed matrix metalloproteinase-9 (0.6?RFI) compared to the control. Combined thermal and tensile stress increased vascular endothelial growth factor (VEGF) secretion into the culture supernatant (1.54-fold increase compared to the control). Therefore, combined thermal and mechanical stress preconditioning can enhance HSP induction and influence protein expression important for bone tissue healing. PMID:25013774

Chung, Eunna; Sampson, Alana Cherrell; Rylander, Marissa Nichole

2014-01-01

353

Using quantitative PCR to identify kinesin-3 genes that are upregulated during growth arrest in mouse NIH3T3 cells.  

PubMed

Most cells in our body form a single primary cilium when entering growth arrest. During the past decade, a number of studies have revealed a key role for primary cilia in coordinating a variety of signaling pathways that control important cellular and developmental processes. Consequently, significant effort has been directed toward the identification of genes involved in ciliary assembly and function. Many candidate ciliary genes and proteins have been identified using large-scale "omics" approaches, including proteomics, transcriptomics, and comparative genomics. Although such large-scale approaches can be extremely informative, additional validation of candidate ciliary genes using alternative "small-scale" approaches is often necessary. Here we describe a quantitative PCR-based method that can be used to screen groups of genes for those that are upregulated during growth arrest in cultured mouse NIH3T3 cells and those that might have cilia-related functions. We employed this method to specifically search for mouse kinesin-3 genes that are upregulated during growth arrest and identified three such genes (Kif13A, Kif13B, and Kif16A). In principle, however, the method can be extended to identify other genes or gene families that are upregulated during growth arrest. PMID:20362085

Thorsteinsson, Rikke I; Christensen, Søren T; Pedersen, Lotte B

2009-01-01

354

Vitronectin Absorbed on Nanoparticles Mediate Cell Viability/Proliferation and Uptake by 3T3 Swiss Albino Mouse Fibroblasts: In Vitro Study  

PubMed Central

We study the interaction of 3T3 Swiss albino mouse fibroblasts with polymeric nanoparticles (NPs) and investigate cellular behaviour in terms of viability/cytotoxicity, cell cycle, NPs uptake, MAP kinase (ERK1/2), and focal adhesion kinase (FAK) activation. After incubation of NPs with cell culture media, western blot analysis showed that Vitronectin is retained by NPs, while Fibronectin is not detected. From cytotoxicity studies (MTT and BrdU methods) an LD50 of about 1.5?mg/mL results for NPs. However, NPs in the range 0.01–0.30?mg/mL are able to trigger a statistically significant increase in proliferation and cell cycle progression in dose and time depending manner. Also, biochemical evaluation of ERK1/2 and FAK clearly shows an increasing phosphorylation in a dose and time depending manner. Finally, we found by transmission electron microscopy that NPs are internalised by cells. Competitively blocking VN-integrin receptors with echistatin (1??g/mL) results in a decrease of viability/proliferation, cell cycle progression, cellular uptake, and FAK/ERK activation showing the involvement of Vitronectin receptors in signal transduction. In conclusion, our results show that cell surface NPs interactions are mediated by absorbed plasma proteins (i.e., Vitronectin) that represent an external stimuli, switched to the nucleus by FAK enzyme, which in turn modulate fibroblasts viability/proliferation. PMID:23710450

Rosso, F.; Marino, G.; Grimaldi, A.; Cafiero, G.; Chiellini, E.; Chiellini, F.; Barbarisi, M.; Barbarisi, A.

2013-01-01

355

The effect of gold nanoparticles on the proliferation and differentiation of murine osteoblast: a study of MC3T3-E1 cells in vitro.  

PubMed

The current study involves in identification and molecular levels characterization of optimal size and concentration of gold nanoparticles (AuNPs). Stable, gold nanoparticles were synthesized and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The concentration and size dependent effects of the gold nanoparticles on proliferation of pre-osteoblast cells MC3T3-E1 was evaluated employing MTT cell proliferation assay. The results revealed that 30 nm diameter gold nanoparticles at a concentration of 10(-11) ppm were the most effective in promoting cell proliferation. Assay for alkaline phosphatase (ALP) activity and ALP staining were also used to confirm the effect of gold nanoparticles on osteoblast proliferation and differentiation. Moreover, reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure the expression of the osteogenic genes Runx2, ALP, OCN and OPN as response gold nanoparticles. The data demonstrated that 30 nm gold nanoparticles at a concentration of 10(-11) ppm was the best combination of size and concentration to promote the proliferation and differentiation of osteoblasts, as indicated by an increase in the ALP activity and expression of the osteogenic genes Runx2, ALP, OCN and OPN. Collectively the results of this study suggest that gold nanoparticles can promote the proliferation and differentiation of osteoblasts and could be used effectively in treatments promoting bone regeneration. PMID:24757953

Yao, Yuanyuan; Shi, Xiujuan; Chen, Fengshan

2014-07-01

356

Isomeric C12-Alkamides from the Roots of Echinacea purpurea Improve Basal and Insulin-Dependent Glucose Uptake in 3T3-L1 Adipocytes.  

PubMed

Echinacea purpurea has been used in traditional medicine as a remedy for the treatment and prevention of upper respiratory tract infections and the common cold. Recent investigations have indicated that E. purpurea also has an effect on insulin resistance. A dichloromethane extract of E. purpurea roots was found to enhance glucose uptake in adipocytes and to activate peroxisome proliferator-activated receptor ?. The purpose of the present study was to identify the bioactive compounds responsible for the potential antidiabetic effect of the dichloromethane extract using a bioassay-guided fractionation approach. Basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes were used to assess the bioactivity of extract, fractions and isolated metabolites. A peroxisome proliferator-activated receptor ? transactivation assay was used to determine the peroxisome proliferator-activated receptor ? activating properties of the extract, active fractions and isolated metabolites. Two novel isomeric dodeca-2E,4E,8Z,10E/Z-tetraenoic acid 2-methylbutylamides together with two known C12-alkamides and ?-linolenic acid were isolated from the active fractions. The isomeric C12-alkamides were found to activate peroxisome proliferator-activated receptor ?, to increase basal and insulin-dependent glucose uptake in adipocytes in a dose-dependent manner, and to exhibit characteristics of a peroxisome proliferator-activated receptor ? partial agonist. PMID:25371981

Kotowska, Dorota; El-Houri, Rime B; Borkowski, Kamil; Petersen, Rasmus K; Fretté, Xavier C; Wolber, Gerhard; Grevsen, Kai; Christensen, Kathrine B; Christensen, Lars P; Kristiansen, Karsten

2014-12-01

357

Screening of dried plant seed extracts for adiponectin production activity and tumor necrosis factor-alpha inhibitory activity on 3T3-L1 adipocytes.  

PubMed

To search for dried plant seeds with potent anti-diabetes activity, we conducted a large scale screening for inhibitory activity 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. komatsuna (Brassica rapa var. perviridis), common bean (Phaseolus vulgaris L.), qing geng cai (Brassica rapa var. chinensis), green soybean (Glycine max), spinach (Spinacia oleracea L.) and sugar snap pea (Pisum sativum L.) markedly enhanced adiponectin production (11.3?~?12.7 ng/ml) but Japanese radish (Raphanus sativus), edible burdock (Arctium lappa L.), bitter melon (Momordica charantia) and broccoli (Brassica oleracea var. italica) did not (0.9?~?2.7 ng/ml). All adiponectin-production-enhancing seeds except spinach (2.7 pg/ml) and okra (Abelmoschus esculentus) (6.6 pg/ml) effectively decreased tumor necrosis factor-alpha levels (0.0 pg/ml). We further examined the effects on free radical scavenging activities in the dried seed extracts. Although scavenging activity correlated well with total phenolic content of samples, no correlation was observed with adiponectin production. These results point to the potential of dried seed extracts as a means to modify the activity of tumor necrosis factor-alpha for the adiponectin production. PMID:20717728

Okada, Yoshinori; Okada, Mizue; Sagesaka, Yumi

2010-09-01

358

Widening the mutation spectrum of EVC and EVC2: ectopic expression of Weyer variants in NIH 3T3 fibroblasts disrupts Hedgehog signaling.  

PubMed

Autosomal recessive Ellis-van Creveld syndrome and autosomal dominant Weyer acrodental dysostosis are allelic conditions caused by mutations in EVC or EVC2. We performed a mutation screening study in 36 EvC cases and 3 cases of Weyer acrodental dysostosis, and identified pathogenic changes either in EVC or in EVC2 in all cases. We detected 40 independent EVC/EVC2 mutations of which 29 were novel changes in Ellis-van Creveld cases and 2 were novel mutations identified in Weyer pedigrees. Of interest one EvC patient had a T>G nucleotide substitution in intron 7 of EVC (c.940-150T>G), which creates a new donor splice site and results in the inclusion of a new exon. The T>G substitution is at nucleotide +5 of the novel 5' splice site. The three Weyer mutations occurred in the final exon of EVC2 (exon 22), suggesting that specific residues encoded by this exon are a key part of the protein. Using murine versions of EVC2 exon 22 mutations we demonstrate that the expression of a Weyer variant, but not the expression of a truncated protein that mimics an Ellis-van Creveld syndrome mutation, impairs Hedgehog signal transduction in NIH 3T3 cells in keeping with its dominant effect. PMID:19810119

Valencia, Maria; Lapunzina, Pablo; Lim, Derek; Zannolli, Raffaella; Bartholdi, Deborah; Wollnik, Bernd; Al-Ajlouni, Othman; Eid, Suhair S; Cox, Helen; Buoni, Sabrina; Hayek, Joseph; Martinez-Frias, Maria L; Antonio, Perez-Aytes; Temtamy, Samia; Aglan, Mona; Goodship, Judith A; Ruiz-Perez, Victor L

2009-12-01

359

Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans  

SciTech Connect

The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with (35S)sulfate and (3H) glucosamine for 24 h and then extracted and analyzed. There was a 1.68 {plus minus} 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of {approximately} 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of {approximately} 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 {plus minus} 0.2-fold in media and 3.2 {plus minus} 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.

Calvo, J.C.; Rodbard, D.; Katki, A.; Chernick, S.; Yanagishita, M. (Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (USA))

1991-06-15

360

Altered subcellular distribution of estrogen receptor alpha is implicated in estradiol-induced dual regulation of insulin signaling in 3T3-L1 adipocytes.  

PubMed

We investigated the mechanisms by which estrogen alters insulin signaling in 3T3-L1 adipocytes. Treatment with 17beta-estradiol (E2) did not affect insulin-induced tyrosine phosphorylation of insulin receptor. E2 enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1/p85 association, phosphorylation of Akt, and 2-deoxyglucose uptake at 10(-8) m, but inhibited these effects at 10(-5) m. A concentration of 10(-5) m E2 enhanced insulin-induced phosphorylation of IRS-1 at Ser(307), which was abolished by treatment with a c-Jun NH(2)-terminal kinase inhibitor. In addition, the effect of E2 was abrogated by pretreatment with a specific estrogen receptor antagonist, ICI182,780. Membrane-impermeable E2, E2-BSA, did not affect the insulin-induced phosphorylation of Akt at 10(-8) m, but inhibited it at 10(-5) m. Furthermore, E2 decreased the amount of estrogen receptor alpha at the plasma membrane at 10(-8) m, but increased it at 10(-5) m. In contrast, the subcellular distribution of estrogen receptor beta was not altered by the treatment. These results indicate that E2 affects the metabolic action of insulin in a concentration-specific manner, that high concentrations of E2 inhibit insulin signaling by modulating phosphorylation of IRS-1 at Ser(307) via a c-Jun NH(2)-terminal kinase-dependent pathway, and that the subcellular redistribution of estrogen receptor alpha in response to E2 may explain the dual effect of E2. PMID:16269459

Nagira, Kiyofumi; Sasaoka, Toshiyasu; Wada, Tsutomu; Fukui, Kazuhito; Ikubo, Mariko; Hori, Satoko; Tsuneki, Hiroshi; Saito, Shigeru; Kobayashi, Masashi

2006-02-01

361

Dataset integration identifies transcriptional regulation of microRNA genes by PPAR? in differentiating mouse 3T3-L1 adipocytes.  

PubMed

Peroxisome proliferator-activated receptor ? (PPAR?) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPAR? binding sites in mouse adipocytes and PPAR? upregulates hundreds of protein-coding genes during adipogenesis. However, no microRNA (miRNA) genes have been identified as primary PPAR?-targets. By integration of four separate datasets of genome-wide PPAR? binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPAR? binding within 50?kb from miRNA transcription start sites. Nineteen mature miRNAs were upregulated ?2-fold during adipogenesis and for six of these miRNA loci the PPAR? binding sites were confirmed by at least three datasets. The upregulation of five miRNA genes miR-103-1 (host gene Pank3), miR-148b (Copz1), miR-182/96/183, miR-205 and miR-378 (Ppargc1b) followed that of Pparg. The PPAR?-dependence of four of these miRNA loci was demonstrated by PPAR? knock-down and the loci of miR-103-1 (Pank3), miR-205 and miR-378 (Ppargc1b) were also responsive to the PPAR? ligand rosiglitazone. Finally, chromatin immunoprecipitation analysis validated in silico predicted PPAR? binding sites at all three loci and H3K27 acetylation was analyzed to confirm the activity of these enhancers. In conclusion, we identified 22 putative PPAR? target miRNA genes, showed the PPAR? dependence of four of these genes and demonstrated three as direct PPAR? target genes in mouse adipogenesis. PMID:22319216

John, Elisabeth; Wienecke-Baldacchino, Anke; Liivrand, Maria; Heinäniemi, Merja; Carlberg, Carsten; Sinkkonen, Lasse

2012-05-01

362

Effects of Coating a Titanium Alloy with Fibronectin on the Expression of Osteoblast Gene Markers in the MC3T3 Osteoprogenitor Cell Line  

PubMed Central

Purpose A number of environmental and patient-related factors contribute to implant failure. A significant fraction of these failures can be attributed to limited osseointegration resulting from poor bone healing responses. The overall goal of this study was to determine whether surface treatment of a titanium-aluminum-vanadium alloy (Ti-6Al-4V) implant material with a biomimetic protein coating could promote the differentiation of attached osteoblastic cells. The specific aims of the study were to investigate whether osteoprogenitor cells cultured on a rigorously cleaned implant specimen showed a normal pattern of differentiation and whether preadsorbed fibronectin accelerated or enhanced osteoblast differentiation. Materials and Methods Ti-6Al-4V disks were rigorously cleaned, passivated in nitric acid, and dry heat–sterilized; some of the disks were then coated with 1 nmol/L fibronectin. MC3T3 osteoprogenitor cells were then cultured on the pretreated disks for several weeks. Quantitative real-time polymerase chain reaction was performed to measure changes over time in the mRNA levels of osteoblast genes. Results Fibronectin increased the peak expression of all analyzed osteoblast gene markers. “Early” genes that normally mark the proliferative phase (0 to 10 days) of osteoblastic development showed peak expression within the first 10 days after cell attachment to the titanium alloy. In contrast, “late” genes that normally mark the differentiation (10 to 20 days) and mineralization (20 to 36 days) phases of osteoblastogenesis achieved peak expression only after approximately 3 to 4 weeks of culture. Conclusions Osteoprogenitors cultured on a rigorously cleaned Ti-6Al-4V alloy were found to demonstrate a normal pattern of osteoblast differentiation. Preadsorbed fibronectin was observed to stimulate osteoblast differentiation during the mineralization phase of osteoblastogenesis. PMID:23057020

Rapuano, Bruce E.; Hackshaw, Kyle M.; Schniepp, Hannes C.; MacDonald, Daniel E.

2013-01-01

363

MicroRNA-1 Participates in Nitric Oxide-Induced Apoptotic Insults to MC3T3-E1 Cells by Targeting Heat-Shock Protein-70  

PubMed Central

Our previous studies showed that nitric oxide (NO) could induce osteoblast apoptosis. MicroRNA-1 (miR-1), a skeletal- and cardiac muscle-specific small non-coding RNA, contributes to the regulation of multiple cell activities. In this study, we evaluated the roles of miR-1 in NO-induced insults to osteoblasts and the possible mechanisms. Exposure of mouse MC3T3-E1 cells to sodium nitroprusside (SNP) increased amounts of cellular NO and intracellular reactive oxygen species. Sequentially, SNP decreased cell survival but induced caspase-3 activation, DNA fragmentation, and cell apoptosis. In parallel, treatment with SNP induced miR-1 expression in a time-dependent manner. Application of miR-1 antisense inhibitors to osteoblasts caused significant inhibition of SNP-induced miR-1 expression. Knocking down miR-1 concurrently attenuated SNP-induced alterations in cell morphology and survival. Consecutively, SNP time-dependently inhibited heat-shock protein (HSP)-70 messenger (m)RNA and protein expressions. A bioinformatic search predicted the existence of miR-1-specific binding elements in the 3'-untranslational region of HSP-70 mRNA. Downregulation of miR-1 expression simultaneously lessened SNP-induced inhibition of HSP-70 mRNA and protein expressions. Consequently, SNP-induced modifications in the mitochondrial membrane potential, caspase-3 activation, DNA fragmentation, and apoptotic insults were significantly alleviated by miR-1 antisense inhibitors. Therefore, this study showed that miR-1 participates in NO-induced apoptotic insults through targeting HSP-70 gene expression.

Lee, Yong-Eng; Hong, Chung-Ye; Lin, Yi-Ling; Chen, Ruei-Ming

2015-01-01

364

Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system  

PubMed Central

Background Tobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity. Findings The findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P?

2014-01-01

365

Assessing the impact of As-Cd-Pb metal mixture on cell transformation by two-stage Balb/c 3T3 cell assay.  

PubMed

Human beings are exposed to metals as a consequence of various industrial activities, including glass production, agrochemical production, metallurgy and battery manufacture. New data about the possible mechanisms involved in the carcinogenic activity of these metals are constantly being reported. Exposure to complex mixtures of metals is more likely to occur than exposure to a single metal alone. Among these elements, arsenic, cadmium and lead are ubiquitous air and water pollutants that continue to threaten the quality of public health around the world. The aim of the present study was to evaluate the capability of a mixture of 2 µM NaAsO2, 2 µM CdCl2 and 5 µM Pb(C2H3O2)2·3H2O at relevant epidemiological concentrations to induce cell transformation processes. Transforming potential was determined by a murine two-stage Balb/c 3T3 cell assay. Cell viability, reactive oxygen species (ROS), DNA damage, cell cycle analysis, senescence, generation time and metallothionein expression were also evaluated. The results showed that the metal mixture induced morphological cell transformation only when acting as initiator stimuli of the process. A decrease in cell viability was observed at the promotion stage, a time during which ROS increase, especially when a metal mixture was applied as a promoter stimulant. Changes in DNA damage were not observed throughout the assay; however, we observed G1 cell cycle arrest. The metal mixture, acting as a promoter, is capable of inducing senescence, but metals employed as initiators with 12-O-tetradecanoylphorbol-13-acetate as a promoter are capable of causing avoidance of senescence and triggering the transformation potential of the cells. PMID:24782466

Rodríguez-Sastre, M A; Rojas, E; Valverde, M

2014-07-01

366

Investigation of the interfacial effects of small chemical-modified TiO2 nanotubes on 3T3 fibroblast responses.  

PubMed

In order to gain insight into how interfacial effects influence cell responses, chemically modified anodized TiO2 nanotubes (ATNs) were used to simultaneously investigate the effects of nanoscale substrate structure and angstrom-scale chemicals on cell morphological change and cell growth. Two small chemicals were used to modify the ATNs, namely, 3-aminopropyltrimethoxysilane (APTMS) and 3-mercaptopropyltrimethoxysilane (MPTMS), resulting in APTMS-modified ATNs (APTMS-ATNs) and MPTMS-modified ATNs (MPTMS-ATNs), respectively. In our in vitro observation of NIH/3T3 fibroblasts, cells thrived on both unmodified and modified ATNs. Quantitative analyses of cell numbers exhibited that APTMS-ATNs effectively facilitated cell proliferation and directed cell orientation owing to full cell-substrate contact caused by positively charged amino groups (-NH3(+)) on the surface. In addition, scanning electron microscopy and fluorescence images showed different cell morphologies on APTMS-ATNs and MPTMS-ATNs. APTMS-ATNs resulted in flat spreading of fibroblasts, while MPTMS-ATNs resulted in fibroblasts with a three-dimensional solid shape and clear contours. The results indicate that the synergistic effects of nanotube surface topology and small chemical modification and, to a lesser extent, surface hydrophilicity, alter the interfacial interactions between cells and substrates, significantly affecting cell morphology, attachment, and growth. Using ATNs with different interfacial effects from various small chemicals, orientation of cells into various patterns can be achieved and investigation of cell fates, such as proliferation or stem cell differentiation, can be performed for future advanced medical or biological applications. PMID:25012464

Lin, Shu-Ping; Huang, Shu-Yen; Chen, Se-Fen; Vinzons, Lester U; Ciou, Jhong-Yi; Wong, Pei-Jie

2014-08-13

367

Long Non-Coding RNA NEAT1 Associates with SRp40 to Temporally Regulate PPAR?2 Splicing during Adipogenesis in 3T3-L1 Cells  

PubMed Central

Long non-coding (lnc) RNAs serve a multitude of functions in cells. NEAT1 RNA is a highly abundant 4 kb lncRNA in nuclei, and coincides with paraspeckles, nuclear domains that control sequestration of paraspeckle proteins. We examined NEAT1 RNA levels and its function in 3T3-L1 cells during differentiation to adipocytes. Levels of NEAT1 transcript, measured by RT-PCR, fluctuated in a temporal manner over the course of differentiation that suggested its role in alternative splicing of PPAR? mRNA, the major transcription factor driving adipogenesis. When cells were induced to differentiate by a media cocktail of insulin, dexamethasone, and isobutylmethyxanthine (IBMX) on Day 0, NEAT1 levels dropped on Day 4, when the PPAR?2 variant was spliced and when terminal differentiation occurs The appearance of PPAR?2 coordinates with the PPAR?1 variant to drive differentiation of adipocytes. SiRNA used to deplete NEAT1 resulted in the inability of cells to phosphorylate the serine/arginine-rich splicing protein, SRp40. SiRNA treatment for SRp40 resulted in dysregulation of PPAR?1 and, primarily, PPAR?2 mRNA levels. SRp40 associated with NEAT1, as shown by RNA-IP on days 0 and 8, but decreased on day 4, and concentrations increased over that of IgG control. Overexpression of SRp40 increased PPAR?2, but not ?1. Although lncRNA MALAT1 has been investigated in SR protein function, NEAT1 has not been shown to bind SR proteins for phosphorylation such that alternative splicing results. The ability of cells to increase phosphorylated SR proteins for PPAR?2 splicing suggests that fluxes in NEAT1 levels during adipogenesis regulate alternative splicing events. PMID:25437750

Cooper, Denise R.; Carter, Gay; Li, Pengfei; Patel, Rehka; Watson, James E.; Patel, Niketa A.

2014-01-01

368

A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK 1/2 pathway  

NASA Technical Reports Server (NTRS)

Physiological mechanical loading is crucial for maintenance of bone integrity and architecture. We have calculated the strain caused by gravity stress on osteoblasts and found that 4-30g corresponds to physiological levels of 40-300 microstrain. Short-term gravity loading (15 minutes) induced a 15-fold increase in expression of growth-related immediate early gene c-fos, a 5-fold increase in egr-1, and a 3-fold increase in autocrine bFGF. The non-growth-related genes EP-1, TGF-beta, and 18s were unaffected by gravity loading. Short-term physiological loading induced extracellular signal-regulated kinase (ERK 1/2) phosphorylation in a dose-dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12g (p < 0.001) with no effect on total ERK. The phosphorylation of focal adhesion kinase (FAK) was unaffected by mechanical force. g-Loading did not activate P38 MAPK or c-jun N-terminal kinase (JNK). Additionally, a gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus; this did not occur in unloaded cells. The induction of c-fos was inhibited 74% by the MEK1/2 inhibitor U0126 (p < 0.001) but was not affected by MEK1 or p38 MAPK-specific inhibitors. The long-term consequence of a single 15-minute gravity pulse was a 64% increase in cell growth (p < 0.001). U0126 significantly inhibited gravity-induced growth by 50% (p < 0.001). These studies suggest that short periods of physiological mechanical stress induce immediate early gene expression and growth in MC3T3-E1 osteoblasts primarily through an ERK 1/2-mediated pathway.

Hatton, Jason P.; Pooran, Milad; Li, Chai-Fei; Luzzio, Chris; Hughes-Fulford, Millie

2003-01-01

369

Individual trans 18:1 Isomers are Metabolised Differently and Have Distinct Effects on Lipogenesis in 3T3-L1 Adipocytes.  

PubMed

The objective of this research was to study the metabolism of individual trans fatty acids (FAs) that can be found in ruminant fat or partially hydrogenated vegetable oils (PHVO) and determine their effects on FA composition and lipogenic gene expression in adipocytes. Differentiated 3T3-L1 adipocytes were treated with 200 µM of either trans-9-18:1, trans-11-18:1, trans-13-18:1, cis-9-18:1 or BSA vehicle control for 120 h. Trans-9-18:1 increased total cell FA content (µmole/well) compared to other FA treatments, which was mainly related to the accumulation of trans-9-18:1 in the cells. Adipocytes were able to desaturate a significant proportion of absorbed trans-11-18:1 and trans-13-18:1 (~20 and 30 % respectively) to cis-9,trans-11-18:2 and cis-9,trans-13-18:2, whereas trans-9-18:1 was mostly incorporated intact resulting in a greater lipophilic index (i.e. decreased mean FA fluidity) of adipocytes. Trans-9-18:1 up-regulated (P < 0.05) the expression of lipogenic genes including acetyl-CoA carboxylase (1.65 fold), FA synthase (1.45 fold), FA elongase-5 (1.52 fold) and stearoyl-CoA desaturase-1 (1.49 fold), compared to the control, whereas trans-11-18:1 and trans-13-18:1 did not affect the expression of these genes compared to control. Our results suggest that the metabolism and lipogenic properties of trans-11-18:1 and trans-13-18:1, typically the most abundant trans FA in beef from cattle fed forage-based diets, are similar and are different from those of trans-9-18:1, the predominant trans FA in PHVO. PMID:25544125

Vahmani, P; Meadus, W J; Turner, T D; Duff, P; Rolland, D C; Mapiye, C; Dugan, M E R

2015-02-01

370

1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine pre-osteoblastic MC3T3-E1 cells by direct regulation of cystathionine ?-synthase  

PubMed Central

High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY due to its negative impact on collagen maturation. HCY is cleared by cystathionine ?-synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3-E1 murine pre-osteoblasts treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH)2D3. Quantitative real-time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH)2D3 treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH)2D3-treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knock-out osteoblasts, in which up-regulation of cbs in response to 1,25(OH)2D3 was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs. To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D3 levels] and HCY levels. In conclusion, this study demonstrated that cbs is a primary 1,25(OH)2D3 target gene which renders HCY metabolism responsive to 1,25(OH)2D3. PMID:21898591

Kriebitzsch, Carsten; Verlinden, Lieve; Eelen, Guy; van Schoor, Natasja M.; Swart, Karin; Lips, Paul; Meyer, Mark B.; Pike, J Wesley; Boonen, Steven; Carlberg, Carsten; Vitvitsky, Victor; Bouillon, Roger; Banerjee, Ruma; Verstuyf, Annemieke

2011-01-01

371

Sulfonate groups grafted on Ti6Al4V favor MC3T3-E1 cell performance in serum free medium conditions.  

PubMed

Ten years ago, we synthesized "bioactive model polymers" bearing sulfonate groups and proposed a mechanism of their modulation effect at different steps of the cell response. Then, we set up the grafting of polymers bearing sulfonate on Ti6Al4V surfaces by a grafting "from" technique making sure of the creation of covalent bonds between the grafted polymer and the Ti6Al4V surface. We have checked and confirmed the positive effect of grafted sulfonate groups on the osteoblastic cell response in vivo and in vitro but we did not elucidate the mechanism. The aim of this basic work consists first in investigating the role of sulfonate groups in the presence and in the absence of proteins at early stages of the osteointegration process on poly(sodium styrene sulfonate) poly(NaSS) grafted and ungrafted Ti6Al4V surfaces, in vitro. To understand the role of poly(NaSS) grafted chains on osteoblast-like cell response and to confirm/elucidate the importance of fetal bovine serum (FBS) proteins in the culture medium, MC3T3-E1 cells were seeded onto poly(NaSS) grafted and non-grafted Ti6Al4V surfaces. Cultures were carried out in a complete (10% FBS) and in a non-complete medium (without FBS). Cell viability assay, cell attachment number and cell adhesion strength were followed up to 3days of culture. The presence of proteins enhanced cell growth and development whatever the surface and the presence of sulfonate groups enhanced the cell attachment even in the absence of proteins, which suggests and confirms that the sulfonate groups can modify the activity of cells such as the secretion of binding proteins. Statistical differences were found in the attachment strength tests on poly(NaSS) grafted and ungrafted surfaces and showed that the sulfonate groups play an important role in the cell resistance to shear stress. PMID:24863216

Felgueiras, Helena; Migonney, Véronique

2014-06-01

372

Titanium is not "the most biocompatible metal" under cathodic potential: The relationship between voltage and MC3T3 preosteoblast behavior on electrically polarized cpTi surfaces.  

PubMed

An electrochemically controlled system has been developed which allows for cell culture directly on electrically polarized metal surfaces with simultaneous control and assessment of the electrochemical current, potential, and impedance of the interface. This system was utilized in this study to assess the interactions between electrochemically polarized commercially pure titanium (cpTi) and MC3T3 preosteoblast cells. Cells were cultured on CpTi for 24 h at static potentials between -1000 mV and +1000 mV vs. Ag/AgCl and cell morphology (SEM and cell area) and viability (MTT and Live-Dead assay) were assessed along with the electrochemical current densities and surface oxide impedance properties. The results indicate that cathodic polarization in the range of -600 mV to -1000 mV markedly reduces the spreading and viability of cells cultured directly on cpTi within 24 h, while anodic polarization (-300 mV to +1000 mV) out to 72 h shows no difference in cell behavior as compared to the OCP condition. Analysis of the relationship between the cell outcomes and the electrochemical current densities and impedance indicated the presence of voltage-dependent electrochemical thresholds (cathodic current density, i(c) > 1.0 microA/cm(2), R(p) < 10(5) Omega cm(2)) which may control the biocompatibility of cpTi. In addition, these outcomes have direct clinical significance for modular orthopedic implants whose potential can shift, via fretting corrosion, down into the range of potentials exhibiting poor cell behavior. PMID:20014293

Ehrensberger, Mark T; Sivan, Shiril;