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Sample records for 3t3 feeder layer

  1. A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations.

    PubMed

    Miyashita, Hideyuki; Shimmura, Shigeto; Higa, Kazunari; Yoshida, Satoru; Kawakita, Tetsuya; Shimazaki, Jun; Tsubota, Kazuo

    2008-07-01

    Corneal epithelial cell sheets co-cultivated with feeder cells are used to reconstruct the ocular surface in stem cell-depleted eyes. The present study was conducted to investigate the optimal method of using feeder cells in the interest of preserving progenitor cells in cultivated sheets. We compared the phenotype and secondary colony forming efficiency (CFE) of cell sheets that were engineered using 3T3 feeder cells as a separate layer or as a contact layer. We also devised a novel "duplex feeder" system that consists of two separate layers of feeder cells. After cells reached confluence, cells were cultured at the air-liquid interface to allow full stratification. Stratified sheets were then analyzed using immunohistochemistry and secondary colony formation. Contact feeder cultures and duplex feeder cultures yielded epithelial sheets with small, cuboid basal cells with strong expression of keratin (K)3, K12, and K 15. Furthermore, only duplex feeder layers reproduced the basal K 15, suprabasal K12 limbal phenotype where epithelial stem cells reside. A similar effect was observed when cornea stroma-derived progenitor cells were used as feeder cells. Duplex feeder sheets also produced significantly more secondary colonies than cells dissociated from single layer sheets, suggesting that the duplex feeder system produces transplantable sheets with a higher yield of progenitor cells. PMID:18433313

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

    PubMed Central

    Bisson, Francis; Rochefort, Éloise; Lavoie, Amélie; Larouche, Danielle; Zaniolo, Karine; Simard-Bisson, Carolyne; Damour, Odile; Auger, François A.; Guérin, Sylvain L.; Germain, Lucie

    2013-01-01

    A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes. PMID:23443166

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

    PubMed

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

    2015-06-01

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

  4. Induction of pluripotency in human umbilical cord mesenchymal stem cells in feeder layer-free condition.

    PubMed

    Daneshvar, Nasibeh; Rasedee, Abdullah; Shamsabadi, Fatemeh Tash; Moeini, Hassan; Mehrboud, Parvaneh; Rahman, Heshu Sulaiman; Boroojerdi, Mohadeseh Hashem; Vellasamy, Shalini

    2015-12-01

    Induced Pluripotent Stem Cells (iPSCs) has been produced by the reprogramming of several types of somatic cells through the expression of different sets of transcription factors. This study consists of a technique to obtain iPSCs from human umbilical cord mesenchymal stem cells (UC-MSCs) in a feeder layer-free process using a mini-circle vector containing defined reprogramming genes, Lin28, Nanog, Oct4 and Sox2. The human MSCs transfected with the minicircle vector were cultured in iPSCs medium. Human embryonic stem cell (ESC)-like colonies with tightly packed domelike structures appeared 7-10 days after the second transfection. In the earliest stages, the colonies were green fluorescence protein (GFP)-positive, while upon continuous culture and passage, genuine hiPSC clones expressing GFP were observed. The induced cells, based on the ectopic expression of the pluripotent markers, exhibited characteristics similar to the embryonic stem cells. These iPSCs demonstrated in vitro capabilities for differentiation into the three main embryonic germ layers by embryoid bodies formation. There was no evidence of transgenes integration into the genome of the iPSCs in this study. In conclusion, this method offers a means of producing iPSCs without viral delivery that could possibly overcome ethical concerns and immune rejection in the use of stem cells in medical applications. PMID:26471847

  5. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

    PubMed

    Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

    2015-09-01

    Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers. PMID:26246271

  6. Vaspin promotes 3T3-L1 preadipocyte differentiation.

    PubMed

    Liu, Ping; Li, Guoliang; Wu, Jine; Zhou, Xin; Wang, Liping; Han, Wenqi; Lv, Ying; Sun, Chaofeng

    2015-11-01

    Vaspin, a novel adipocyte factor secreted from visceral adipose tissues, is associated with obesity and insulin resistance and can regulate glucose and lipid metabolism, increase insulin sensitivity, and suppress inflammation; however, the underlying mechanisms remain unknown. Proliferation and maladaptive differentiation are important pathological mechanisms underlying obesity. This study aimed to evaluate the effects of vaspin on the proliferation and differentiation of preadipocyte 3T3-L1 cells and to explore the likely mechanisms responsible for 3T3-L1 differentiation. Vaspin was added to cultured 3T3-L1 cells, and the differentiation of adipocytes was evaluated using Oil Red O staining. The AKT signaling pathway and specific differentiation factors related to the differentiation of preadipocyte 3T3-L1 cells, peroxisome proliferator-activated ? and the CCAAT/enhancer-binding protein (C/EBP) family, were evaluated using reverse transcription polymerase chain reaction (RT-PCR) and western blot analyses during the early phase of differentiation. Additionally, adiponectin mRNA, interleukin-6 mRNA (IL-6 mRNA), and glucose transporter-4 (GLUT4) protein levels were measured in the differentiated adipocytes. The results indicated that vaspin promotes the intracellular accumulation of lipids and increases differentiation-related factors, including peroxisome proliferator-activated receptor ?, C/EBP?, and free fatty acid-binding protein 4 (FABP4), in a dose-dependent manner. Additionally, vaspin (200?ng/mL) increased the mRNA and protein levels of C/EBP?, peroxisome proliferator-activated ?, C/EBP?, and FABP4. Moreover, compared with the control, significantly smaller eight-day differentiated adipocytes were observed, and these cells exhibited decreased IL-6 mRNA and increased GLUT4 mRNA levels; these results also indicated the potential of vaspin to promote the insulin-mediated AKT signaling pathway during the early phase of differentiation. In conclusion, vaspin is able to promote the differentiation of 3T3-L1 preadipocytes and may increase their sensitivity to insulin and suppress obesity. PMID:25585626

  7. Topiramate effects lipolysis in 3T3-L1 adipocytes

    PubMed Central

    MARTINS, GABRIELA POLTRONIERI CAMPAGNARO; SOUZA, CAMILA OLIVEIRA; MARQUES, SCHEROLIN; LUCIANO, THAIS FERNANDES; DA SILVA PIERI, BRUNO LUIZ; ROSA, JOSÉ CÉSAR; DA SILVA, ADELINO SANCHEZ RAMOS; PAULI, JOSÉ RODRIGO; CINTRA, DENNYS ESPER; ROPELLE, EDUARDO ROCHETE; RODRIGUES, BRUNO; DE LIRA, FABIO SANTOS; DE SOUZA, CLAUDIO TEODORO

    2015-01-01

    Studies have shown that topiramate (TPM)-induced weight loss can be dependent on the central nervous system (CNS). However, the direct action of TPM on adipose tissue has not been tested previously. Thus, the present study aimed to examine whether TPM modulates lipolysis in 3T3-L1. The 3T3-L1 cells were incubated in 50 µM TPM for 30 min. The ?-adrenergic stimulator, isoproterenol, was used as a positive control. The release of lactate dehydrogenase, non-esterified fatty acid, glycerol and incorporation of 14C-palmitate to lipid were analyzed. The phosphorylation of protein kinase A (PKA), hormone-sensitive lipase (HSL), adipocyte triglyceride lipase (ATGL) and perilipin A, as well as the protein levels of comparative genetic identification 58 (CGI-58) were assessed. The levels of glycerol and non-esterified fatty acid increased markedly when the cells were treated with TPM. The TPM effects were similar to the isoproterenol positive control. Additionally, TPM reduced lipogenesis. These results were observed without any change in cell viability. Finally, the phosphorylation of PKA, HSL, ATGL and perilipin A, as well as the protein levels of CGI-58 were increased compared to the control cells. These results were similar to those observed in the cells treated with isoproterenol. The present results show that TPM increased the phosphorylation of pivotal lipolytic enzymes, which induced lipolysis in 3T3-L1 adipocytes, suggesting that this drug may act directly in the adipose tissue independent from its effect on the CNS. PMID:26623024

  8. Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

    SciTech Connect

    Turley, E.A.; Moore, D.; Hayden, L.J.

    1987-06-02

    A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.

  9. Precision powder feeder

    DOEpatents

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

    2001-07-10

    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.

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

    PubMed

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

    2000-10-24

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

  11. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    SciTech Connect

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

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

    SciTech Connect

    Liu, Te; Shanghai Geriatric Institute of Chinese Medicine, Shanghai 200031 ; Cheng, Weiwei; Huang, Yongyi; Huang, Qin; Jiang, Lizhen; Guo, Lihe

    2012-02-15

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

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

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; Department of Ophthalmology, Chiayi Christian Hospital, Chiayi 600, Taiwan, ROC ; Tseng, Min-Jen

    2013-01-18

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

  14. MicroRNA let-7 Regulates 3T3-L1 Adipogenesis

    PubMed Central

    Sun, Tingwan; Fu, Mingui; Bookout, Angie L.; Kliewer, Steven A.; Mangelsdorf, David J.

    2009-01-01

    Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of events including clonal expansion, growth arrest, and terminal differentiation. The mechanisms coordinating these different steps are not yet fully understood. Here we investigated whether microRNAs (miRNAs) play a role in this process. Microarray analysis was performed to detect miRNA expression during 3T3-L1 preadipocyte differentiation. Several miRNAs, including let-7, were up-regulated during 3T3-L1 adipogenesis. Ectopic introduction of let-7 into 3T3-L1 cells inhibited clonal expansion as well as terminal differentiation. The mRNA encoding high-mobility group AT-hook 2 (HMGA2), a transcription factor that regulates growth and proliferation in other contexts, was inversely correlated with let-7 levels during 3T3-L1 cell adipogenesis, and let-7 markedly reduced HMGA2 concentrations. Knockdown of HMGA2 inhibited 3T3-L1 differentiation. These results suggest that let-7 plays an important role in adipocyte differentiation and that it does so in part by targeting HMGA2, thereby regulating the transition from clonal expansion to terminal differentiation. PMID:19324969

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

  16. The effect of myostatin on proliferation and lipid accumulation in 3T3-L1 preadipocytes.

    PubMed

    Zhu, Hui Juan; Pan, Hui; Zhang, Xu Zhe; Li, Nai Shi; Wang, Lin Jie; Yang, Hong Bo; Gong, Feng Ying

    2015-06-01

    Myostatin is a critical negative regulator of skeletal muscle development, and has been reported to be involved in the progression of obesity and diabetes. In the present study, we explored the effects of myostatin on the proliferation and differentiation of 3T3-L1 preadipocytes by using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide spectrophotometry, intracellular triglyceride (TG) assays, and real-time quantitative RT-PCR methods. The results indicated that recombinant myostatin significantly promoted the proliferation of 3T3-L1 preadipocytes and the expression of proliferation-related genes, including Cyclin B2, Cyclin D1, Cyclin E1, Pcna, and c-Myc, and IGF1 levels in the medium of 3T3-L1 were notably upregulated by 35.2, 30.5, 20.5, 33.4, 51.2, and 179% respectively (all P<0.01) in myostatin-treated 3T3-L1 cells. Meanwhile, the intracellular lipid content of myostatin-treated cells was notably reduced as compared with the non-treated cells. Additionally, the mRNA levels of Ppar?, Cebp?, Gpdh, Dgat, Acs1, Atgl, and Hsl were significantly downregulated by 22-76% in fully differentiated myostatin-treated adipocytes. Finally, myostatin regulated the mRNA levels and secretion of adipokines, including Adiponectin, Resistin, Visfatin, and plasminogen activator inhibitor-1 (PAI-1) in 3T3-L1 adipocytes (all P<0.001). Above all, myostatin promoted 3T3-L1 proliferation by increasing the expression of cell-proliferation-related genes and by stimulating IGF1 secretion. Myostatin inhibited 3T3-L1 adipocyte differentiation by suppressing Ppar? and Cebp? expression, which consequently deceased lipid accumulation in 3T3-L1 cells by inhibiting the expression of critical lipogenic enzymes and by promoting the expression of lipolytic enzymes. Finally, myostatin modulated the expression and secretion of adipokines in fully differentiated 3T3-L1 adipocytes. PMID:25878062

  17. Filopodia of spreading 3T3 cells. Do they have a substrate-exploring function?

    PubMed Central

    1976-01-01

    Freshly plated 3T3 cells send out radial projections or filopodia. We observed cells which happended to settle on glass near the borderline of a gold-plated area. When some of the filopodia contacted the gold- plated area and others the glass substratum and remained attached for a few minutes, lamellipodia then extended preferentially toward the gold- plated area. 1-2 h later, most of the cells were found in the gold- plated area. When the filopodia of a spreading 3T3 cell contacted another already spread 3T3 cell and also the glass substratum, the first lamellipodia extended preferentially towards the glass. These observations suggest a directionally differentiated extension of lamellipodia after the filopodia of a spreading 3T3 cell have contacted different substrates in their environment. Before filopodia contact a substrate, they perform a rapid "scanning" motion. Therefore, we suggest that the filopodia of a spreading 3T3 cell serve as organs which explore the nonfluid environment and react to a certain quality of the substrate that is presently unknown. Subsequently, they mediate the extension of lamellipodia into the direction in which this quality is found. The described phenomena are reversibly inhibited by Cytochalasin B at concentrations above 5 mug/ml although filopodia are produced. PMID:1262391

  18. Heat shock induces the release of fibroblast growth factor 1 from NIH 3T3 cells.

    PubMed

    Jackson, A; Friedman, S; Zhan, X; Engleka, K A; Forough, R; Maciag, T

    1992-11-15

    Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene encoding FGF-1 secrete FGF-1 into their conditioned medium in response to heat shock. The form of FGF-1 released by NIH 3T3 cells in response to increased temperature (42 degrees C, 2 hr) in vitro is not biologically active and does not associate with either heparin or the extracellular NIH 3T3 monolayer matrix. However, it was possible to derive biologically active FGF-1 from the conditioned medium of heat-shocked NIH 3T3 cell transfectants by ammonium sulfate fractionation. The form of FGF-1 exposed by ammonium sulfate fractionation is similar in size to cytosolic FGF-1 and can bind and be eluted from immobilized heparin similarly to the recombinant human FGF-1 polypeptide. Further, the release of FGF-1 by NIH 3T3 cell transfectants in response to heat shock is reduced significantly by both actinomycin D and cycloheximide. These data indicate that increased temperature may upregulate the expression of a factor responsible for the secretion of FGF-1 as a biologically inactive complex that requires an activation step to exhibit the biological activity of the extracellular polypeptide mitogen. PMID:1279690

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

    SciTech Connect

    Dumas, L.; Page, M.

    1986-01-01

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

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

    PubMed

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

    2014-04-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    1988-03-01

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

  2. Ginsenoside Rb1 promotes browning through regulation of PPAR? in 3T3-L1 adipocytes.

    PubMed

    Mu, Qianqian; Fang, Xin; Li, Xiaoke; Zhao, Dandan; Mo, Fangfang; Jiang, Guangjian; Yu, Na; Zhang, Yi; Guo, Yubo; Fu, Min; Liu, Jun-Li; Zhang, Dongwei; Gao, Sihua

    2015-10-23

    Browning of white adipocyte tissue (WAT) has received considerable attention due to its potential implication in preventing obesity and related comorbidities. Ginsenoside Rb1 is reported to improve glycolipid metabolism and reduce body weight in obese animals. However whether the body reducing effect mediates by browning effect remains unclear. For this purpose, 3T3-L1 adipocytes were used to study the effect of ginsenoside Rb1 on browning adipocytes specific genes and oxygen consumptions. The results demonstrate that 10 ?M of ginsenoside Rb1 increases basal glucose uptake and promoted browning evidenced by significant increases in mRNA expressions of UCP-1, PGC-1? and PRDM16 in 3T3-L1 mature adipocytes. Further, ginsenoside Rb1 also increases PPAR? activity. And the browning effect is abrogated by GW9692, a PPAR? antagonist. In addition, ginsenoside Rb1 increases basal respiration rate, ATP production and uncoupling capacity in 3T3-L1 adipocytes. Those effects are also blunted by GW9692. The results suggest that ginsenoside Rb1 promote browning of 3T3-L1 adipocytes through induction of PPAR?. Our finding offer a new source to discover browning agonists and also useful to understand and extend the applications of ginseng and its constituents. PMID:26381176

  3. Isoproterenol Increases Uncoupling, Glycolysis, and Markers of Beiging in Mature 3T3-L1 Adipocytes

    PubMed Central

    Miller, Colette N.; Yang, Jeong-Yeh; England, Emily; Yin, Amelia; Rayalam, Srujana

    2015-01-01

    Beta-adrenergic activation stimulates uncoupling protein 1 (UCP1), enhancing metabolic rate. In vitro, most work has studied brown adipocytes, however, few have investigated more established adipocyte lines such as the murine 3T3-L1 line. To assess the effect of beta-adrenergic activation, mature 3T3-L1s were treated for 6 or 48 hours with or without isoproterenol (10 and 100 ?M) following standard differentiation supplemented with thyroid hormone (T3; 1 nM). The highest dose of isoproterenol increased lipid content following 48 hours of treatment. This concentration enhanced UCP1 mRNA and protein expression. The increase in UCP1 following 48 hours of isoproterenol increased oxygen consumption rate. Further, coupling efficiency of the electron transport chain was disturbed and an enhancement of glycolytic rate was measured alongside this, indicating an attempt to meet the energy demands of the cell. Lastly, markers of beige adipocytes (protein content of CD137 and gene transcript of CITED1) were also found to be upregulated at 48 hours of isoproterenol treatment. This data indicates that mature 3T3-L1 adipocytes are responsive to isoproterenol and induce UCP1 expression and activity. Further, this finding provides a model for further pharmaceutical and nutraceutical investigation of UCP1 in 3T3-L1s. PMID:26390217

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    E-print Network

    Gu, Tingyue

    Effects of growth hormone antagonists on 3T3-F442A preadipocyte differentiation B C Xu, W Y Chen. lournal of Endocrinology (1995) 146, 131-1 39 Introductiun Growth hormone (GH) exerts &verse growth expression of the proto-oncogene c-fos and c-jun RNAs and synthesis of c-f& and c-jun proteins which act

  6. 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

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

    SciTech Connect

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

    1986-10-01

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

  8. Expression of Nanog gene promotes NIH3T3 cell proliferation

    SciTech Connect

    Zhang Jingyu; Wang Xia; Chen Bing; Suo Guangli; Zhao Yanhong; Duan Ziyuan; Dai Jianwu . E-mail: jwdai@genetics.ac.cn

    2005-12-16

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

  9. Sparstolonin B inhibits lipopolysaccharide-induced inflammation in 3T3-L1 adipocytes.

    PubMed

    Wang, Ming; Xiu, Liangchang; Diao, Jianxin; Wei, Lianbo; Sun, Jia

    2015-12-15

    Sparstolonin B (SsnB), an isocoumarin compound isolated from the tubers of both Sparganium stoloniferum and Scirpus yagara, has been reported to have anti-inflammatory effects. However, whether SsnB has anti-inflammatory effects on LPS-stimulated 3T3-L1 adipocytes remains unclear. In this study, we investigated the effects of SsnB on adipocyte inflammation in 3T3-L1 adipocytes and anti-obesity properties in high fat diet (HFD)-induced obese rats. 3T3-L1 adipocytes were pretreated with SsnB 1h before LPS treatment. The expression of MCP-1, IL-6, TNF-?, and IL-10 were measured by qRT-PCR and ELISA. The expression of PPAR-?, TLR4 and NF-?B were detected by western blotting. SsnB was administered to HFD-induced obese rats to confirm its effects in vivo. Our results showed that SsnB dose-dependently inhibited LPS-induced MCP-1, IL-6, and TNF-? production. SsnB was found to inhibit LPS-induced TLR4 expression and NF-?B activition. Furthermore, SsnB was found to activate PPAR-? and the inhibitory effects of SsnB on MCP-1, IL-6, and TNF-? production can be reversed by PPAR-? antagonist GW9662. In vivo, SsnB was found to reduce the body weight of rats fed with HFD. SsnB also inhibited the levels of serum triglyceride (TG) and cholesterol (TC) induced by HFD. In conclusion, the results suggested that SsnB could reduce HFD-induced obesity in rats and inhibited LPS-induced cytokines production in 3T3-L1 adipocytes by activating PPAR-?. PMID:26522926

  10. Rutin Stimulates Adipocyte Differentiation and Adiponectin Secretion in 3T3-L1 Adipocytes.

    PubMed

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

    2015-04-01

    Rutin is aflavonoid, which is found in many plants. It has been shown to reduce blood glucose and increase insulin levels in diabetic rats. In the present study, the authors aimed to elucidate the molecular basis for the observed antidiabetic activity using murine 3T3-L1 preadipocyte cultures. The treatment of differentiating 3T3-L1 cells with rutin at concentrations of 3, 10, 30 and 100 µM significantly increased lipid accumulation and mRNA expression of transcription factors, such as peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein alpha, and adipocyte fatty acid-binding protein. Furthermore, rutin at concentrations of 10, 30 and 100 µM increased adiponectin mRNA expression together with stimulating the secretion of adiponectin in differentiating 3T3-L1 cells. These results indicate that the stimulatory effect of rutin on adipocyte differentiation likely occurs through up-regulation of adipogenic transcription factors and downstream adipocyte-specific gene expression. Such effects of rutin on adiponectin secretion and adipocyte activity may account for, at least in part, the antidiabetic effects of consumption of food containing rutin. PMID:26387381

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

    PubMed Central

    Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

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

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

    SciTech Connect

    Takahashi, Nobuhiko; Division of Gastroenterology and Hematology Yoshizaki, Takayuki; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayoshi; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2013-02-01

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

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

    PubMed Central

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

    2001-01-01

    Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development. PMID:11513731

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  16. Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway

    PubMed Central

    Liou, Chian-Jiun; Lai, Xuan-Yu; Chen, Ya-Ling; Wang, Chia-Ling; Wei, Ciao-Han; Huang, Wen-Chung

    2015-01-01

    Ginkgolide C, isolated from Ginkgo biloba leaves, is a flavone reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the antiadipogenic effect of ginkgolide C in 3T3-L1 adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to adipogenesis and lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated adipocytes. It also decreased adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK), resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK inhibitor (compound C), ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing lipolysis and inhibiting adipogenesis in adipocytes through the activated AMPK pathway. PMID:26413119

  17. Enzymatic fragments of hyaluronan inhibit adipocyte differentiation in 3T3-L1 pre-adipocytes.

    PubMed

    Park, Byong-Gon; Lee, Chang Won; Park, Joo Woong; Cui, Yuan; Park, Yoon-Sun; Shin, Woon-Seob

    2015-11-27

    Hyaluronan has diverse biological activities depending on its molecular size. High molecular weight hyaluronan (2000 kDa) is a major component of extracellular matrix, and has been used in wounding healing, extracellular matrix regeneration, and in the treatment of osteoarthritis. Hyaluronan fragments can stimulate inflammation or induce loss of extracellular matrix. Hyaluronan is expressed during adipocyte differentiation, and down regulation of hyaluronan synthesis can reduce adipogenic differentiation. However, the direct effects of hyaluronan fragments on adipocyte differentiation have not been elucidated. Therefore, we prepared hyaluronan fragments by enzymatic digestion, and examined the inhibitory effects of these hyaluronan fragments on the accumulation of lipid droplets and on adipogenic gene mRNA expression in differentiating 3T3-L1 pre-adipocytes. Medium sized hyaluronan fragments (50 kDa) decreased lipid droplet accumulation in a dose-dependent manner. However, high molecular weight hyaluronan did not inhibit lipid droplet accumulation when used at a concentration of 600 ?g/ml. Two or 4 day treatments with medium molecular weight of hyaluronan resulted in similar inhibitory levels of lipid accumulation as did treatment for 8 days. Medium sized hyaluronan inhibited the differentiation of 3T3-L1 pre-adipocytes during the early stages of adipogenesis. When 3T3-L1 cells were treated with 180 ?g/ml of medium sized hyaluronan, the mRNAs for the master adipogenic transcription factors PPAR-? and C/EBP-? were inhibited. Additionally, medium molecular weight hyaluronan suppressed mRNA expression of PPAR-? target genes, including aP2 and FAS. This study is the first to report that medium molecular weight hyaluronan fragments can inhibit adipocyte differentiation. PMID:26525853

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

    SciTech Connect

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

    2006-07-01

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

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

    SciTech Connect

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai; Yang, Ying; Shen, Weili

    2013-08-16

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

  20. Suppressive Effects of an Ishige okamurae extract on 3T3-L1 Preadipocyte Differentiation

    PubMed Central

    Cha, Sun-yeong; Cheon, Yong-Pil

    2013-01-01

    The biological activity of tissue specific stem cell is under the control of their specific microenvironment and the exogenous chemicals derived from digestive tract can be one of the constructing factors of that. It is suggested that the extract of brown algae Ishige okamurae has antioxidant-, apoptosis induction-, and antiinflammatory- effects. On the other hand, a few studies have shown that antioxidant assist inhibition of accumulation of fat. So we studied the effect of the extract of I. okamura on the cellular activity and differentiation of 3T3-L1 preadipocyte to adipose cell. The viability of cell was analyzed using 3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Adipogenesis of 3T3-L1 cell was analyzed after induction in the induction medium containing the I. okamurae extract. The cellular activity was high compared with the vehicle and 0.05 mM caffeine in all groups of I. okamurae extract treated cells. The extract of I. okamura inhibited accumulation of lipids in 10 and 50 ?g/ml. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. These results suggest that the extract of I. okamurae increases the cellular viability of adipose precursor cells. On the other hand, it suppresses the differentiation of preadipocyte to adipocyte and accumulation of lipids in concentration-dependent manners. It may be possible that the major component of the extract can be applied in the control of adipose tissuegenesis. PMID:25949162

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

    PubMed

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

    2015-03-01

    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

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

    PubMed

    Quinlan, D C; Hochstadt, J

    1977-11-01

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

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

    SciTech Connect

    Zachary, I.; Gil, J.; Lehmann, W.; Sinnett-Smith, J.; Rozengurt, E. )

    1991-06-01

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

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed Central

    Ward, Meliza G.; Ajuwon, Kolapo M.

    2012-01-01

    Obesity promotes increased secretion of a number of inflammatory factors from adipose tissue. These factors include cytokines and very lately, extracellular matrix components (ECM). Biglycan, a small leucine rich proteoglycan ECM protein, is up-regulated in obesity and has recently been recognized as a pro-inflammatory molecule. However, it is unknown whether biglycan contributes to adipose tissue dysfunction. In the present study, we characterized biglycan expression in various adipose depots in wild-type mice fed a low fat diet (LFD) or obesity-inducing high fat diet (HFD). High fat feeding induced biglycan mRNA expression in multiple adipose depots. Adiponectin is an adipokine with anti-inflammatory and insulin sensitizing effects. Due to the importance of adiponectin, we examined the effect of biglycan on adiponectin expression. Comparison of adiponectin expression in biglycan knockout (bgn?/0) and wild-type (bgn+/0) reveals higher adiponectin mRNA and protein in epididymal white adipose tissue in bgn?/0 mice, as well higher serum concentration of adiponectin, and lower serum insulin concentration. On the contrary, knockdown of biglycan in 3T3-L1 adipocytes led to decreased expression and secretion of adiponectin. Furthermore, treatment of 3T3-L1 adipocytes with conditioned medium from biglycan treated macrophages resulted in an increase in adiponectin mRNA expression. These data suggest a link between biglycan and adiponectin expression. However, the difference in the pattern of regulation between in vivo and in vitro settings reveals the complexity of this relationship. PMID:23189205

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

    PubMed

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

    2008-12-01

    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

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

    PubMed Central

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

    2014-01-01

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

  8. Regulation of pyruvate carboxylase in 3T3-L1 cells.

    PubMed Central

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

    1995-01-01

    When 3T3-L1 fibroblasts differentiate to adipocytes, the specific activity of pyruvate carboxylase (PC) increases about 25-fold in parallel with its intracellular protein concentration. The increase in PC protein concentration is accompanied by a 9-10-fold increase in the relative abundance of 4.2 kb PC mRNA measured by Northern-blot analysis using a cDNA probe encoding a segment of the PC gene of 3T3-L1 adipocytes. The effects of cyclic AMP (cAMP) alone and together with insulin on levels of cellular protein, PC activity, PC protein and on the relative abundance of PC mRNA were examined in mature 3T3-L1 adipocytes. Adipocytes exposed to cAMP for 24 h exhibited a 25% decrease in cellular protein and marked decreases in enzyme activity (88%) and PC mRNA abundance (98%) compared with untreated adipocyte controls. After 48 h of exposure to cAMP, PC activity and PC mRNA diminished to levels approaching their detection limits. When exposed to medium containing cAMP plus insulin, adipocyte enzyme activity and PC mRNA declined more slowly during the first 24 h exposure (about 20% decrease) but after 48 h fell to values comparable with those of adipocytes exposed to cAMP alone. Despite these decreases in enzyme activity, the PC protein content of adipocytes treated with cAMP alone or cAMP plus insulin are nearly identical with that of control adipocytes. The inactivation of PC in cAMP-treated adipocytes does not involve loss of the prosthetic group from the holoenzyme. Cross-linking experiments suggest that the spatial arrangement of protomers in inactive PC may differ from that in the active tetrameric enzyme. Data presented suggest that, in addition to inducing inactivation, cAMP may also regulate adipocyte PC by decreasing transcription of the PC gene and/or enhancing the rate of degradation of PC mRNA. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7864811

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

    PubMed Central

    2014-01-01

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

  10. Effect of water-soluble chitosan on the osteoblast function in MC3T3-E1 cells.

    PubMed

    Hou, Jian-Wen; Qian, Li; Kou, Jian-Ming; Zhang, Cui-Wen; Jia, Xue-Jun; Tian, Wei

    2015-01-01

    In the present study, water-soluble chitosan (WSC) was prepared by hydrolysis of chitosan with commercial ?-amylase, and the effect of WSC on osteoblast function, i.e. alkaline phosphatase activity (ALP), cell viability, mineralization, and reactive oxygen species (ROS) in osteoblastic MC3T3-E1 cells was investigated. Osteoblastic MC3T3-E1 cells were cultured in various concentrations of WSC solutions (1-5 mg/mL) for designated time and then the ALP, cell viability, mineralization, and ROS in the cells were evaluated. Treatments of osteoblastic with WSC caused a significant increase in ALP, cell viability, and mineralization in osteoblastic MC3T3-E1 cells. Moreover, treatments of osteoblastic with WSC decreased ROS level in osteoblastic MC3T3-E1 cells. The results demonstrate that the WSC may reduce or prevent osteoblasts degeneration through antioxidant activity. PMID:25453282

  11. Photovoltaics support distribution feeder

    SciTech Connect

    Barker, P.P.; Bailey, B.; Peterson, A.J. Jr.

    1997-03-01

    The concept of supporting the transmission and distribution (T&D) system with a photovoltaic (PV) distributed energy source has gained increasing attention as the cost of PV energy has declined. Locating a PV system at a strategic point on the distribution feeder can enhance the overall T&D system performance and provide a source of renewable power generation. In such applications, the PV system peak output ranges from a few percent up to about 20 percent of the peak feeder load. A good example of one such project on a line supplied by the Pacific Gas & Electric Co.`s Kerman Substation near Fresno, California. Given the success of this and other projects, Niagara Mohawk Power Corp. (NMPC) will be testing a 100 kW ac output system interconnected with a 13.2 kV distribution feeder to demonstrate PV T&D support concepts in its service territory. The demonstration system construction and operation is to be funded by NMPC, Utility Photovoltaics Group (UPVG) and New York State Energy Research and Development Authority (NYSERDA). AWS Scientific will manage the site construction and be responsible for maintaining, operating and monitoring the performance of the system. As a prerequisite to construction of the system, the NMPC research and development department funded AWS Scientific Inc. (Albany, N.Y.) and Power Technologies Inc. (Schenectady, N.Y.) to investigate the use of PV energy for T&D support applications on its system. The study involved reviewing a large number of distribution circuits throughout NMPC`s service territory to find candidate locations for the 100 kW demonstration project. A key focus of the study was to find a feeder whereby the injection of PV energy provided maximum dispersed generation benefits.

  12. Human Induced Pluripotent Stem Cells on Autologous Feeders

    PubMed Central

    Takahashi, Kazutoshi; Narita, Megumi; Yokura, Midori; Ichisaka, Tomoko; Yamanaka, Shinya

    2009-01-01

    Background For therapeutic usage of induced Pluripotent Stem (iPS) cells, to accomplish xeno-free culture is critical. Previous reports have shown that human embryonic stem (ES) cells can be maintained in feeder-free condition. However, absence of feeder cells can be a hostile environment for pluripotent cells and often results in karyotype abnormalities. Instead of animal feeders, human fibroblasts can be used as feeder cells of human ES cells. However, one still has to be concerned about the existence of unidentified pathogens, such as viruses and prions in these non-autologous feeders. Methodology/Principal Findings This report demonstrates that human induced Pluripotent Stem (iPS) cells can be established and maintained on isogenic parental feeder cells. We tested four independent human skin fibroblasts for the potential to maintain self-renewal of iPS cells. All the fibroblasts tested, as well as their conditioned medium, were capable of maintaining the undifferentiated state and normal karyotypes of iPS cells. Furthermore, human iPS cells can be generated on isogenic parental fibroblasts as feeders. These iPS cells carried on proliferation over 19 passages with undifferentiated morphologies. They expressed undifferentiated pluripotent cell markers, and could differentiate into all three germ layers via embryoid body and teratoma formation. Conclusions/Significance These results suggest that autologous fibroblasts can be not only a source for iPS cells but also be feeder layers. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder layers of iPS cells. This is an important step toward the establishment of clinical grade iPS cells. PMID:19956543

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

    PubMed Central

    1991-01-01

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

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

    PubMed Central

    Raman, Aparna; Gawalt, Ellen S.

    2010-01-01

    Inhibiting the non-specific adhesion of cells and proteins to biomaterials such as stents, catheters and guide wires is an important interfacial issue that needs to be addressed in order to reduce surface-related implant complications. Medical grade stainless steel 316L was used as a model system to address this issue. To alter the interfacial property of the implant, self assembled monolayers of long chain phosphonic acids with ?CH3, ?COOH, ?OH tail groups were formed on the native oxide surface of medical grade stainless steel 316L. The effect of varying the tail groups on 3T3 fibroblast adhesion was investigated. The methyl terminated phosphonic acid significantly prevented cell adhesion however presentation of hydrophilic tail groups at the interface did not significantly reduce cell adhesion when compared to the control stainless steel 316L. PMID:21461313

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

    PubMed

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

    2008-01-01

    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

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

    SciTech Connect

    Kazi, Julhash U.; Soh, Jae-Won

    2007-12-14

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

  17. Tannic acid stimulates glucose transport and inhibits adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Liu, Xueqing; Kim, Jae-kyung; Li, Yunsheng; Li, Jing; Liu, Fang; Chen, Xiaozhuo

    2005-02-01

    Obesity is a major risk factor for Syndrome X and type II diabetes (T2D). However, most antidiabetic drugs that are hypoglycemic also promote weight gain, thus alleviating one symptom of T2D while aggravating a major risk factor that leads to T2D. Adipogenesis, the differentiation and proliferation of adipocytes, is a major mechanism leading to weight gain and obesity. It is highly desirable to develop pharmaceuticals and treatments for T2D that reduce blood glucose levels without inducing adipogenesis in patients. Previously, we reported that an extract from Lagerstroemia speciosa L. (banaba) possessed activities that both stimulated glucose transport and inhibited adipocyte differentiation in 3T3-L1 cells. Using glucose uptake assays and Western/Northern blot analyses as major tools and 3T3-L1 cells as a model, we showed that the banaba extract (BE) with tannin removed was devoid of the 2 activities, and tannic acid (TA), a major component of tannins, had the same 2 activities as BE. Inhibitors known to abolish insulin-induced glucose transport also blocked TA-induced glucose transport. We further detected that TA induced phosphorylation of the insulin receptor (IR) and Akt, as well as translocation of glucose transporter 4 (GLUT 4), the protein factors involved in the signaling pathway of insulin-mediated glucose transport. We also demonstrated that TA inhibited the expression of key genes for adipogenesis. Differences between samples with or without TA in all of the quantitative assays were significant (P < 0.05). These results suggest that TA may be useful for the prevention and treatment of T2D and its associated obesity. TA may have the potential to become the lead compound in the development of new types of antidiabetic pharmaceuticals that are able to reduce blood glucose levels without increasing adiposity. PMID:15671208

  18. Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress

    PubMed Central

    Lambert, Ian Henry; Enghoff, Maria Stine; Brandi, Marie-Luise; Hoffmann, Else Kay

    2015-01-01

    The aim of this project was to analyze the regulation of p53 expression in NIH3T3 fibroblasts under the influence of increasing hyperosmotic stress. Expression of p53 showed a biphasic response pattern in NIH3T3 cells under increasing osmotic stress (337 mOsm to 737 mOsm) with a maximum at 587 mOsm. Under isotonic conditions p53 expression increased after addition of the proteasome inhibitor MG132 indicating that cellular p53 levels in unperturbed cells is kept low by proteasomal degradation. However, under hypertonic conditions p53 synthesis as well as p53 degradation were significantly reduced and it is demonstrated that the increase in p53 expression observed when tonicity is increased from 337 to 587 mOsm reflects that degradation is more inhibited than synthesis, whereas the decrease in p53 expression at higher tonicities reflects that synthesis is more inhibited than degradation. The activity of the p53 regulating proteins p38 MAP kinase and the ubiquitin ligase MDM2 were studied as a function of increasing osmolarity. MDM2 protein expression was unchanged at all osmolarities, whereas MDM2 phosphorylation (Ser166) increased at osmolarities up to 537 mOsm and remained constant at higher osmolarities. Phosphorylation of p38 increased at osmolarities up to 687 mOsm which correlated with an increased phosphorylation of p53 (Ser15) and the decreased p53 degradation. Caspase-3 activity increased gradually with hypertonicity and at 737 mOsm both Caspase-3 activity and annexin V binding are high even though p53 expression and activity are low, indicating that initiation of apoptosis under severe hypertonic conditions is not strictly controlled by p53. PMID:26056062

  19. Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation.

    PubMed

    Yang, Jung Yoon; Park, Min Young; Park, Sam Young; Yoo, Hong Il; Kim, Min Seok; Kim, Jae Hyung; Kim, Won Jae; Jung, Ji Yeon

    2015-11-01

    Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells. PMID:26557017

  20. Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

    PubMed Central

    Yang, Jung Yoon; Park, Min Young; Park, Sam Young; Yoo, Hong Il; Kim, Min Seok; Kim, Jae Hyung

    2015-01-01

    Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells. PMID:26557017

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

    SciTech Connect

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

    2012-04-27

    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.

  2. Purple Sweet Potato Leaf Extract Induces Apoptosis and Reduces Inflammatory Adipokine Expression in 3T3-L1 Differentiated Adipocytes

    PubMed Central

    Lee, Shou-Lun; Chin, Ting-Yu; Tu, Ssu-Chieh; Wang, Yu-Jie; Hsu, Ya-Ting; Kao, Ming-Ching; Wu, Yang-Chang

    2015-01-01

    Background. Purple sweet potato leaves (PSPL) are widely grown and are considered a healthy vegetable in Taiwan. PSPL contain a high content of flavonoids, and the boiling water-extracted PSPL (PSPLE) is believed to prevent metabolic syndrome. However, its efficacy has not yet been verified. Therefore, we investigated the effect of PSPLE on adipocytes. Methods. The differentiated 3T3-L1 cells used in this study were derived from preadipocytes that were differentiated into adipocytes using an adipogenic agent (insulin, dexamethasone, and 3-isobutyl-1-methylxanthine); approximately 90% of the cells were differentiated using this method. Results. Treating the differentiated 3T3-L1 cells with PSPLE caused a dose-dependent decrease in the number of adipocytes rather than preadipocytes. In addition, treatment with PSPLE resulted in apoptosis of the differentiated 3T3-L1 cells as determined by DAPI analysis and flow cytometry. PSPLE also increased the expression of cleaved caspase-3 and poly ADP-ribose polymerase (PARP). Furthermore, PSPLE induced downregulation of interleukin-6 (IL-6) and tumor necrosis factor-? (TNF-?) gene expression in the differentiated 3T3-L1 cells. Conclusions. These results suggest that PSPLE not only induced apoptosis but also downregulated inflammation-associated genes in the differentiated 3T3-L1 cells. PMID:26170870

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  4. Synthesis and release of hyaluronic acid by Swiss 3T3 fibroblasts.

    PubMed Central

    Kitchen, J R; Cysyk, R L

    1995-01-01

    Hyaluronic acid (HA) and its synthesis were studied in intact Swiss 3T3 mouse fibroblasts and isolated membranes. HA chains in culture medium, attached to cells and in isolated membranes, were determined to possess average M(r) values of 5.2 x 10(6), 1.8 x 10(6) and 0.14 x 10(6) respectively. Log cells were determined to possess 680,000 HA molecules/cell, and to release 120,000 HA chains/h. The time required for intact cells to synthesize and release a complete HA chain was approximately 4 h, with elongation proceeding at a rate of 57 dimers/min. The amount of cell-associated HA of various cell populations correlated strongly with their rate of HA release into culture media and with the HA synthetase activity determined for their membranes. Prevention of protein synthesis with cycloheximide decreased the rate of HA synthesis of log cells and HA synthetase activity of isolated membranes by 50% within 2-3 h. Because of the similarity between the biological lifetime of HA synthetase and the time required to synthesize a HA chain, we propose a model where each synthetase makes only one HA chain; after synthesis of a complete HA chain, HA synthetase activity is terminated as its HA chain is released from the cell. Images Figure 1 PMID:7626032

  5. Fisetin induces Sirt1 expression while inhibiting early adipogenesis in 3T3-L1 cells.

    PubMed

    Kim, Sang Chon; Kim, Yoo Hoon; Son, Sung Wook; Moon, Eun-Yi; Pyo, Suhkneung; Um, Sung Hee

    2015-11-27

    Fisetin (3,7,3',4'-tetrahydroxyflavone) is a naturally found flavonol in many fruits and vegetables and is known to have anti-aging, anti-cancer and anti-viral effects. However, the effects of fisetin on early adipocyte differentiation and the epigenetic regulator controlling adipogenic transcription factors remain unclear. Here, we show that fisetin inhibits lipid accumulation and suppresses the expression of PPAR? in 3T3-L1 cells. Fisetin suppressed early stages of preadipocyte differentiation, and induced expression of Sirt1. Depletion of Sirt1 abolished the inhibitory effects of fisetin on intracellular lipid accumulation and on PPAR? expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPAR? and FoxO1, and enhanced the association of Sirt1 with the PPAR? promoter, leading to suppression of PPAR? transcriptional activity, thereby repressing adipogenesis. Lowering Sirt1 levels reversed the effects of fisetin on deacetylation of PPAR? and increased PPAR? transactivation. Collectively, our results suggest the effects of fisetin in increasing Sirt1 expression and in epigenetic control of early adipogenesis. PMID:26499075

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

    SciTech Connect

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

    1987-03-01

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

  7. MicroRNA-23a regulates 3T3-L1 adipocyte differentiation.

    PubMed

    Shen, Linyuan; Zhang, Yi; Du, Jingjing; Chen, Li; Luo, Jia; Li, Xuewei; Li, Mingzhou; Tang, Guoqing; Zhang, Shunhua; Zhu, Li

    2016-01-10

    MicroRNAs (miRNAs) are small, non-coding RNAs, which are involved in regulation of a variety of biological processes. Since previous studies regarding the role of miRNAs in the regulation of adipogenic differentiation have shown that miRNA-27a, one member of miRNA-23a?27a?24 cluster, could suppress adipogenesis. We now investigated whether miRNA-23a regulates adipogenic differentiation. In the present study, we showed that the expression of miRNA-23a is decreased during the process of adipogenic differentiation. Over-expression of miRNA-23a decreased lipid accumulation and triglyceride content in 3T3-L1 adipocytes. Our results also demonstrated that miRNA-23a decreases mRNA levels of adipocyte-specific genes involved in lipogenic transcription, fatty acid synthesis and fatty acid transport. These findings suggested miRNA-23a to be a new type of adipogenic depressor and to play an important role in regulating adipocyte differentiation. PMID:26415879

  8. Inhibition by the fungicide fenpropimorph of cholesterol biosynthesis in 3T3 fibroblasts.

    PubMed Central

    Corio-Costet, M F; Gerst, N; Benveniste, P; Schuber, F

    1988-01-01

    Fenpropimorph (N-[3-(p-t-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine), a morpholine fungicide known to be an inhibitor of sterol biosynthesis in fungi and in higher plants, was demonstrated to be an efficient inhibitor of cholesterol biosynthesis in cultured Swiss 3T3 fibroblasts. Treatment of the mammalian cells with fenpropimorph resulted in a dose-dependent inhibition of [14C]acetate incorporation into the C27 sterols [IC50 (concentration causing half-maximal inhibition) = 0.5 microM], which was accompanied by an accumulation of polar sterols and a decrease in cellular hydroxymethylglutaryl-CoA reductase activity. Exposure of the cells to the drug affected cell growth. Analysis of the sterols in the growth-arrested and in the pulse-labelled cells indicate that fenpropimorph has, in the sterol-biosynthetic pathway, target enzymes in mammalian cells different from those in the other phyla. Whereas in plants and fungi fenpropimorph mainly affects sterol isomerases and reductases, in the fibroblasts its main target seems to be the demethylation of lanosterol. Images Fig. 2. PMID:3223956

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

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

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

    PubMed

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

    2015-09-01

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

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

    SciTech Connect

    Saito, Takeshi; Abe, Daigo; Sekiya, Keizo . E-mail: ksekiya@affrc.go.jp

    2007-06-01

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

  12. Protein Kinase C ? (PKC?) Splice Variants Modulate Apoptosis Pathway in 3T3L1 Cells during Adipogenesis

    PubMed Central

    Patel, Rekha; Apostolatos, André; Carter, Gay; Ajmo, Joanne; Gali, Meghanath; Cooper, Denise R.; You, Min; Bisht, Kirpal S.; Patel, Niketa A.

    2013-01-01

    Increased food intake and lack of physical activity results in excess energy stored in adipocytes, and this imbalance contributes to obesity. New adipocytes are required for storage of energy in the white adipose tissue. This process of adipogenesis is widely studied in differentiating 3T3L1 preadipocytes in vitro. We have identified a key signaling kinase, protein kinase C delta (PKC?), whose alternative splice variant expression is modulated during adipogenesis. We demonstrate that PKC?II splice variant promotes survival in differentiating 3T3L1 cells through the Bcl2 pathway. Here we demonstrate that resveratrol, a naturally occurring polyphenol, increases apoptosis and inhibits adipogenesis along with disruption of PKC? alternative splicing during 3T3L1 differentiation. Importantly, we have identified a PKC?II splice variant inhibitor. This inhibitor may be a valuable tool with therapeutic implications in obesity. PMID:23902767

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

    PubMed

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

    2012-08-01

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

  14. Electrochemical characterization of MC3T3-E1 cells cultured on ?TiAl and Ti-6Al-4V alloys.

    PubMed

    Bueno-Vera, J A; Torres-Zapata, I; Sundaram, P A; Diffoot-Carlo, N; Vega-Olivencia, C A

    2015-12-01

    Electrochemical impedance spectroscopy (EIS) was used to study the behavior of MC3T3-E1 cells cultured in an ?MEM+FBS solution on two Ti-based alloys (Ti-6Al-4V and ?TiAl) for 4, 7 and 14 days. EIS measurements were carried out at an open-circuit potential in a 1 mHz to 100 kHz frequency range. Results indicate a general increase in impedance on the Ti alloy surfaces with cells as a function of time. Bode plots indicate changes corresponding to the passive oxide film, adsorption of proteins and cell tissue on surfaces with the passage of time. Normal cellular activity based on the polygonal morphology, with long and fine cytoplasmic prolongations of the cells on Ti-6Al-4V and ?TiAl was observed from SEM images. Similarly, mineralization nodules corresponding to cell differentiation associated with the osseogenetic process were observed confirmed by Alizarin Red S staining. Immunofluorescence analysis to detect the presence of collagen Type I showed an increase in the segregation of collagen as a function of time. The impedance values obtained from EIS testing are indicative of the corrosion protection offered to the Ti alloy substrates by the cell layer. This study shows that ?TiAl has better corrosion resistance than that of Ti-6Al-4V in the ?MEM+FBS environment in the presence of MC3T3-E1 cells. PMID:26145813

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

    NASA Astrophysics Data System (ADS)

    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

    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.

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

    SciTech Connect

    Sagara, Chiaki; Takahashi, Katsuhiko; Kagechika, Hiroyuki; Takahashi, Noriko

    2013-03-29

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

  17. MUC4, a multifunctional transmembrane glycoprotein, induces oncogenic transformation of NIH3T3 mouse fibroblast cells

    PubMed Central

    Bafna, Sangeeta; Singh, Ajay P; Moniaux, Nicolas; Eudy, James D; Meza, Jane L; Batra, Surinder K.

    2008-01-01

    Numerous studies have established the association of MUC4 with the progression of cancer and metastasis. An aberrant expression of MUC4 is reported in precancerous lesions indicating its early involvement in the disease process; however, its precise role in cellular transformation has not been explored. MUC4 contains many unique domains and is proposed to impact on cell signaling pathways and behavior of the tumor cells. In the present study, to decipher its oncogenic potential of MUC4, we stably expressed the MUC4 mucin in NIH3T3 mouse fibroblast cells. Stable ectopic expression of MUC4 resulted in increased growth, colony formation and motility of NIH3T3 cells in vitro and tumor formation in nude mice, when cells were injected subcutaneously. Microarray analysis demonstrated increased expression of several growth- and mitochondrial energy production-associated genes in MUC4-expressing NIH3T3 cells. In addition, expression of MUC4 in NIH3T3 cells resulted in enhanced levels of oncoprotein ErbB2 and its phosphorylated form (pY1248-ErbB2). In conclusion, our studies provide the first evidence that MUC4 alone induces cellular transformation and indicates a novel role of MUC4 in cancer biology. PMID:19010895

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

    SciTech Connect

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

    2013-11-01

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

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

    SciTech Connect

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2014-02-15

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

  20. Multiarray formation of CHO spheroids cocultured with feeder cells for highly efficient protein production in serum-free medium.

    PubMed

    Kutsuzawa, Koichi; Takahashi, Chihiro; Sato, Ryohei; Suzuki, Toshihiro; Kishimoto, Hidehiro; Murakami, Akiichi; Azuma, Takachika; Abe, Ryo; Otsuka, Hidenori

    2013-01-01

    Functional proteins like antibody, cytokine and growth factor have been widely used for basic biological research, diagnosis and cancer therapy. Particularly, antibody drugs as attractive biopharmaceuticals will be expected to create an enormous new market. Chinese hamster ovay (CHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins including antibody drugs. Although three-dimensional culture is preferred to two-dimensional monolayer culture for the efficient large scale culture of CHO cells and subsequent mass production of recombinant proteins, it has the limitation of low protein production. Therefore, a new cell culture em essentially required for an efficient protein production. Here we report on a new three-dimensional cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein in CHO cells. Furthermore, cocultivation of CHO spheroids with feeder cells including bovine aortic endothelial cells (BAEC) and NIH 3T3 was essential to more increase a protein production. The results indicated that CHO heterospheroids cocultured with BAECs were much superior to either CHO monolayers or CHO homospheroids in protein production. Significantly, the above cocultured spheroids in the serum-free medium drastically enhanced protein expression level up to 3-fold compared with CHO spheroids in serum medium, suggesting that a coculture of spheroid system with feeder layer cells is a promising method to enhance protein production under serum-free condition. The spheroid array constructed here is highly usuful as a platform of biopharmaceutical manufacturing as well as tissue and cell based biosensors to detect a wide variety of clinically active compounds through a cellular physiological response. PMID:23646721

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed Central

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

    2009-01-01

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

  3. S-resistin, a non secretable resistin isoform, impairs the insulin signalling pathway in 3T3-L1 adipocytes.

    PubMed

    Rodríguez, María; Moltó, Eduardo; Aguado, Lidia; Gallardo, Nilda; Andrés, Antonio; Arribas, Carmen

    2015-09-01

    S-resistin is a non-secretable resistin spliced variant, which is expressed mainly in the white adipose tissue from Wistar rats. Previous results confirmed that 3T3-L1 cells expressing s-resistin (3T3-L1-s-res) showed an inflammatory response and exhibited a decrease in glucose transport, both basal and insulin-stimulated. Here we present evidences demonstrating for the first time that s-resistin, like resistin, blocks insulin signalling pathway by inhibiting insulin receptor, insulin receptor substrate 1, protein kinase B/Akt and the mammalian target of rapamycin phosphorylation, and increasing the suppressor of cytokine signalling 3 levels being the later probably due to augmented of leptin expression. Thus, our data suggest that s-resistin could act by a still unknown intracrine pathway as an intracellular sensor, regulating the adipocyte insulin sensitivity. PMID:26036220

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

    PubMed

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

    2009-11-01

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

  5. Hydroxytyrosol Inhibits Cannabinoid CB1 Receptor Gene Expression in 3T3-L1 Preadipocyte Cell Line.

    PubMed

    Tutino, Valeria; Orlando, Antonella; Russo, Francesco; Notarnicola, Maria

    2016-02-01

    The 3T3-L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3-L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR-? receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity-associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3-L1 pre-adipocytes the effects of n-3-PUFA, ?-Linolenic acid (OM-3), n-6-PUFA, Linoleic acid (OM-6), and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis-related genes PPAR-?, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3-L1 cell differentiation by down-regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti-adipogenic effects, whereas OM-3 and OM-6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM-3 and OM-6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription. J. Cell. Physiol. 231: 483-489, 2016. © 2015 Wiley Periodicals, Inc. PMID:26189725

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

    SciTech Connect

    Yuan, Guoyue; Jia, Jue; Di, Liangliang; Zhou, Libin; Dong, Sijing; Ye, Jingjing; Wang, Dong; Yang, Ling; Wang, Jifang; Li, Lianxi; Yang, Ying; Mao, Chaoming; Chen, Mingdao

    2012-08-03

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

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

    PubMed

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

    1989-05-25

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

  8. Inhibition of adipogenesis and leptin production in 3T3-L1 adipocytes by a derivative of meridianin C

    SciTech Connect

    Park, Yu-Kyoung; Lee, Tae-Yoon; Choi, Jong-Soon; Hong, Victor Sukbong; Lee, Jinho; Park, Jong-Wook; Jang, Byeong-Churl

    2014-10-03

    Highlights: • Compound 7b, a meridianin C derivative, inhibits adipogenesis. • Compound 7b inhibits C/EBP-?, PPAR-?, FAS, STAT-3, and STAT-5 in 3T3-L1 adipocytes. • Compound 7b inhibits leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Compound 7b thus may have therapeutic potential against obesity. - Abstract: Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a–7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases’ inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of these meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-? (C/EBP-?), peroxisome proliferator-activated receptor-? (PPAR-?), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-?, PPAR-?, FAS, STAT-3 and STAT-5.

  9. Effects of Pueraria lobata Root Ethanol Extract on Adipogenesis and Lipogenesis During 3T3-L1 Differentiation into Adipocytes

    PubMed Central

    Lee, Chae Myoung; Yoon, Mi Sook

    2015-01-01

    We evaluated the inhibitory effect of Pueraria lobata root ethanol extract (PLREE) on lipid accumulation during 3T3-L1 differentiation to adipocytes by measuring the intracellular expression of adipogenic, lipogenic, and lipolytic markers and lipid accumulation. The total polyphenol and flavonoid content of PLREE were 47 and 29 mg/g, respectively. The electron donating capacity of PLREE at 1,000 ?g/mL was 48.8%. Treatment of 3T3-L1 preadipocytes with 100, 250, or 500 ?g/mL PLREE for 8 days dose-dependently promoted the differentiation of 3T3-L1 cells. In contrast, the lipid content of PLREE-treated cells was significantly reduced by 7.8% (p < 0.05), 35.6% (p < 0.001), and 42.2% (p < 0.001) following treatment with 100, 250, and 500 ?g/mL PLREE, respectively, as compared to differentiated control cells. PLREE upregulated peroxisome proliferator-activated receptor ? mRNA and protein, and sterol regulator element-binding protein-1c mRNA levels, but did not affect CCAAT/enhancer binding-protein ? and ? mRNA levels. PLREE also downregulated acetyl-CoA carboxylase mRNA and protein, fatty acid synthase (FAS) protein, and leptin mRNA levels, but did not affect FAS mRNA expression. PLREE upregulated adipose triglyceride lipase mRNA and protein expression, and hormone-sensitive lipase (HSL) protein expression, but did not affect HSL mRNA expression. In conclusion, we found that PLREE enhanced adipogenesis, but reduced lipogenesis, resulting in decreased lipid accumulation in 3T3-L1 cells. PMID:26191386

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  12. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    SciTech Connect

    Kimira, Yoshifumi; Ogura, Kana; Taniuchi, Yuri; Kataoka, Aya; Inoue, Naoki; Sugihara, Fumihito; Nakatani, Sachie; Shimizu, Jun; Wada, Masahiro; Mano, Hiroshi

    2014-10-24

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1?1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1?1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Coll?1 gene expression.

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

    SciTech Connect

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

    2014-10-31

    Highlights: • Aculeatin promoted adipocyte differentiation. • Aculeatin improved glucose uptake. • Aculeatin enhanced adipocyte lipolysis. - Abstract: 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.

  14. Strategies of NF-?B signaling modulation by ectromelia virus in BALB/3T3 murine fibroblasts.

    PubMed

    Struzik, Justyna; Szulc-D?browska, Lidia; Winnicka, Anna; Niemia?towski, Marek

    2015-10-01

    Nuclear factor ?B (NF-?B) is a pleiotropic transcription factor that regulates the expression of immune response genes. NF-?B signaling can be disrupted by pathogens that prevent host immune response. In this work, we examined the influence of ectromelia (mousepox) virus (ECTV) on NF-?B signaling in murine BALB/3T3 fibroblasts. Activation of NF-?B via tumor necrosis factor (TNF) receptor 1 (TNFR1) in these cells induces proinflammatory cytokine secretion. We show that ECTV does not recruit NF-?B to viral factories or induce NF-?B nuclear translocation in BALB/3T3 cells. Additionally, ECTV counteracts TNF-?-induced p65 NF-?B nuclear translocation during the course of infection. Inhibition of TNF-?-induced p65 nuclear translocation was also observed in neighboring cells that underwent fusion with ECTV-infected cells. ECTV inhibits the key step of NF-?B activation, i.e. Ser32 phosphorylation and degradation of inhibitor ?B? (I?B?) induced by TNF-?. We also observed that ECTV prevents TNF-?-induced Ser536 of p65 phosphorylation in BALB/3T3 cells. Studying TNFR1 signaling provides information about regulation of inflammatory response and cell survival. Unraveling poxviral immunomodulatory strategies may be helpful in drug target identification as well as in vaccine development. PMID:26232502

  15. Isoflavones in Chickpeas Inhibit Adipocyte Differentiation and Prevent Insulin Resistance in 3T3-L1 Cells.

    PubMed

    Gao, Yue; Yao, Yang; Zhu, Yinging; Ren, Guixing

    2015-11-11

    Diabetes mellitus is a metabolic disease characterized by hyperglycemia arising from defects in insulin secretion. This study investigated the effects of isoflavones in chickpea sprouts germinated in light (IGL) and isoflavones in chickpea seeds (ICS) on insulin resistance through their role in suppression of 3T3-L1 adipocyte differentiation. Results showed that IGL and ICS inhibit the differentiation of 3T3-L1 pre-adipocytes induced by differentiation medium in a dose-dependent manner, and the suppressive effect of IGL was stronger (p < 0.05) than that of ICS, evidenced by a decrease of Oil Red O staining and intracellular triacylglycerol content in the mature adipocytes. IGL and ICS also stimulated glucose uptake significantly (p < 0.05). Besides, IGL and ICS treatment caused a significant decrease in mRNA and protein expression levels of adipogenesis-related transcription factors peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT-enhancer-binding protein ? (C/EBP?). Furthermore, the mRNA and protein expression levels of adipocyte fatty acid-binding protein (ap2), lipoprotein lipase (LPL), uncoupling protein-2 (UCP-2), and glucose transporter 4 (Glut4) in 3T3-L1 cells were also markedly down-regulated (p < 0.05). PMID:26494490

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

    SciTech Connect

    Smith, M.R.; Ryu, Sungho; Suh, Panghill; Rhee, Suegoo; Kung, Hsiangfu )

    1989-05-01

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

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

    SciTech Connect

    Hashimoto, Takeshi; Yokokawa, Takumi; Endo, Yuriko; Iwanaka, Nobumasa; Higashida, Kazuhiko; Faculty of Sport Science, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192 ; Taguchi, Sadayoshi

    2013-10-11

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

  18. Conditions Determining Initiation of DNA Synthesis in 3T3 Cells*

    PubMed Central

    Dulbecco, R.; Stoker, M. G. P.

    1970-01-01

    Experiments were designed to discriminate between inhibition of growth due to contacts or exhaustion of serum factors. The cell layer was wounded and the migrating cells were followed by time-lapse cinematography; DNA synthesis in the same cells was recognized by means of 3H-thymidine labeling and radioautography. In this way, the complete history of individual cells migrating to the wound could be described. The results show that topographical relationships between cells play an important role in controlling initiation of DNA synthesis. It is still unclear whether initiation is promoted by release from contacts or by the increased ability of the cells to utilize serum factors because of their changes in shapes and activities. PMID:5273897

  19. Widdrol-induced lipolysis is mediated by PKC and MEK/ERK in 3T3-L1 adipocytes.

    PubMed

    Jeong, Hyun Young; Yun, Hee Jung; Kim, Byung Woo; Lee, Eun Woo; Kwon, Hyun Ju

    2015-12-01

    Obesity is a serious medical condition causing various diseases such as heart disease, type-2 diabetes, and cancer. Fat cells (adipocytes) play an important role in the generation of energy through hydrolysis of lipids they accumulate. Therefore, induction of lipolysis (breakdown of lipids into fatty acids and glycerol), is one of the ways to treat obesity. In the present study, we investigated the lipolytic effect of widdrol in 3T3-L1 adipocytes and its mechanism. Widdrol considerably increased the amount of glycerol released from 3T3-L1 adipocytes into the medium in a time- and dose-dependent manner. To determine the mechanism of this effect, we investigated the alterations in glycerol release and protein expression in 3T3-L1 adipocytes treated with widdrol alone or widdrol and inhibitors of proteins involved in the cAMP-dependent pathway or cAMP-independent PKC-MAPK pathway, which are known to induce lipolysis in adipocytes. The adenylyl cyclase inhibitor SQ-22536, PLA2 inhibitor dexamethasone, PI3K inhibitor wortmannin, and PKA inhibitor H-89, which were used to investigate the involvement of the cAMP-dependent pathway, did not affect the lipolytic effect of widdrol. Widdrol-induced phosphorylation of PKC, MEK, and ERK, which are related to the PKC-MAPK pathway, and their phosphorylation was inhibited by their inhibitors (H-7, U0126, and PD-98059, respectively). Moreover, the increase in glycerol release induced by widdrol was almost completely blocked by PKC, MEK, and ERK inhibitors. These results suggest that widdrol induces lipolysis through activation of the PKC-MEK-ERK pathway. PMID:26359088

  20. Effect of botulinum neurotoxin type A (BoNTA) on the morphology and viability of 3T3 murine fibroblasts

    PubMed Central

    Bandala, Cindy; Terán-Melo, Juan Luis; Anaya-Ruiz, Maricruz; Mejía-Barradas, Cesar Miguel; Domínguez-Rubio, Rene; la Garza-Montano, Paloma De; Alfaro-Rodríguez, Alfonso; Lara-Padilla, Eleazar

    2015-01-01

    Aim: BoNTA is used in the treatment of ophthalmological disorders, muscular hyperactivity and pain. In recent years it has been described that BoNTA reduces cellular viability and induces apoptosis in prostate cells lines. Studies about the effect of BoNTA are no well known. There have been studies about the effect of BoNTA on the expression levels of collagenase in fibroblasts, but not on its morphological impact on these cells. The aim of this study was to determine the effect of BoNTA on the morphology and viability of the 3T3 fibroblast cell line. Material and methods: The 3T3 fibroblast cell line was cultured and the experimental group received 10 U BoNTA added to a 0.9% sterile saline solution in a reconstituted vial. The control group received saline solution only. Cultured cells were observed and photographed at 5, 10, 15 and 20 h. Cell viability was evaluated by means of the trypan blue test, and cell proliferation with the Proliferation Assay kit (PROMEGA). Results: The application of BoNTA to 3T3 fibroblast cells induced morphological changes, such as a loss of normal fibroblast morphology. Additionally, we observed the cytoplasmic retraction and spread phenomena. The nuclei showed other important changes with Giemsa staining. Conclusion: The results indicate that BoNTA induced a loss of spindle form, increase in cytoplasmic vesicles, and the presence of nuclear vesicles (compacted chromatin surrounded by a nuclear envelope). This suggests an apoptotic process and decreased cell viability. Further studies are needed to explore the mechanisms of these alterations. PMID:26464704

  1. Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

    SciTech Connect

    Foster, C.M.; Shafer, J.A.; Rozsa, F.W.; Wang, X.; Lewis, S.D.; Renken, D.A.; Natale, J.E.; Schwartz, J.; Carter-Su, C.

    1988-01-12

    Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. /sup 125/I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained in M/sub r/ 134,000 /sup 125/I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of fibroblast form. O-Phosphotyrosine prevented /sup 125/I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with (/sup 32/P)P/sub i/, GH was shown to stimulate formation of a /sup 32/P-labeled protein which bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114,000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. These observations provide strong evidence that binding of GH to its receptor stimulates phosphorylation of tyrosyl residues in the GH receptor.

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

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

    SciTech Connect

    He, Yonghan; Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3; State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 ; Li, Ying; Zhang, Shuocheng; Perry, Ben; Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 ; Zhao, Tiantian; Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 ; Wang, Yanwen; Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 ; Sun, Changhao

    2013-06-28

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

  4. [Dynamic changes and effect of primary cilia on adipogenesis during 3T3-L1 cell differentiation into adipocytes].

    PubMed

    Qiu, Ni; Wei, Xuemei; Fang, Weijin; Liu, Jiaxi; Xiong, Yan

    2015-11-01

    To investigate the dynamic changes and effect of primary cilia on adipogenesis during 3T3-L1 cell differentiation into adipocyte. Methods Chloral hydrate was applied to inhibit primary cilia on day 0 and 4 of 3T3-L1 cell differentiation into adipocyte. The expression levels of primary cilia key proteins like kinesin family member 3a (Kif3a), intraflagellar transport protein 88 (IFT88), acetylated ?-tubulin (acAT) and adipogenic key protein peroxisome proliferator activated receptor ? (PPAR?) were detected by Western blotting. The number of primary cilia was analyzed by immunofluorescence assay. The promoter activity of PPAR? gene was measured with dual-luciferase reporter assay system. The lipid accumulation was observed by oil red O staining. Results The levels of primary cilia key proteins, including Kif3a, IFT88, acAT, and the number of the primary cilia were higher on day 0 of cell differentiation, decreased on day 2, and then increased to the beginning levels on day 4, and significantly decreased again on day 8. Incubation with chloral hydrate on day 0 of differentiation significantly reduced the levels of Kif3a, IFT88, acAT proteins and the number of primary cilia, dramatically enhanced the promoter activity of PPAR? gene, level of PPAR ? protein and lipid accumulation. However, although primary cilia was significantly inhibited by the incubation with chloral hydrate on day 4 of differentiation, the promoter activity and protein expression of PPAR?, lipid accumulation had no obvious changes compared with vehicle groups. Conclusion Primary cilia presented a dynamic change during 3T3-L1 cell differentiation into adipocytes. Few primary cilia in the early differentiation led to significantly enhanced adipogenesis, but once the differentiation was under way, the decrease of primary cilia would not affect adipogenesis. PMID:26522354

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

    PubMed Central

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

    2003-01-01

    Endothelin-1 (ET-1) and tumor necrosis factor ? (TNF?) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNF? induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNF?-induced iNOS expression and to determine whether or not ET-1 could influence TNF?-induced iNOS expression and NO production. In differentiated 3T3-F442A, treatment with TNF? (20 ng ml?1) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription–polymerase chain reaction and Northern blot analysis. TNF?-induced iNOS expression requires nuclear factor ?B activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38–MAP kinase pathways. ET-1, but not ET-3, inhibited the TNF?-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA2 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNF?, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNF?-induced iNOS expression. PMID:12839867

  6. Bioconversion of Citrus unshiu peel extracts with cytolase suppresses adipogenic activity in 3T3-L1 cells

    PubMed Central

    Lim, Heejin; Yeo, Eunju; Song, Eunju; Chang, Yun-Hee; Han, Bok-Kyung; Choi, Hyuk-Joon

    2015-01-01

    BACKGROUND/OBJECTIVES Citrus flavonoids have a variety of physiological properties such as anti-oxidant, anti-inflammation, anti-cancer, and anti-obesity. We investigated whether bioconversion of Citrus unshiu with cytolase (CU-C) ameliorates the anti-adipogenic effects by modulation of adipocyte differentiation and lipid metabolism in 3T3-L1 cells. MATERIALS/METHODS Glycoside forms of Citrus unshiu (CU) were converted into aglycoside forms with cytolase treatment. Cell viability of CU and CU-C was measured at various concentrations in 3T3L-1 cells. The anti-adipogenic and lipolytic effects were examined using Oil red O staining and free glycerol assay, respectively. We performed real time-polymerase chain reaction and western immunoblotting assay to detect mRNA and protein expression of adipogenic transcription factors, respectively. RESULTS Treatment with cytolase decreased flavanone rutinoside forms (narirutin and hesperidin) and instead, increased flavanone aglycoside forms (naringenin and hesperetin). During adipocyte differentiation, 3T3-L1 cells were treated with CU or CU-C at a dose of 0.5 mg/ml. Adipocyte differentiation was inhibited in CU-C group, but not in CU group. CU-C markedly suppressed the insulin-induced protein expression of CCAAT/enhancer-binding protein ? (C/EBP?) and peroxisome proliferator-activated receptor gamma (PPAR?) as well as the mRNA levels of CEBP?, PPAR?, and sterol regulatory element binding protein 1c (SREBP1c). Both CU and CU-C groups significantly increased the adipolytic activity with the higher release of free glycerol than those of control group in differentiated 3T3-L1 adipocytes. CU-C is particularly superior in suppression of adipogenesis, whereas CU-C has similar effect to CU on stimulation of lipolysis. CONCLUSIONS These results suggest that bioconversion of Citrus unshiu peel extracts with cytolase enhances aglycoside flavonoids and improves the anti-adipogenic metabolism via both inhibition of key adipogenic transcription factors and induction of adipolytic activity. PMID:26634048

  7. THERMOREGULATORY RESPONSES OF FEEDER CATTLE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat stress in feedlot cattle causes reduced performance and in the most severe cases death, thus resulting in the loss of millions of dollars in revenue to the cattle industry. A study was designed to investigate the thermoregulatory responses of feeder cattle to both acute and chronic exposures t...

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

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

    Takahashi, Nobuhiko; Division of Gastroenterology and Hematology Yoshizaki, Takayuki; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2011-11-11

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

  10. PPAR? partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes.

    PubMed

    Milton, Flora Aparecida; Cvoro, Aleksandra; Amato, Angelica A; Sieglaff, Douglas H; Filgueira, Carly S; Arumanayagam, Anithachristy Sigamani; de Lima, Maria do Carmo Alves; Pitta, Ivan Rocha; de Assis Rocha Neves, Francisco; Webb, Paul

    2015-08-28

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPAR?) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPAR? partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPAR? target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPAR? activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1 adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPAR? partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPAR? responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. PMID:26168725

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

    PubMed

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

    2008-09-01

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

  12. Yin Yang 1 is a multi-functional regulator of adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Han, Younho; Choi, You Hee; Lee, Sung Ho; Jin, Yun-Hye; Cheong, Heesun; Lee, Kwang Youl

    2015-09-15

    Yin Yang 1 (YY1) is an ubiquitously distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins. The mechanism by which YY1 regulates adipocyte differentiation remains unclear. In this study, we investigated the functional role of YY1 during adipocyte differentiation. During the early stage, YY1 gene and protein expression was transiently downregulated upon the induction of differentiation, however, it was consistently induced during the later stage. YY1 overexpression decreased adipocyte differentiation and blocked cell differentiation at the preadipocyte stage, while YY1 knockdown by RNA interference increased adipocyte differentiation. YY1 physically interacted with PPAR? (Peroxisome proliferator-activated receptor gamma) and C/EBP? (CCAAT/enhancer-binding protein beta) respectively in 3T3-L1 cells. Through its interaction with PPAR?, YY1 directly decreased PPAR? transcriptional activity. YY1 ectopic expression prevented C/EBP? from binding to the PPAR? promoter, resulting in the downregulation of PPAR? transcriptional activity. These results indicate that YY1 repressed adipocyte differentiation by repressing the activity of adipogenic transcriptional factors in 3T3-L1 cells. PMID:26159900

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

    PubMed

    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

    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

  14. 1'-acetoxychavicol acetate inhibits adipogenesis in 3T3-L1 adipocytes and in high fat-fed rats.

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2013-01-01

    Objective Due to the low bioavailability of resveratrol, determining whether its metabolites exert any beneficial effect is an interesting issue. Methods 3T3-L1 maturing pre-adipocytes were treated during differentiation with 25 µM of resveratrol or with its metabolites and 3T3-L1 mature adipocytes were treated for 24 hours with 10 µM resveratrol or its metabolites. The gene expression of adiponectin, leptin, visfatin and apelin was assessed by Real Time RT-PCR and their concentration in the incubation medium was quantified by ELISA. Results Resveratrol reduced mRNA levels of leptin and increased those of adiponectin. It induced the same changes in leptin secretion. Trans-resveratrol-3-O-glucuronide and trans-resveratrol-4?-O-glucuronide increased apelin and visfatin mRNA levels. Trans-resveratrol-3-O-sulfate reduced leptin mRNA levels and increased those of apelin and visfatin. Conclusions The present study shows for the first time that resveratrol metabolites have a regulatory effect on adipokine expression and secretion. Since resveratrol has been reported to reduce body-fat accumulation and to improve insulin sensitivity, and considering that these effects are mediated in part by changes in the analyzed adipokines, it may be proposed that resveratrol metabolites play a part in these beneficial effects of resveratrol. PMID:23717508

  16. [Mechanical stretching inhibits adipocyte differentiation of 3T3-L1 cells: the molecular mechanism and pharmacological regulation].

    PubMed

    Tanabe, Yoshiyuki; Nakayama, Koichi

    2004-11-01

    Obesity frequently promotes a variety of cardiovascular diseases including atherosclerosis, hypertension, and type 2 diabetes. In a view of both the preventive and therapeutic aspects of the abovementioned diseases, most intensive clinical interventions have been primarily directed at decreasing excessive amounts of fat tissue by a change in the balance between intake and expenditure of energy; such changes are typically effected via daily exercise and diet control. Mechanical stimuli such as stretching and rubbing of fat tissues using gymnastic exercises or massage are believed to decrease obesity; however, there is no report concerning the direct effect of the mechanical stimulation on adipocytes. Here, we demonstrated that cyclic stretch inhibited adipocyte differentiation of mouse 3T3-L1 cells, which was attributable to a reduced expression of adipogenic transcription factor peroxisome proliferator-activated receptor (PPAR)gamma(2) via the activation of an extracellular signal-regulated protein kinase (ERK) pathway. The inhibitory effect of the cyclic stretching on the differentiation of 3T3-L1 cells could be restored by troglitazone, a synthetic ligand for PPARgamma. Our results provide a molecular basis for the physiological significance of the local application of mechanical stimuli to fat tissues, which is totally independent of a mechanism for systemic energy consumption. PMID:15502399

  17. Panax notoginseng stimulates alkaline phosphatase activity, collagen synthesis, and mineralization in osteoblastic MC3T3-E1 cells.

    PubMed

    Ji, Zhe; Cheng, Yizhao; Yuan, Puwei; Dang, Xiaoqian; Guo, Xiong; Wang, Weizhuo

    2015-10-01

    Total Panax notoginseng saponin (PNS) has been extensively used to treat a variety of diseases, such as bone fractures, soft tissue injuries, etc. In this study, mouse calvaria-original osteoblastic MC3T3-E1 cells were cultured in various concentrations of PNS (0.005-5 mg/mL) during the period (1, 5, 14, and 23 d). At the endpoint, the osteogenic capacity of MC3T3-E1 cells was investigated by measuring the alkaline phosphatase (ALP) activity, the deposited calcium, and the expression of osteogenic-related markers, including bone collagen type 1 (Col1) and osteocalcin (OCN). Compared with all groups in each period, the most pronounced effect was observed at the concentration range between 0.05 and 0.5 mg/mL (P?

  18. Non-histone protein HMGB1 inhibits the repair of cisplatin damaged DNA in NIH-3T3 murine fibroblasts.

    PubMed

    Yusein-Myashkova, Shazie; Ugrinova, Iva; Pasheva, Evdokia

    2013-12-12

    The nuclear non-histone protein high mobility group box (HMGB) 1 demonstrated an inhibitory effect on the repair of DNA damaged by the antitumor drug cisplatin in vitro. To investigate the role of HMGB1 in living cells we studied the DNA repair of cisplatin damages in mouse fibroblast cell line NIH-3T3. We evaluated the effect of the post-synthetic acetylation and C-terminal domain of the protein by overexpression of the parental and mutant GFP fused forms. The results revealed that HMGB1 had also an inhibitory effect on the repair of cisplatin damaged DNA in vivo. The silencing of HMGB1 in NIH-3T3 cells increases the cellular DNA repair potential. The increased levels of repair synthesis could be "rescued" and returned to less than normal levels if the knockdown cells were transfected with plasmids encoding HMGB1 and HMGB1 K2A. In this case the truncated form also exhibited slight inhibitory effect. PMID:24325815

  19. Structural analysis of ITER magnet feeders

    SciTech Connect

    Ilyin, Yuri; Gung, Chen-Yu; Bauer, Pierre; Chen, Yonghua; Jong, Cornelis; Devred, Arnaud; Mitchell, Neil; Lorriere, Philippe; Farek, Jaromir; Nannini, Matthieu

    2012-06-15

    This paper summarizes the results of the static structural analyses, which were conducted in support of the ITER magnet feeder design with the aim of validating certain components against the structural design criteria. While almost every feeder has unique features, they all share many common constructional elements and the same functional specifications. The analysis approach to assess the load conditions and stresses that have driven the design is equivalent for all feeders, except for particularities that needed to be modeled in each case. The mechanical analysis of the feeders follows the sub-modeling approach: the results of the global mechanical model of a feeder assembly are used as input for the detailed models of the feeder' sub-assemblies or single components. Examples of such approach, including the load conditions, stress assessment criteria and solutions for the most critical components, are discussed. It has been concluded that the feeder system is safe in the referential operation scenarios. (authors)

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

    PubMed Central

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

    2013-01-01

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

  1. Healthy Stocker and Feeder Calves. 

    E-print Network

    Barron, H. T.; Maddox, L. A. Jr.

    1975-01-01

    rather than drylot. I Feed hay and concentrates in troughs unless calves have learned to eat from self-feeders. For the ultimate in premium calf production, the Preconditioning for Health Program outlined by the American Association of Bovine... 30 days before weaning. Simultaneously vacci- nate for "red nose" (infectious bovine rhino- tracheitis), bovine virus diarrhea and para- influenza 3 (SF-4). If no early vaccination was practiced, give these vaccines and repeat at least...

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

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

    E-print Network

    Safi, Malak; 10.1016/j.phpro.2010.11.059

    2011-01-01

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

  4. Differential effects of eicosapentaenoic acid and docosahexaenoic acid in promoting the differentiation of 3T3-L1 preadipocytes.

    PubMed

    Murali, Ganesan; Desouza, Cyrus V; Clevenger, Michelle E; Ramalingam, Ramesh; Saraswathi, Viswanathan

    2014-01-01

    The objective of this study was to determine the effects of enrichment with n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the differentiation of 3T3-L1 preadipocytes. Enrichment with DHA but not EPA significantly increased the differentiation markers compared to control differentiated cells. DHA compared to EPA treatment led to a greater increase in adiponectin secretion and, conditioned media collected from DHA treated cells inhibited monocyte migration. Moreover, DHA treatment resulted in inhibition of pro-inflammatory signaling pathways. DHA treated cells predominantly accumulated DHA in phospholipids whereas EPA treatment led to accumulation of both EPA and its elongation product docosapentaenoic acid (DPA), an n-3 fatty acid. Of note, adding DPA to DHA inhibited DHA-induced differentiation. The differential effects of EPA and DHA on preadipocyte differentiation may be due, in part, to differences in their intracellular modification which could impact the type of n-3 fatty acids incorporated into the cells. PMID:24332315

  5. Increased NIH 3T3 fibroblast functions on cell culture dishes which mimic the nanometer fibers of natural tissues

    PubMed Central

    Bhardwaj, Garima; Webster, Thomas J

    2015-01-01

    Traditional flat tissue cell culture dishes have consisted of polystyrene treated with plasma gases for growing, subculturing, and studying cell behavior in vitro. However, increasingly it has been observed that mimicking natural tissue properties (such as chemistry, three-dimensional structure, mechanical properties, etc) in vitro can lead to a better correlation of in vitro to in vivo cellular functions. The following studies compared traditional NIH 3T3 fibroblasts’ functions on XanoMatrix scaffolds to standard tissue culture polystyrene. Results found significantly greater fibroblast adhesion and proliferation on XanoMatrix cell culture dishes which mimic the nanoscale geometry of natural tissue fibers with true, tortuous fiber beds creating a robust, consistent, and versatile growth platform. In this manner, this study supports that cell culture dishes which mimic features of natural tissues should be continually studied for a wide range of applications in which mimicking natural cellular functions are important. PMID:26345155

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

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

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

    SciTech Connect

    Chung, S.S.; Choi, H.H.; Cho, Y.M.; Lee, H.K.; Park, K.S. . E-mail: kspark@snu.ac.kr

    2006-09-15

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

  10. Cranberries (Oxycoccus quadripetalus) inhibit pro-inflammatory cytokine and chemokine expression in 3T3-L1 adipocytes.

    PubMed

    Kowalska, Katarzyna; Olejnik, Anna

    2016-04-01

    Oxidative stress and inflammation are involved in the development of obesity, type 2 diabetes and vascular complications. Systemic inflammation, as seen in obesity, is associated with high plasmatic levels of pro-inflammatory, pro-atherogenic and pro-thrombotic adipokines. Here we studied the effects of lyophilized cranberries (LCB) on the secretion and expression of PAI-1, IL-6, MCP-1 and leptin in mature 3T3-L1 adipocytes under baseline conditions and excessive inflammatory response elicitation by stimulation with H2O2. Our data demonstrated that LCB significantly reduced the expression and secretion of IL-6, MCP-1 and leptin, as well as suppressed the overexpression of PAI-1 induced by H2O2. Our findings suggested that LCB counteracted the stimulatory effect of H2O2 on secretion and expression of pro-inflammatory adipokines, implying a potential anti-inflammatory effect during the inflammatory process induced via oxidative stress in adipose tissue. PMID:26593599

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

    SciTech Connect

    Kang, Seong-Il; Ko, Hee-Chul; Shin, Hye-Sun; Kim, Hyo-Min; Hong, Youn-Suk; Lee, Nam-Ho; Kim, Se-Jae; Jeju Sasa Industry Development Agency, Jeju National University, Jejusi, Jeju 690-756

    2011-06-17

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

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

    SciTech Connect

    Bick, T.; Frick, G.P.; Leonard, D.

    1994-12-31

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

  13. Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes

    PubMed Central

    Robinson, Delia B.; Harris, Carla M.; Johnson, Joyce E.; Mohler, Peter J.; Jerome, W. Gray; Swift, Larry L.

    2015-01-01

    Lipid droplets are intracellular energy storage organelles composed of a hydrophobic core of neutral lipid, surrounded by a monolayer of phospholipid and a diverse array of proteins. The function of the vast majority of these proteins with regard to the formation and/or turnover of lipid droplets is unknown. Our laboratory was the first to report that microsomal triglyceride transfer protein (MTP), a lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins, was expressed in adipose tissue of humans and mice. In addition, our studies suggested that MTP was associated with lipid droplets in both brown and white fat. Our observations led us to hypothesize that MTP plays a key role in lipid droplet formation and/or turnover. The objective of these studies was to gain insight into the function of MTP in adipocytes. Using molecular, biochemical, and morphologic approaches we have shown: 1) MTP protein levels increase nearly five-fold as 3T3-L1 cells differentiate into adipocytes. 2) As 3T3-L1 cells undergo differentiation, MTP moves from the juxtanuclear region of the cell to the surface of lipid droplets. MTP and perilipin 2, a major lipid droplet surface protein, are found on the same droplets; however, MTP does not co-localize with perilipin 2. 3) Inhibition of MTP activity has no effect on the movement of triglyceride out of the cell either as a lipid complex or via lipolysis. 4) MTP is found associated with lipid droplets within hepatocytes from human fatty livers, suggesting that association of MTP with lipid droplets is not restricted to adipocytes. In summary, our data demonstrate that MTP is a lipid droplet-associated protein. Its location on the surface of the droplet in adipocytes and hepatocytes, coupled with its known function as a lipid transfer protein and its increased expression during adipocyte differentiation suggest a role in lipid droplet biology. PMID:26267806

  14. Role of Sandhika: a polyherbal formulation on MC3T3-E1 osteoblast-like cells.

    PubMed

    Tripathi, Yamini B; Tripathi, Pratibha; Korlagunta, Kiranmayi; Chai, Sheau Ching; Smith, Brenda J; Arjmandi, Bahram H

    2008-02-01

    Sandhika is a polyherbal formulation, (water soluble fraction of Commiphora mukul, Boswellia serrata, Semecarpus anacardium and Strychnos nux vomica), which has been in clinical use in India for last 20 years. Its modified formulation BHUx has shown specific inhibition of cyclooxygenase (COX)-2 and lipoxygenase (LOX)-15 and has prevented diet-induced atherosclerosis in rabbits. In order to explore the possibility of the use of Sandhika for the management of osteoporosis, we have examined its influence on MC3T3-E1 osteoblast-like cells in presence of lipopolysaccharide (1 microg/ml) in terms of calcium nodule formation and alkaline phosphatase activity. MC3T3-E1 osteoblast-like cells (80% confluence in 6-well plates) were treated with water extract of Sandhika, for 10 days, in the concentration range of 0.5 to 16 mg/ml final concentration, in presence of LPS. Media was changed on every third day and culture supernatant was collected after every change to assess the alkaline phosphatase activity and on the tenth day, cells were washed and stained with "Alizarin S" for visualization of calcium nodules by using Meta Morph software (Universal Imaging, Downingtown, PA). The results showed significant enhancement in calcium nodule formation in the dose dependent manner up to 2 mg/ml, followed by gradual decrease at higher concentrations. This change was accompanied with the increase in the alkaline phosphatase activity in these plates, indicating a potential anabolic effect of this polyherbal formulation on osteoblast-like cells under inflammatory conditions induced by LPS. PMID:17687634

  15. Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation, collagen synthesis and gene expression

    PubMed Central

    Liu, Cheng-Hai; Hu, Yi-Yang; Wang, Xiao-Ling; Xu, Lie-Ming; Liu, Ping

    2000-01-01

    AIM: To investigate the mechanisms of salvianolic acid A (SA-A) against liver fibrosis in vitro. METHODS: NIH/3T3 fibroblasts were cultured routinely, and incubated with 10-4 mol/L-10-7 mol/L SA-A for 22 h. The cell viability was assayed by [3H]proline incorporation, cell proliferation by [3H]TdR incorporation, cell collagen synthetic rate was measured with [3H]proline impulse and collagenase digestion method. The total RNA was prepared from the control cells and the drug treated cells respectively, and ? (1) I pro-collagen mRNA expression was semi-quantitatively analyzed with RT-PCR. RESULTS: 10-4 mol/L SA-A decreased cell viability and exerted some cytotoxiciy, while 10-5 mol/L-10-7 mol/L SA-A did not affect cell viability, but inhibited cell proliferation significantly, and 10-6 mol/L SA-A had the best effect on cell viability among these concentrations of drugs. 10-5 mol/L-10-6 mol/L SA-A inhibited intracellular collagen synthetic rate, but no significant influence on extracellular collagen secretion. Both 10-5 mol/L and 10-6 mol/L SA-A could decrease ? (1) I pro-collagen mRNA expression remarkably. CONCLUSION: SA-A had potent action against liver fibrosis. It inhibited NIH/3T3 fibroblast proliferation, intracellular collagen synthetic rate and type I pro-collagen gene expression, which may be one of the main mechanisms of the drug. PMID:11819598

  16. Depletion of mitoferrins leads to mitochondrial dysfunction and impairment of adipogenic differentiation in 3T3-L1 preadipocytes.

    PubMed

    Chen, Y-C; Wu, Y-T; Wei, Y-H

    2015-11-01

    Dysregulation of iron homeostasis is a potential risk factor for type 2 diabetes mellitus (T2DM) and insulin resistance. Iron transported into mitochondria by mitoferrins is mainly utilized for the biosynthesis of iron-sulfur clusters, heme, and other cofactors. Recent studies revealed that mitochondrial dysfunction leads to impaired adipogenesis and insulin insensitivity in adipocytes. However, it is unknown whether mitochondrial iron import and iron status affect the biogenesis and function of mitochondria during adipogenic differentiation. In this study, we used double knockdown of mitoferrin 1 and mitoferrin 2 (Mfrn1/2) to investigate the role of mitochondrial iron homeostasis in mitochondrial bioenergetic function and adipogenic differentiation. The results showed that depletion of Mfrn1/2 in 3T3-L1 preadipocytes impaired the biosynthesis of iron-sulfur proteins in mitochondria due to a decrease in mitochondrial iron content. This was associated with a decrease in mitochondrial oxygen consumption rate and intracellular ATP level in adipocytes with Mfrn1/2 knockdown. Remarkably, Mfrn1/2 deficiency reduced the expression of adipogenic genes and lipid production during adipogenic differentiation. Moreover, insulin-induced glucose uptake and Akt phosphorylation at the Ser473 residue were decreased concurrently in adipocytes differentiated from 3T3-L1 preadipocytes after knockdown of Mfrn1/2. These findings suggest that dysregulation of mitochondrial iron metabolism elicited by knockdown of Mfrn1/2 results in mitochondrial dysfunction, which culminates in the compromise of differentiation and insulin insensitivity of adipocytes. This scenario may explain the recent findings that iron deficiency or alterations in iron metabolism are associated with the pathogenesis of T2DM. PMID:26118715

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    2014-01-01

    Background Ivy gourd (Coccinia grandis L. Voigt) is a tropical plant widely distributed throughout Asia, Africa, and the Pacific Islands. The anti-obesity property of this plant has been claimed but still remains to be scientifically proven. We therefore investigated the effects of ivy gourd leaf, stem, and root on adipocyte differentiation by employing cell culture model. Methods Dried roots, stems, and leaves of ivy gourd were separately extracted with ethanol. Each extract was then applied to 3T3-L1 pre-adipocytes upon induction with a mixture of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone, for anti-adipogenesis assay. The active extract was further fractionated by a sequential solvent partitioning method, and the resulting fractions were examined for their abilities to inhibit adipogenesis in 3T3-L1 cells. Differences in the expression of adipogenesis-related genes between the treated and untreated cells were determined from their mRNA and protein levels. Results Of the three ivy gourd extracts, the root extract exhibited an anti-adipogenic effect. It significantly reduced intracellular fat accumulation during the early stages of adipocyte differentiation. Together with the suppression of differentiation, expression of the genes encoding PPAR?, C/EBP?, adiponectin, and GLUT4 were down-regulated. Hexane-soluble fraction of the root extract also inhibited adipocyte differentiation and decreased the mRNA levels of various adipogenic genes in the differentiating cells. Conclusions This is the first study to demonstrate that ivy gourd root may prevent obesity based mainly on the ability of its active constituent(s) to suppress adipocyte differentiation in vitro. Such an inhibitory effect is mediated by at least down-regulating the expression of PPAR?-the key transcription factor of adipogenesis in pre-adipocytes during their early differentiation processes. PMID:24884680

  19. Temperature distribution during ICG-dye-enhanced laser photocoagulation of feeder vessels in treatment of AMD-related choroidal neovascularization.

    PubMed

    Zhu, Liang; Banerjee, Rupak K; Salloum, Maher; Bachmann, Albert; Flower, Robert W

    2008-06-01

    Laser photocoagulation of the feeder vessels of age-related macula degeneration-related choroidal neovascularization (CNV) membranes is a compelling treatment modality, one important reason being that the treatment site is removed from the fovea in cases of sub- or juxtafoveal CNV. To enhance the energy absorption in a target feeder vessel, an indocyanine green dye bolus is injected intravenously, and the 805 nm wavelength diode laser beam is applied when the dye bolus transits the feeder vessel; this tends to reduce concomitant damage to adjacent tissue. A 3D theoretical simulation, using the Pennes bioheat equation, was performed to study the temperature distribution in the choroidal feeder vessel and its vicinity during laser photocoagulation. The results indicate that temperature elevation in the target feeder vessel increases by 20% in dye-enhanced photocoagulation, compared to just photocoagulation alone. The dye bolus not only increases the laser energy absorption in the feeder vessel but also shifts the epicenter of maximum temperature away from the sensitive sensory retina and retinal pigment epithelial layers and toward the feeder vessel. Two dominant factors in temperature elevation of the feeder vessel are location of the feeder vessel and blood flow velocity through it. Feeder vessel temperature elevation becomes smaller as distance between it and the choriocapillaris layer increases. The cooling effect of blood flow through the feeder vessel can reduce the temperature elevation by up to 21% of the maximum that could be produced. Calculations were also performed to examine the effect of the size of the laser spot. To achieve the same temperature elevation in the feeder vessel when the laser spot diameter is doubled, the laser power level has to be increased by only 60%. In addition, our results have suggested that more studies are needed to measure the constants in the Arrhenius integral for assessing thermal damage in various tissues. PMID:18532859

  20. Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor. beta

    SciTech Connect

    Kuratomi, Y.; Ono, M.; Yasutake, C.; Mawatari, M.; Kuwano, M.

    1987-01-01

    A mutant clone (MO-5) was originally isolated as a clone resistant to Na/sup +//K/sup +/ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF):binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-..beta.. efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-..beta.. while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-..beta.. showed low colony formation capacity in soft agar in the absence of TGF-..beta... Colonies of MO-5 formed by TGF-..beta.. in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-..beta... Pretreatment of MO-5 with TGF-..beta.. induced secretion of TGF-..beta..-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-..beta..-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the transformed phenotype in MO-5 is discussed.

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

    PubMed

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

    2013-04-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-01-01

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

  4. High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts

    PubMed Central

    Angius, Fabrizio; Spolitu, Stefano; Uda, Sabrina; Deligia, Stefania; Frau, Alessandra; Banni, Sebastiano; Collu, Maria; Accossu, Simonetta; Madeddu, Clelia; Serpe, Roberto; Batetta, Barbara

    2015-01-01

    High density lipoproteins (HDLs) play a crucial role in removing excess cholesterol from peripheral tissues. Although their concentration is lower during conditions of high cell growth rate (cancer and infections), their involvement during cell proliferation is not known. To this aim, we investigated the replicative cycles in synchronised Swiss 3T3 fibroblasts in different experimental conditions: i) contact-inhibited fibroblasts re-entering cell cycle after dilution; ii) scratch-wound assay; iii) serum-deprived cells induced to re-enter G1 by FCS, HDL or PDGF. Analyses were performed during each cell cycle up to quiescence. Cholesterol synthesis increased remarkably during the replicative cycles, decreasing only after cells reached confluence. In contrast, cholesteryl ester (CE) synthesis and content were high at 24?h after dilution and then decreased steeply in the successive cycles. Flow cytometry analysis of DiO-HDL, as well as radiolabeled HDL pulse, demonstrated a significant uptake of CE-HDL in 24?h. DiI-HDL uptake, lipid droplets (LDs) and SR-BI immunostaining and expression followed the same trend. Addition of HDL or PDGF partially restore the proliferation rate and significantly increase SR-BI and pAKT expression in serum-deprived cells. In conclusion, cell transition from G0 to G1/S requires CE-HDL uptake, leading to CE-HDL/SR-BI pathway activation and CEs increase into LDs. PMID:26640042

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

    SciTech Connect

    Shimada, Koichi; Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo ; Ikeda, Kyoko; Ito, Koichi; Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo

    2009-12-18

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  8. The Effect of OSM on MC3T3-E1 Osteoblastic Cells in Simulated Microgravity with Radiation

    PubMed Central

    Goyden, Jake; Tawara, Ken; Hedeen, Danielle; Willey, Jeffrey S.; Thom Oxford, Julia; Jorcyk, Cheryl L.

    2015-01-01

    Bone deterioration is a challenge in long-term spaceflight with significant connections to patients experiencing disuse bone loss. Prolonged unloading and radiation exposure, defining characteristics of space travel, have both been associated with changes in inflammatory signaling via IL-6 class cytokines in bone. While there is also evidence for perturbed IL-6 class signaling in spaceflight, there has been scant examination of the connections between microgravity, radiation, and inflammatory stimuli in bone. Our lab and others have shown that the IL-6 class cytokine oncostatin M (OSM) is an important regulator of bone remodeling. We hypothesize that simulated microgravity alters osteoblast OSM signaling, contributing to the decoupling of osteolysis and osteogenesis in bone homeostasis. To test this hypothesis, we induced OSM signaling in murine MC3T3-E1 pre-osteoblast cells cultured in modeled microgravity using a rotating wall vessel bioreactor with and without exposure to radiation typical of a solar particle event. We measured effects on inflammatory signaling, osteoblast activity, and mineralization. Results indicated time dependent interactions among all conditions in the regulation of IL-6 production. Furthermore, OSM induced the transcription of OSM receptor ß, IL 6 receptor ? subunits, collagen ?1(I), osteocalcin, sclerostin, RANKL, and osteoprotegerin. Measurements of osteoid mineralization suggest that the spatial organization of the osteoblast environment is an important consideration in understanding bone formation. Taken together, these results support a role for altered OSM signaling in the mechanism of microgravity-induced bone loss. PMID:26030441

  9. Effects of SMYD3 overexpression on transformation, serum dependence, and apoptosis sensitivity in NIH3T3 cells.

    PubMed

    Luo, Xue-Gang; Xi, Tao; Guo, Shu; Liu, Zhi-Peng; Wang, Nan; Jiang, Yong; Zhang, Tong-Cun

    2009-06-01

    The SET and MYND domain-containing protein 3 (SMYD3) gene was found to encode a novel histone methyltransferase involved in human cancer cells. It could specifically methylate histone H3 at lysine 4 and activate the transcription of a set of downstream genes, including of several oncogenes (e.g., N-Myc, CrkL, Wnt10b, RIZ, and hTERT) and genes involved in the control of cell cycle (e.g., Cyclin G1 and CDK2) and signal transduction (e.g., STAT1, MAP3K11, and PIK3CB). To determine the effects of SMYD3 overexpression on cell transformation, serum dependence and apoptosis sensitivity, we expressed SMYD3 in NIH3T3 cells, and these cells showed several transformed phenotypes as demonstrated by foci formation and colony growth in soft agar. Besides, these transfectants also showed increased serum dependence and depression of sensitivity to apoptosis induced by dexamethasone. These findings lend further understanding to the role of SMYD3 in the genesis of human cancers and might throw light on the development of novel therapeutic approaches to human cancers. PMID:19472189

  10. Nck? Adapter Regulates Actin Polymerization in NIH 3T3 Fibroblasts in Response to Platelet-Derived Growth Factor bb

    PubMed Central

    Chen, Min; She, Hongyun; Kim, Airie; Woodley, David T.; Li, Wei

    2000-01-01

    The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nck? (Nck) and Nck? (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nck? in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nck? but not Nck? blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nck? abolishes its interfering effect. Nck? binds at Tyr-1009 in human PDGF receptor ? (PDGFR-?) which is different from Nck?'s binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nck? but not an anti-Nck? antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nck? but not Nck? blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nck? acts in parallel to or downstream of Rac1. This is the first report of Nck?'s role in receptor tyrosine kinase signaling to the actin cytoskeleton. PMID:11027258

  11. Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase beta family in NIH 3T3 cells.

    PubMed

    Cocco, L; Rubbini, S; Manzoli, L; Billi, A M; Faenza, I; Peruzzi, D; Matteucci, A; Artico, M; Gilmour, R S; Rhee, S G

    1999-05-18

    Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCbeta1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCbeta1 is present, but also PLCbeta2, PLCbeta3 and PLCbeta4. The more abounding isoform is PLCbeta1 followed by PLCbeta3, PLCbeta2 and PLCbeta4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCbeta isozymes is in the inner nuclear compartment. PMID:10320812

  12. MC3T3-E1 cell response to stainless steel 316L with different surface treatments.

    PubMed

    Zhang, Hongyu; Han, Jianmin; Sun, Yulong; Huang, Yongling; Zhou, Ming

    2015-11-01

    In the present study, stainless steel 316L samples with polishing, aluminum oxide blasting, and hydroxyapatite (HA) coating were prepared and characterized through a scanning electron microscope (SEM), optical interferometer (surface roughness, Sq), contact angle, surface composition and phase composition analyses. Osteoblast-like MC3T3-E1 cell adhesion on the samples was investigated by cell morphology using a SEM (4h, 1d, 3d, 7d), and cell proliferation was assessed by MTT method at 1d, 3d, and 7d. In addition, adsorption of bovine serum albumin on the samples was evaluated at 1h. The polished sample was smooth (Sq: 1.8nm), and the blasted and HA coated samples were much rougher (Sq: 3.2?m and 7.8?m). Within 1d of incubation, the HA coated samples showed the best cell morphology (e.g., flattened shape and complete spread), but there was no significant difference after 3d and 7d of incubation for all the samples. The absorbance value for the HA coated samples was the highest after 1d and 3d of incubation, indicating better cell viability. However, it reduced to the lowest value at 7d. Protein adsorption on the HA coated samples was the highest at 1h. The results indicate that rough stainless steel surface improves cell adhesion and morphology, and HA coating contributes to superior cell adhesion, but inhibits cell proliferation. PMID:26249561

  13. Curcumin induces brown fat-like phenotype in 3T3-L1 and primary white adipocytes.

    PubMed

    Lone, Jameel; Choi, Jae Heon; Kim, Sang Woo; Yun, Jong Won

    2016-01-01

    Recent advances have been made in the understanding of pharmacological and dietary agents that contribute to browning of white adipose tissue in order to combat obesity by promoting energy expenditure. Here, we show that curcumin induces browning of 3T3-L1 and primary white adipocytes via enhanced expression of brown fat-specific genes. Curcumin-induced browning in white adipocytes was investigated by determining expression levels of brown adipocyte-specific genes/proteins by real-time reverse transcriptase polymerase chain reaction, immunoblot analysis and immunocytochemical staining. Curcumin increased mitochondrial biogenesis, as evidenced by transmission electronic microscopic detection and enhanced expression of proteins involved in fat oxidation. Cucurmin also increased protein levels of hormone-sensitive lipase and p-acyl-CoA carboxylase, suggesting its possible role in augmentation of lipolysis and suppression of lipogenesis. Increased expression of UCP1 and other brown adipocyte-specific markers was possibly mediated by curcumin-induced activation of AMP-activated protein kinase (AMPK) based on the fact that inhibition of AMPK by dorsomorphin abolished expression of PRDM16, UCP1 and peroxisome proliferator-activated receptor gamma co-activator 1-alpha while the activator 5-Aminoimidazole-4-carboxamide ribonucleotide elevated expression of these brown marker proteins. Our findings suggest that curcumin plays a dual modulatory role in inhibition of adipogenesis as well as induction of the brown fat-like phenotype and thus may have potential therapeutic implications for treatment of obesity. PMID:26456563

  14. Effect of zinc oxide nanoparticles on the function of MC3T3-E1 osteoblastic cells.

    PubMed

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

    2013-11-01

    Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 ?g/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01~1 ?g/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 ?g/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells (P < 0.05). Moreover, pretreatment with ZnO NPs (0.01~1 ?g/ml) significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Measurement of reactive oxygen species (ROS) indicated decrease in ROS level upon exposure to ZnO nanoparticles (0.01 ?g/ml). Hence, our study indicated that ZnO nanoparticles can have protective effects on osteoblasts at low concentrations where there are little or no observable cytotoxic effects. PMID:23900646

  15. Suppressive effects of saponin-enriched extracts from quinoa on 3T3-L1 adipocyte differentiation.

    PubMed

    Yao, Yang; Zhu, Yingying; Gao, Yue; Shi, Zhenxing; Hu, Yibo; Ren, Guixing

    2015-10-01

    This study was performed to investigate the effect of quinoa saponins (QS) on the differentiation of 3T3-L1 preadipocytes. QS inhibited triglyceride (TG) accumulation in the mature adipocytes, evidenced by oil-red O staining and intracellular quantification. Real time-PCR analysis and western blot analysis showed that QS significantly down-regulated the mRNA and protein expression of key adipogenic transcription factors, peroxisome proliferator-activated receptor ? (PPAR?), and CCAAT/enhancer-binding protein alpha (C/EBP?), however, they had no significant effect on CCAAT/enhancer-binding protein beta (C/EBP?) and CCAAT/enhancer-binding protein delta (C/EBP?) which are the upstream regulators for adipogenesis compared with mature adipocytes. QS also reduced mRNA and protein expression of sterol regulatory element-binding protein-1c (SREBP-1c) related to the late stage of adipogenesis. Furthermore, lipoprotein lipase (LPL), adipocyte protein 2 (aP2) and glucose transporter 4 (Glut4), as adipocyte specific genes, were decreased in mature adipocytes by QS treatment. These findings indicate that QS are capable of suppressing adipogenesis and therefore they seem to be natural bioactive factors effective in adipose tissue mass modulation. PMID:26242624

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

    PubMed

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

    2013-03-01

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

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

    PubMed

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

    2014-01-01

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

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

    SciTech Connect

    Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty; Patel, Yashomati M.

    2008-10-15

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

  19. Dietary polyphenols preconditioning protects 3T3-L1 preadipocytes from mitochondrial alterations induced by oxidative stress.

    PubMed

    Baret, Pascal; Septembre-Malaterre, Axelle; Rigoulet, Michel; Lefebvre d'Hellencourt, Christian; Priault, Muriel; Gonthier, Marie-Paule; Devin, Anne

    2013-01-01

    Numerous studies indicate that an increase in reactive oxygen species (ROS) significantly affects white adipose tissue biology and leads to an inflammatory profile and insulin resistance, which could contribute to obesity-associated diabetes and cardiovascular diseases. Mitochondria play a key role in adipose tissue energy metabolism and constitute the main source of cellular ROS such as H(2)O(2). Polyphenols constitute the most abundant antioxidants provided by the human diet. Indeed, they are widely distributed in fruits, vegetables and some plant-derived beverages such as coffee and tea. Thus, the biological effects of dietary polyphenols that may increase the antioxidant capacity of the body against obesity-induced oxidative stress are of high interest. Here, we studied the capacity of polyphenols to modulate the impact of oxidative stress on the mitochondria of preadipocytes, which are important cells governing the adipose tissue development for energy homeostasis. Whereas H(2)O(2) treatment induces a proliferation arrest associated with an increase in mitochondrial content in 3T3-L1 preadipocytes, preconditioning with some major dietary polyphenols totally or partially protects the cells against oxidative stress consequences. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy. PMID:23103716

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

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1999-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  2. High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts.

    PubMed

    Angius, Fabrizio; Spolitu, Stefano; Uda, Sabrina; Deligia, Stefania; Frau, Alessandra; Banni, Sebastiano; Collu, Maria; Accossu, Simonetta; Madeddu, Clelia; Serpe, Roberto; Batetta, Barbara

    2015-01-01

    High density lipoproteins (HDLs) play a crucial role in removing excess cholesterol from peripheral tissues. Although their concentration is lower during conditions of high cell growth rate (cancer and infections), their involvement during cell proliferation is not known. To this aim, we investigated the replicative cycles in synchronised Swiss 3T3 fibroblasts in different experimental conditions: i) contact-inhibited fibroblasts re-entering cell cycle after dilution; ii) scratch-wound assay; iii) serum-deprived cells induced to re-enter G1 by FCS, HDL or PDGF. Analyses were performed during each cell cycle up to quiescence. Cholesterol synthesis increased remarkably during the replicative cycles, decreasing only after cells reached confluence. In contrast, cholesteryl ester (CE) synthesis and content were high at 24?h after dilution and then decreased steeply in the successive cycles. Flow cytometry analysis of DiO-HDL, as well as radiolabeled HDL pulse, demonstrated a significant uptake of CE-HDL in 24?h. DiI-HDL uptake, lipid droplets (LDs) and SR-BI immunostaining and expression followed the same trend. Addition of HDL or PDGF partially restore the proliferation rate and significantly increase SR-BI and pAKT expression in serum-deprived cells. In conclusion, cell transition from G0 to G1/S requires CE-HDL uptake, leading to CE-HDL/SR-BI pathway activation and CEs increase into LDs. PMID:26640042

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

    PubMed Central

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

    2014-01-01

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

  4. Insulinomimetic Zn complex (Zn(opt)2) enhances insulin signaling pathway in 3T3-L1 adipocytes.

    PubMed

    Basuki, Wanny; Hiromura, Makoto; Sakurai, Hiromu

    2007-04-01

    Zinc (Zn) is an essential trace element with multiple regulatory functions, involving insulin synthesis, secretion, signaling and glucose transport. Since 2000, we have proposed that Zn complexes with different coordination environments exhibit high insulinomimetic and antidiabetic activities in type 2 diabetic animals. However, the molecular mechanism for the activities is still unsolved. The purpose of this study was to reveal the molecular mechanism of several types of Zn complexes in 3T3-L1 adipocytes, with respect to insulin signaling pathway. Obtained results shows that bis(1-oxy-2-pyridine-thiolato)Zn(II), Zn(opt)2, with S(2)O(2) coordination environment induced most strongly Akt/protein kinase B (Akt/PKB) phosphorylation, in which the optimal phosphorylation was achieved at a concentration of 25 microM, and this Zn(opt)2-induced Akt/PKB phosphorylation was inhibited by wortmannin at 100 nM. Further, the phosphorylation was maximal at 5-10 min stimulation, in agreement with the Zn uptake which was also maximal at 5-10 min stimulation. The Akt/PKB phosphorylation was in concentration- and time-dependent manners. Zn(opt)2 was also capable to translocate GLUT4 protein to the plasma membrane. We conclude that Zn(opt)2 was revealed to exhibit both insulinomimetic and antidiabetic activities by activating insulin signaling cascade through Akt/PKB phosphorylation, which in turn caused the GLUT4 translocation from the cytosol to the plasma membrane. PMID:17316811

  5. Cranberries (Oxycoccus quadripetalus) inhibit lipid metabolism and modulate leptin and adiponectin secretion in 3T3-L1 adipocytes.

    PubMed

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

    2015-10-15

    It has previously been shown that lyophilized cranberries (LCB) decreased lipid accumulation in 3T3-L1 cells and inhibited preadipocyte differentiation by down-regulation of the expression of key transcription factors (PPAR?, C/EBP?, SREBP1) of the adipogenesis pathway. To elucidate the molecular basis of anti-lipogenic activity of LCB, the expression of several genes involved in lipid metabolism, such as adipocyte fatty acid-binding protein (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), hormone sensitive lipase (HSL) and perilipin 1 (PLIN1), was examined in the present study. Additionally, the effects of LCB on adiponectin and leptin expression and protein secretion were also investigated. LCB reduced lipid accumulation during preadipocyte differentiation by down-regulation of the mRNA level of aP2, FAS, LPL, HSL and PLIN1. Moreover, LCB decreased leptin gene expression and increased adiponectin gene expression and protein secretion in a dose-dependent manner. Therefore cranberries could be considered as bioactive factors, which are effective in the inhibition of adipose tissue mass production. PMID:25952883

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

    SciTech Connect

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

    1986-01-01

    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.

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

    SciTech Connect

    Saito, Takeshi; Abe, Daigo; Sekiya, Keizo

    2009-03-06

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

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

    Auld, Corinth A.; Hopkins, Robin G.; Fernandes, Karishma M.; Morrison, Ron F. . E-mail: ron_morrison@uncg.edu

    2006-07-21

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

  10. 7 CFR 1280.107 - Feeder.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.107 Feeder. Feeder means any person who acquires ownership of lambs and feeds such lambs in the U.S. until they reach...

  11. 7 CFR 1280.107 - Feeder.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.107 Feeder. Feeder means any person who acquires ownership of lambs and feeds such lambs in the U.S. until they reach...

  12. 7 CFR 1280.107 - Feeder.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.107 Feeder. Feeder means any person who acquires ownership of lambs and feeds such lambs in the U.S. until they reach...

  13. 7 CFR 1280.107 - Feeder.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.107 Feeder. Feeder means any person who acquires ownership of lambs and feeds such lambs in the U.S. until they reach...

  14. 7 CFR 1280.107 - Feeder.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.107 Feeder. Feeder means any person who acquires ownership of lambs and feeds such lambs in the U.S. until they reach...

  15. TEXAS FEEDER SCHOOLS SUMMER/FALL 2014

    E-print Network

    Pillow, Jonathan

    TEXAS FEEDER SCHOOLS SUMMER/FALL 2014 September 23, 2014 HS Code HS Name HS City Total 2440010 Austin 35 1 of 17 2014-0149 #12;TEXAS FEEDER SCHOOLS SUMMER/FALL 2014 September 23, 2014 HS Code HS Name ACE ACADEMY Austin 2 2440069 LIBERAL ARTS & SCIENCE ACAD HS Austin 30 2440294 L C ANDERSON HIGH SCHOOL

  16. Structural Assessment of Small Bore Feeder Piping

    E-print Network

    Sun, Yu

    STRESSES ON THE INTE- RIOR SURFACE OF THE PIPE AT THE LOCALLY THINNED FLAW. FURTHER TESTINGBACKGROUND Structural Assessment of Small Bore Feeder Piping Kathryn Tang, Janos Mann, Skerdi. Supervisor: A. N. Sinclair CASE ONE CANDU REACTORS HAVE 380+ SMALL BORE FEEDER PIPES. THE PIPES

  17. Ginsenoside Rc promotes anti-adipogenic activity on 3T3-L1 adipocytes by down-regulating C/EBP? and PPAR?.

    PubMed

    Yang, Ji-Won; Kim, Sung Soo

    2015-01-01

    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

  18. ITER Magnet Feeder: Design, Manufacturing and Integration

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  19. Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into "osteocyte-like" cells.

    PubMed

    Alvares, Keith; Ren, Yinshi; Feng, Jian Q; Veis, Arthur

    2016-01-01

    P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, syncytium formation, and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP, a protein common to all vertebrate teeth, and crucial for their mineralization. Here, we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture, the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18-fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN), and ?-catenin decreased by 70%, 64%, and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an "osteocyte-like" phenotype, an important process in bone biology. The mechanisms involved are presently under study. J. Exp. Zool. (Mol. Dev. Evol.) 326B:38-46, 2016. © 2015 Wiley Periodicals, Inc. PMID:26581835

  20. Real Time Monitoring of Inhibition of Adipogenesis and Angiogenesis by (?)-Epigallocatechin-3-Gallate in 3T3-L1 Adipocytes and Human Umbilical Vein Endothelial Cells

    PubMed Central

    Tang, Wenjing; Song, Huanlei; Cai, Wei; Shen, Xiuhua

    2015-01-01

    Little is known about the effect of (?)-epigallocatechin-3-gallate (EGCG) on angiogenesis in adipocytes. We aimed to test the effect of EGCG on the expression of vascular endothelial growth factor (VEGF) in adipocytes. The levels of VEGF secretion, the expression of VEGF message ribonucleic acid (mRNA) and VEGF protein in 3T3-L1 cells were measured by enzyme linked immunosorbent assay (ELISA), real time polymerase chain reaction (PCR), and immunofluorescence staining, respectively. The xCELLigence real time cell analysis system was used to study the growth and differentiation of 3T3-L1 preadipocytes. A coculture system was used to test the effects of 3T3-L1 cells on proliferation of human umbilical vein endothelial cells (HUVECs). The conditioned media derived from 3T3-L1 cells treated with or without EGCG was used to culture the HUVECs for a tube formation assay. Peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer binding protein ? (C/EBP?), two transcription factors related to both adipogenesis and angiogenesis, were examined to explore the potential mechanism. We found that all the three measurements of VEGF expression in adipocytes (mRNA, protein and secretion in media) were reduced after EGCG treatment. The growth of HUVECs co-cultured with 3T3-L1 cells was significantly increased and the conditioned media from EGCG treated 3T3-L1 adipocytes inhibited tube formation in HUVECs. Both PPAR? and C/EBP? expression in adipocytes were decreased with EGCG treatment. In conclusion, findings from this study suggest that EGCG may inhibit angiogenesis by regulating VEGF expression and secretion in adipocytes. PMID:26516907

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

    SciTech Connect

    An, Lei; Pang, Yun-Wei; Gao, Hong-Mei; 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 ; Tao, Li; College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin 130118 ; Miao, Kai; Wu, Zhong-Hong; and others

    2012-11-23

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

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

    PubMed Central

    Shim, Dong Hwi

    2015-01-01

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

  3. Preferential role of intracellular Ca2+ stores in regulation of isometric force in NIH 3T3 fibroblast fibres

    PubMed Central

    Nobe, Koji; Nobe, Hiromi; Obara, Kazuo; Paul, Richard J

    2000-01-01

    Fibroblast contraction plays a major role in wound repair, but the regulatory mechanisms are not well known. We investigated the relations between isometric force and intracellular calcium concentration ([Ca2+]i) in fibroblast fibres. These fibres were made with mouse NIH 3T3 fibroblasts cultured with native collagen in a three-dimensional matrix. Calf serum (CS; 30 %) elicited a monotonic increase in force that attained a maximum within 15 min and could be sustained indefinitely. In contrast, [Ca2+]i increased to a peak at 3 min after CS stimulation, then returned to baseline levels by 10 min. Pretreatment with Ca2+-free medium or the Ca2+-channel antagonist nicardipine (10 ?M) blocked the CS-induced [Ca2+]i increase, but force was not affected. KCl (50 mM) stimulation on the other hand, elicited a prolonged increase in [Ca2+]i but did not increase force. Inhibition of the endoplasmic reticulum Ca2+ release with Ca2+-ATPase inhibitors cyclopiazonic acid (5 ?M) or thapsigargin (5 ?M) nearly abolished (< 20 % control) the increase in [Ca2+]i and force response to CS. Treatment with ryanodine (10 ?M) and caffeine (20 mM) had a similar effect. The phospholipase C inhibitor U73122 (3 ?M) reduced the CS-induced increases in [Ca2+]i and force by 70 and 40 %, respectively. We conclude that fibroblast isometric force is not coupled to Ca2+ arising from transmembrane influx but is correlated with the transient [Ca2+]i increase due to release from intracellular stores. Store-released Ca2+ may initiate activation pathways for fibroblast force development, but is not required for force maintenance. PMID:11118497

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

    PubMed

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

    2013-04-01

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

  5. An RFID Based Smart Feeder for Hummingbirds.

    PubMed

    Ibarra, Vicente; Araya-Salas, Marcelo; Tang, Yu-Ping; Park, Charlie; Hyde, Anthony; Wright, Timothy F; Tang, Wei

    2015-01-01

    We present an interdisciplinary effort to record feeding behaviors and control the diet of a hummingbird species (Phaethornis longirostris, the long-billed hermit or LBH) by developing a Radio Frequency Identification (RFID) based smart feeder. The system contains an RFID reader, a microcontroller, and a servo-controlled hummingbird feeder opener; the system is presented as a tool for studying the cognitive ability of the LBH species. When equipped with glass capsule RFID tags (which are mounted on the hummingbird), the smart feeder can provide specific diets for predetermined sets of hummingbirds at the discretion of biologists. This is done by reading the unique RFID tag on the hummingbirds and comparing the ID number with the pre-programmed ID numbers stored in the smart feeder. The smart feeder records the time and ID of each hummingbird visit. The system data is stored in a readily available SD card and is powered by two 9 V batteries. The detection range of the system is approximately 9-11 cm. Using this system, biologists can assign the wild hummingbirds to different experimental groups and monitor their diets to determine if they develop a preference to any of the available nectars. During field testing, the smart feeder system has demonstrated consistent detection (when compared to detections observed by video-recordings) of RFID tags on hummingbirds and provides pre-designed nectars varying water and sugar concentrations to target individuals. The smart feeder can be applied to other biological and environmental studies in the future. PMID:26694402

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2013-09-01

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

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

    PubMed Central

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

    2013-01-01

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

  9. Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts.

    PubMed

    Uboldi, Chiara; Urbán, Patricia; Gilliland, Douglas; Bajak, Edyta; Valsami-Jones, Eugenia; Ponti, Jessica; Rossi, François

    2016-03-01

    The wide use of titanium dioxide nanoparticles (TiO2 NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO2 NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO2 NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO2 NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO2 NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials. In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO2 NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size. PMID:26571344

  10. Soluble extract of soybean fermented with Aspergillus oryzae GB107 inhibits fat accumulation in cultured 3T3-L1 adipocytes

    PubMed Central

    So, Kyoung-Ha; Suzuki, Yasuki; Yonekura, Shinichi; Suzuki, Yutaka; Lee, Chan Ho; Kim, Sung Woo; Katoh, Kazuo

    2015-01-01

    BACKGROUND/OBJECTIVES This study was conducted to investigate the effects of fermented soybean (FS) extract on adipocyte differentiation and fat accumulation using cultured 3T3-L1 adipocytes. MATERIALS/METHODS 3T3-L1 adipocytes were treated with FS and nonfermented soybean (NFS) extract during differentiation for 10 days in vitro. Oil red O staining was performed and glycerol-3-phosphate dehydrogenase (GPDH) activity was measured for analysis of fat accumulation. Expressions of adipogenic genes were measured. RESULTS Soluble extract of soybean fermented with Aspergillus oryzae GB107 contained higher levels of low-molecular-weight protein than conventional soybean protein did. FS extract (50 µg/ml) inhibited adipocyte differentiation and fat accumulation during differentiation of 3T3-L1 preadipocytes for 10 days in vitro. Significantly lower GPDH activity was observed in differentiated adipocytes treated with the FS extract than those treated with NFS extract. Treatment with FS extract resulted in decreased expression levels of leptin, adiponectin, and adipogenin genes, which are associated with adipogenesis. CONCLUSIONS This report is the first to demonstrate that the water-soluble extract from FS inhibits fat accumulation and lipid storage in 3T3-L1 adipocytes. Thus, the soybean extract fermented with A. oryzae GB107 could be used to control lipid accumulation in adipocytes. PMID:26244085

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

    SciTech Connect

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

    2012-03-02

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

  12. [Research on construction of sheep lung adenomas virus pEGFP-C1/exJSRV-env and induction of malignant transformation in NIH3T3].

    PubMed

    Zhang, Yu-Fei; Liu, Yue; Wang, Zhuan-Jia; Sun, Xiao-Lin; Liu, Shu-Ying

    2014-05-01

    This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein. PMID:25118382

  13. Flavonoids from persimmon (Diospyros kaki) leaves (FPL) attenuate H2O2-induced apoptosis in MC3T3-E1 cells via the NF-?B pathway.

    PubMed

    Sun, Lijun; Zhang, Jianbao; Fang, Kun; Ding, Yan; Zhang, Liyu; Zhang, Yali

    2014-03-01

    The leaves of persimmon (Diospyros kaki L.) have long been used in Chinese medicine for the treatment of paralysis, frostbite, burns, and to stop bleeding. Flavonoids of persimmon leaves (FPL) are known for their antioxidant activity in murine osteoblast MC3T3-E1 cells, but their mechanisms in osteoblast cells injured by oxidative stress are unknown. In this study, the effects of FPL on oxidative damage were investigated by addressing their potential therapeutic or toxic effects on H2O2-stimulated MC3T3-E1 cells. MC3T3-E1 cells were pretreated with FPL (1.25, 2.5 and 5 ?g mL(-1)) for 24 h and were then exposed to 250 ?M H2O2 for an additional 6 h. FPL pre-incubated with MC3T3-E1 cells did not present any cytotoxicity, instead they increased cell viability and ??m in a dose-dependent manner when challenged with H2O2. Treatment with this pro-incubated FPL also significantly suppressed the production of MDA and NO and the activity of iNOS. The mRNA expression of iNOS, COX-2, Bax, Bcl-2, and caspase-3 and the protein expression of NF-?B/p65 showed that FPL significantly inhibited apoptosis in H2O2-stimulated MC3T3-E1 cells. These results suggest that the molecular mechanism of FPL in anti-apoptosis was associated with the suppression of the translocation of NF-?B/p65 into the nucleus. The protective effect of FPL could provide a promising approach for the treatment of osteoporosis. PMID:24488014

  14. Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction

    PubMed Central

    Kunoh, Tatsuki; Wang, Weixiang; Kobayashi, Hiroaki; Matsuzaki, Daisuke; Togo, Yuki; Tokuyama, Masahiro; Hosoi, Miho; Koseki, Koichi; Wada, Shu-ichi; Nagai, Nobuo; Nakamura, Toshinobu; Nomura, Shintaro; Hasegawa, Makoto; Sasaki, Ryuzo; Mizukami, Tamio

    2015-01-01

    Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell—cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy. PMID:26284361

  15. Manually Operated Welding Wire Feeder

    NASA Technical Reports Server (NTRS)

    Rybicki, Daniel J. (Inventor)

    2001-01-01

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

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

    SciTech Connect

    St?pnik, Maciej; Arkusz, Joanna; Smok-Pieni??ek, Anna; Bratek-Skicki, Anna; Salvati, Anna; Lynch, Iseult; Dawson, Kenneth A.; Gromadzi?ska, Jolanta; De Jong, Wim H.; Rydzy?ski, Konrad

    2012-08-15

    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.

  17. Phase balancing optimization for radial feeder systems

    NASA Astrophysics Data System (ADS)

    Zhu, Jinxiang

    In the power distribution systems, unbalanced feeder causes deteriorating power quality and increases investment and operating costs. There are two approaches to balance a feeder system. One approach is feeder reconfiguration, the other is phase swapping. Feeder reconfiguration is an on-line operation of sectionalizing switches. However, feeder reconfiguration is difficult to find the phase balancing solution for a unbalanced feeder system. On the other hand, phase swapping is a direct and effective approach for phase balancing. Phase swapping is an off-line operation of re-tapping loads/laterals to the phase lines during maintenance and restoration periods. Unfortunately, phase balancing problem has not received its deserved importance. Deregulation in power industry has arisen phase balancing issue because it can improve power quality, improve service reliability, and reduce total costs. Thus, phase balancing can enhance utility competitive capability. This research provides utilities the optimization tools to maximize the benefits of phase balancing and minimize the costs. This dissertation proposed several mathematical formulations, including Mixed-integer programming (MIP), Dynamic programming (DP), Simulated annealing (SA), and Fuzzy logic (FL), to perform phase swapping to balance phase in a radial feeder. Due to the discrete nature of phase swapping, a MIP model is proposed to find the optimal phase swapping scheme for small-sized feeder. Candidate sets and monitored branches are then introduced to solve the large-scale feeder systems. Possibilistic integer programming (PIP) is proposed to incorporate load uncertainties in phase swapping problems. The tests show that the load uncertainties may change the optimal phase swapping. DP computation is significant reduced by introducing the tighter upper/lower bounds. The upper bound can be quickly obtained by assignment algorithm. Even though SA is a time-consuming heuristic method, it can provide better solution than other heuristic methods. FL can efficiently identify the most effective phase swapping for large-scale feeder systems. The results can be easily fine-tuned to match the expectation of decision-maker. Finally, the computation efforts and optimality are compared between the proposed methods. Several examples are used to illustrate the effectiveness of the proposed methods.

  18. Substance P enhances the activation of AMPK and cellular lipid accumulation in 3T3?L1 cells in response to high levels of glucose.

    PubMed

    Dubon, Maria Jose; Byeon, Yeji; Park, Ki-Sook

    2015-12-01

    The rescue of glucose tolerance and insulin?sensitivity in peripheral tissues, including adipose tissue, is essential in therapeutic strategies for diabetes. The present study demonstrated that substance P (SP) increases the accumulation of lipids in 3T3?L1 cells during their differentiation into adipocytes in response to a high concentration of glucose. SP reciprocally regulated the activities of AMP?activated protein kinase (AMPK) and Akt: SP enhanced the activation of AMPK, although the activity of Akt was downregulated. Notably, SP induced an increase in the expression level of glucose transporter 4 in the 3T3?L1 adipocytes. Therefore, it is possible that SP leads to an increase in glucose uptake and the accumulation of lipids in adipocytes, and may contribute towards the rescue of insulin?sensitivity in diabetes. PMID:26499365

  19. Inhibition of ASCT2 is essential in all-trans retinoic acid-induced reduction of adipogenesis in 3T3-L1 cells

    PubMed Central

    Takahashi, Katsuhiko; Uchida, Natsumi; Kitanaka, Chisato; Sagara, Chiaki; Imai, Masahiko; Takahashi, Noriko

    2015-01-01

    Vitamin A has preventive effects on obesity. All-trans retinoic acid (ATRA), the active form of vitamin A, inhibits lipid accumulation in 3T3-L1 cells in an experimental adipogenesis model. We found that ATRA suppressed up-regulation of the amino acid transporter, Asct2, in adipogenerating 3T3-L1 cells. We observed that Asct2 was up-regulated at 1 day after adipogenesis stimuli. The Asct2 inhibitor l-?-glutamyl-p-nitroanilide (GPNA) decreased lipid accumulation. Glutamine-free conditions also suppressed adipogenesis. Suppression of adipogenesis by ATRA may be through Asct2 reduction. These results indicate that Asct2 could be a target for obesity prevention and treatment. PMID:26236584

  20. Insulin resistance by TNF-? is associated with mitochondrial dysfunction in 3T3-L1 adipocytes and is ameliorated by punicic acid, a PPAR? agonist.

    PubMed

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

    2015-09-15

    Punicic acid (PA), a poly unsaturated fatty acid found abundantly in pomegranate seed oil is reported to have PPAR? agonist property. TNF-? mediated insulin resistance plays an important role in the pathogenesis of diabetes and is associated with severe mitochondrial impairment. In this study, PA was evaluated for its ability to ameliorate TNF-? induced mitochondrial dysfunctions in 3T3-L1 adipocytes. For this, we examined the alterations in mitochondrial energetics, biogenesis, transmembrane potential and dynamics in TNF-? induced insulin resistant model of 3T3-L1 adipocytes. PA improved glucose uptake, ROS accumulation, mitochondrial biogenesis and energetics in TNF-? treated cells. In addition, treatment with PA was found to ameliorate TNF-? induced alterations in proteins associated with mitochondrial dynamics like FIS1 and OPA1. These findings suggest that PA can be considered as an active lead for the management of insulin resistance and associated mitochondrial dysfunctions. PMID:26116231

  1. Pentacyclic triterpenoids from spikes of Prunella vulgaris L. inhibit glycogen phosphorylase and improve insulin sensitivity in 3T3-L1 adipocytes.

    PubMed

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

    2015-01-01

    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. PMID:25278372

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

    PubMed

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

    2014-12-01

    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

  3. The micosporine-like amino acids-rich aqueous methanol extract of laver (Porphyra yezoensis) inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes

    PubMed Central

    Kim, Hyunhee; Lee, Yunjung; Han, Taejun

    2015-01-01

    BACKGROUND/OBJECTIVES Increased mass of adipose tissue in obese persons is caused by excessive adipogenesis, which is elaborately controlled by an array of transcription factors. Inhibition of adipogenesis by diverse plant-derived substances has been explored. The aim of the current study was to examine the effects of the aqueous methanol extract of laver (Porphyra yezoensis) on adipogenesis and apoptosis in 3T3-L1 adipocytes and to investigate the mechanism underlying the effect of the laver extract. MATERIALS/METHODS 3T3-L1 cells were treated with various concentrations of laver extract in differentiation medium. Lipid accumulation, expression of adipogenic proteins, including CCAAT enhancer-binding protein ?, peroxisome proliferator-activated receptor ?, fatty acid binding protein 4, and fatty acid synthase, cell viability, apoptosis, and the total content and the ratio of reduced to oxidized forms of glutathione (GSH/GSSG) were analyzed. RESULTS Treatment with laver extract resulted in a significant decrease in lipid accumulation in 3T3-L1 adipocytes, which showed correlation with a reduction in expression of adipogenic proteins. Treatment with laver extract also resulted in a decrease in the viability of preadipocytes and an increase in the apoptosis of mature adipocytes. Treatment with laver extract led to exacerbated depletion of cellular glutathione and abolished the transient increase in GSH/GSSG ratio during adipogenesis in 3T3-L1 adipocytes. CONCLUSION Results of our study demonstrated that treatment with the laver extract caused inhibition of adipogenesis, a decrease in proliferation of preadipocytes, and an increase in the apoptosis of mature adipocytes. It appears that these effects were caused by increasing oxidative stress, as demonstrated by the depletion and oxidation of the cellular glutathione pool in the extract-treated adipocytes. Our results suggest that a prooxidant role of laver extract is associated with its antiadipogenic and proapoptotic effects. PMID:26634047

  4. Osteogenic differentiation of MC3T3-E1 cells on poly(l-lactide)/Fe3O4 nanofibers with static magnetic field exposure.

    PubMed

    Cai, Qing; Shi, Yuzhou; Shan, Dingying; Jia, Wenkai; Duan, Shun; Deng, Xuliang; Yang, Xiaoping

    2015-10-01

    Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1mT-1T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(l-lactide) (PLLA) and ferromagnetic Fe3O4 nanoparticles (NPs). The PLLA/Fe3O4 composite nanofibers demonstrated homogeneous dispersion of Fe3O4 NPs, and their magnetism depended on the contents of Fe3O4 NPs. SMF of 100mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe3O4 composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe3O4 NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering. PMID:26117751

  5. Protein Kinase B/AKT 1 Plays a Pivotal Role in Insulin-like Growth Factor-1 Receptor Signaling Induced 3T3-L1

    E-print Network

    Tian, Weidong

    Protein Kinase B/AKT 1 Plays a Pivotal Role in Insulin-like Growth Factor-1 Receptor Signaling upstream of protein kinase B/Akt 1, such as IGF-1 receptor and insulin receptor substrate-1, were normally-protein kinase B/Akt, in 3T3-L1 cells can be activated by IGF-1 receptor signaling. The function of the MAP

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

    PubMed

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

    2001-09-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2009-06-01

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

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

    PubMed

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

    2015-02-01

    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

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

    PubMed Central

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

    2014-01-01

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

  11. Effects of intermedin on proliferation, apoptosis and the expression of OPG/RANKL/M-CSF in the MC3T3-E1 osteoblast cell line.

    PubMed

    Ren, Hongfei; Ren, Hongyu; Li, Xue; Yu, Dongdong; Mu, Shuai; Chen, Zhiguang; Fu, Qin

    2015-11-01

    Bone remodeling is a vital physiological process of healthy bone tissue in humans. It is characterized by the formation of bone by osteoblasts and its resorption by osteoclasts, and the bone resorbed by osteoclasts is replaced through the differentiation and activity of osteoblasts. Imbalances in this vital process lead to pathological conditions, including osteoporosis. Intermedin (IMD) as a newly discovered peptide in the calcitonin (CT) family of peptides, which shares similar functions with CT, calcitonin gene?related peptide and amylin in bone resorption. However, the mechanism underlying its effect remains to be elucidated. This was investigated in the present study using the osteoblastic MC3T3?E1 cell line, which was treated with different doses of IMD (0, 1, 10 and 100 nM). Cell proliferation, apoptosis and the expression of receptor activator of NF??B ligand (RANKL), osteoprotegerin (OPG) and macrophage colony?stimulating factor (M?CSF) were measured following treatment using multiple detection techniques, including an MTT assay, flow cytometry, reverse transcription?quantitative polymerase chain reaction and western blot analysis. The resulting data demonstrated that IMD significantly inhibited the apoptosis of MC3T3?E1 cells induced by serum?free culture and dexamethasone, however, no significant effects on MC3T3?E1 cell proliferation were observed. IMD had additional functions on the MC3T3?E1 cells, including inhibition of the expression of RANKL and M?CSF, and promotion of the expression of OPG. Previous studies have also demonstrated that RANKL and M?CSF are two vital factor produced by osteoblasts to promote the maturation and differentiation of osteoclasts, and it has been reported that IMD can inhibit the osteoclast formation stimulated by RANKL and M?CSF. Together with these findings, the present study concluded that IMD reduces bone resorption by inhibiting osteoblast apoptosis, decreasing the RANKL/OPG ratio and the expression of M-CSF, and inhibiting osteoclast maturation and differentiation. PMID:26398911

  12. Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.

    PubMed Central

    Goruppi, S; Ruaro, E; Varnum, B; Schneider, C

    1997-01-01

    Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival. PMID:9234702

  13. Effect of surface roughness on the initial response of MC3T3-E1 cells cultured on polished titanium alloy.

    PubMed

    Wu, Chunya; Chen, Mingjun; Zheng, Ting; Yang, Xiaonan

    2015-08-17

    Surface roughness has been considered as an important influencing factor for cell response. The aim of this study was to find out whether MC3T3-E1 cells, a mouse osteoblast-like cell line, can sense the amplitudes of surface topography of titanium alloy (Ti6Al4V), and if surface-dependent cell morphology would be presented on the substrata with varied roughness. A series of polished samples (Ra: 0.30~1.80 ?m) were prepared to produce macroscopically parallel grooves using different grades of silicon carbide sandpaper (#100, #320, #600, #1000 and #2000). The experimental results indicated that the behavior and morphology of cells largely depended on the substrata where they were cultured. More efficient proliferation of MC3T3-E1 cells was shown on the surfaces with Ra of 0.50~1.00 ?m, with respect to either the rougher or the smoother specimens. Furthermore, MC3T3-E1 cells seeded on the Ti6Al4V surfaces within this narrow range responded to the increasing surface roughness with increased proliferation. Contact guidance of cells could be observed on the rougher specimens (Ra: 0.80~1.00 ?m), whereas more random orientations were exhibited for the adsorbed cells on the smoother surfaces (Ra: 0.50~0.60 ?m). PMID:26405920

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed Central

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

    2014-01-01

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

  16. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line

    PubMed Central

    Czyrek, Aleksandra; Basinska, Katarzyna; Trynda, Justyna; Skaradzi?ska, Aneta; Siudzi?ska, Anna; Marycz, Krzysztof

    2015-01-01

    Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1?mM, 5?mM, and 10?mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10?mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5?mM and increased after 10?mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure. PMID:26064951

  17. A new lectin in red kidney beans called PvFRIL stimulates proliferation of NIH 3T3 cells expressing the Flt3 receptor.

    PubMed

    Moore, J G; Fuchs, C A; Hata, Y S; Hicklin, D J; Colucci, G; Chrispeels, M J; Feldman, M

    2000-07-26

    A new legume lectin has been identified by its ability to specifically stimulate proliferation of NIH 3T3 fibroblasts expressing the Flt3 tyrosine kinase receptor. The lectin was isolated from conditioned medium harvested from human peripheral blood mononuclear cells activated to secrete cytokines by a crude red kidney bean extract containing phytohemagglutinin (PHA). Untransfected 3T3 cells and 3T3 cells transfected with the related Fms tyrosine kinase receptor do not respond to this lectin, which we called PvFRIL (Phaseolus vulgaris Flt3 receptor-interacting lectin). When tested on cord blood mononuclear cells enriched for Flt3-expressing progenitors, purified PvFRIL fractions maintained a small population of cells that continued to express CD34 after 2 weeks in suspension cultures containing IL3. These cultures did not show the effects of IL3's strong induction of proliferation and differentiation (high cell number and exhausted medium); instead, low cell number at the end of the culture period resulted in persistence of cells in the context of cell death. These observations led to the hypothesis that PvFRIL acts in a dominant manner to preserve progenitor viability and prevent proliferation and differentiation. PMID:10913819

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

    PubMed

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

    2012-11-01

    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

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

    PubMed Central

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

    2014-01-01

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

  20. Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation.

    PubMed

    Gil, J; Higgins, T; Rozengurt, E

    1991-05-01

    Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis. PMID:1709171

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

    SciTech Connect

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka; Kawada, Teruo

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  2. Inhibition of O-GlcNAcase Using a Potent and Cell-Permeable Inhibitor Does Not Induce Insulin Resistance in 3T3-L1 Adipocytes

    PubMed Central

    Macauley, Matthew S.; He, Yuan; Gloster, Tracey M.; Stubbs, Keith A.; Davies, Gideon J.; Vocadlo, David J.

    2010-01-01

    Summary To probe increased O-GlcNAc levels as an independent mechanism governing insulin resistance in 3T3-L1 adipocytes, a new class of O-GlcNAcase (OGA) inhibitor was studied. 6-Acetamido-6-deoxy-castanospermine (6-Ac-Cas) is a potent inhibitor of OGA. The structure of 6-Ac-Cas bound in the active site of an OGA homolog reveals structural features contributing to its potency. Treatment of 3T3-L1 adipocytes with 6-Ac-Cas increases O-GlcNAc levels in a dose-dependent manner. These increases in O-GlcNAc levels do not induce insulin resistance functionally, measured using a 2-deoxyglucose (2-DOG) uptake assay, or at the molecular level, determined by evaluating levels of phosphorylated IRS-1 and Akt. These results, and others described, provide a structural blueprint for improved inhibitors and collectively suggest that increased O-GlcNAc levels, brought about by inhibition of OGA, does not by itself cause insulin resistance in 3T3-L1 adipocytes. PMID:20851343

  3. "The good into the pot, the bad into the crop!"--a new technology to free stem cells from feeder cells.

    PubMed

    Schneider, Annette; Spitkovsky, Dimitry; Riess, Peter; Molcanyi, Marek; Kamisetti, Naidu; Maegele, Marc; Hescheler, Jürgen; Schaefer, Ute

    2008-01-01

    A variety of embryonic and adult stem cell lines require an initial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines. PMID:19023443

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

    NASA Technical Reports Server (NTRS)

    Stelian, J.

    1977-01-01

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

  5. 46 CFR 113.25-8 - Distribution of general emergency alarm system feeders and branch circuits.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... (a) Each system must have a feeder distribution panel to divide the system into the necessary...feeder is necessary; then a branch circuit distribution panel or feeder distribution panel must be used. (b) The feeder...

  6. 46 CFR 113.25-8 - Distribution of general emergency alarm system feeders and branch circuits.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... (a) Each system must have a feeder distribution panel to divide the system into the necessary...feeder is necessary; then a branch circuit distribution panel or feeder distribution panel must be used. (b) The feeder...

  7. 46 CFR 113.25-8 - Distribution of general emergency alarm system feeders and branch circuits.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... (a) Each system must have a feeder distribution panel to divide the system into the necessary...feeder is necessary; then a branch circuit distribution panel or feeder distribution panel must be used. (b) The feeder...

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

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... (a) Each system must have a feeder distribution panel to divide the system into the necessary...feeder is necessary; then a branch circuit distribution panel or feeder distribution panel must be used. (b) The feeder...

  9. 46 CFR 113.25-8 - Distribution of general emergency alarm system feeders and branch circuits.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... (a) Each system must have a feeder distribution panel to divide the system into the necessary...feeder is necessary; then a branch circuit distribution panel or feeder distribution panel must be used. (b) The feeder...

  10. A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test.

    PubMed

    Spielmann, H; Balls, M; Dupuis, J; Pape, W J; de Silva, O; Holzhütter, H G; Gerberick, F; Liebsch, M; Lovell, W W; Pfannenbecker, U

    1998-01-01

    In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1?/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100?/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1?g/ml and 10?g/ml, and that false positive results can be obtained at concentrations greater than 100?g/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100?g/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account. PMID:26042493

  11. Phenyllactic Acid from Lactobacillus plantarum PromotesAdipogenic Activity in 3T3-L1 Adipocyte via Up-Regulationof PPAR-?2.

    PubMed

    Ilavenil, Soundharrajan; Kim, Da Hye; Valan Arasu, Mariadhas; Srigopalram, Srisesharam; Sivanesan, Ravikumar; Choi, Ki Choon

    2015-01-01

    Synthetic drugs are commonly used to cure various human ailments at present. However, the uses of synthetic drugs are strictly regulated because of their adverse effects. Thus, naturally occurring molecules may be more suitable for curing disease without unfavorable effects. Therefore, we investigated phenyllactic acid (PLA) from Lactobacillus plantarum with respect to its effects on adipogenic genes and their protein expression in 3T3-L1 pre-adipocytes by qPCR and western blot techniques. PLA enhanced differentiation and lipid accumulation in 3T3-L1 cells at the concentrations of 25, 50, and 100 ?M. Maximum differentiation and lipid accumulation were observed at a concentration of 100 ?M of PLA, as compared with control adipocytes (p < 0.05). The mRNA and protein expression of PPAR-?2, C/EBP??, adiponectin, fatty acid synthase (FAS), and SREBP-1 were increased by PLA treatment as compared with control adipocytes (p < 0.05). PLA stimulates PPAR-? mRNA expression in a concentration dependent manner, but this expression was lesser than agonist (2.83 ± 0.014 fold) of PPAR-?2. Moreover, PLA supplementation enhances glucose uptake in 3T3-L1 pre-adipocytes (11.81 ± 0.17 mM) compared to control adipocytes, but this glucose uptake was lesser than that induced by troglitazone (13.75 ± 0.95 mM) and insulin treatment (15.49 ± 0.20 mM). Hence, we conclude that PLA treatment enhances adipocyte differentiation and glucose uptake via activation of PPAR-?2, and PLA may thus be the potential candidate for preventing Type 2 Diabetes Mellitus (T2DM). PMID:26305241

  12. N-Oleoyl glycine, a lipoamino acid, stimulates adipogenesis associated with activation of CB1 receptor and Akt signaling pathway in 3T3-L1 adipocyte.

    PubMed

    Wang, Songbo; Xu, Qi; Shu, Gang; Wang, Lina; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Jiang, Qingyan; Zhu, Xiaotong

    2015-10-23

    Adipose tissue plays a vital role in the development of obesity and related diseases. The aim of the present study was to investigate the effects of N-Oleoyl glycine (OLGly), a lipoamino acid, on 3T3-L1 adipogenesis and to explore the likely mechanisms underlying this process. Lipid accumulation were evaluated using Oil Red O staining and triglyceride content assay. The mRNA expressions of cannabinoid receptors and the protein expressions of adipogenic genes and intracellular signaling pathway were determined by real-time quantitative PCR and western blot, respectively. The results indicated that OLGly itself, but not its degradation products, stimulated lipid accumulation and significantly increased adipogenic genes (PPAR? and aP2), in a dose- and time-dependent manner. Additionally, OLGly markedly increased the mRNA expression of CB1 receptor (CB1R) and the inhibition of CB1R by its antagonist SR141716 abolished the promotive effects of OLGly on lipid accumulation and the protein expression of PPAR? and aP2. Furthermore, OLGly increased the ratio of p-Akt/Akt and p-FoxO1/FoxO1, which could be reversed by SR141716. Moreover, OLGly-induced enhancement of adipogenesis, activation of insulin-mediated Akt signaling pathway and inactivation of FoxO1 were effectively blocked by Wortmannin, a specific PI3K/Akt inhibitor, indicating the essential role of Akt signaling pathway in the process of OLGly-stimulated 3T3-L1 adipogenesis. In conclusion, OLGly, a lipoamino acid, was able to promote 3T3-L1 adipogenesis through the activation of CB1 receptor and the enhancement of insulin-mediated Akt signaling pathway. These findings suggested the potential role of OLGly in increasing insulin sensitivity and suppressing obesity and diabetes. PMID:26365347

  13. Anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidea and var. angustifolia on 3T3-L1 adipocytes*

    PubMed Central

    Woon, Shiau Mei; Seng, Yew Wei; Ling, Anna Pick Kiong; Chye, Soi Moi; Koh, Rhun Yian

    2014-01-01

    Objective: This study examined the anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidia and var. angustifolia, a natural slimming aid, on 3T3-L1 adipocytes. Methods: Methanol and water extracts of leaves of the F. deltoidea varieties were analyzed to determine their total flavonoid content (TFC) and total phenolic content (TPC), respectively. The study was initiated by determining the maximum non-toxic dose (MNTD) of the methanol and water extracts for 3T3-L1 preadipocytes. Possible anti-adipogenic effects were then examined by treating 2-d post confluent 3T3-L1 preadipocytes with either methanol extract or water extract at MNTD and half MNTD (½MNTD), after which the preadipocytces were induced to form mature adipocytes. Visualisation and quantification of lipid content in mature adipocytes were carried out through oil red O staining and measurement of optical density (OD) at 520 nm, respectively. Results: The TFCs of the methanol extracts were 1.36 and 1.97 g quercetin equivalents (QE)/100 g dry weight (DW), while the TPCs of the water extracts were 5.61 and 2.73 g gallic acid equivalents (GAE)/100 g DW for var. deltoidea and var. angustilofia, respectively. The MNTDs determined for methanol and water extracts were (300.0±28.3) and (225.0±21.2) ?g/ml, respectively, for var. deltoidea, while much lower MNTDs [(60.0±2.0) ?g/ml for methanol extracts and (8.0±1.0) ?g/ml for water extracts] were recorded for var. angustifolia. Studies revealed that the methanol extracts of both varieties and the water extracts of var. angustifolia at either MNTD or ½MNTD significantly inhibited the maturation of preadipocytes. Conclusions: The inhibition of the formation of mature adipocytes indicated that leaf extracts of F. deltoidea could have potential anti-obesity effects. PMID:24599694

  14. Sustained release of Semaphorin 3A from ?-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

    NASA Astrophysics Data System (ADS)

    Wang, Jin-Ning; Pi, Bin; Wang, Peng; Li, Xue-Feng; Yang, Hui-Lin; Zhu, Xue-Song

    2015-09-01

    The reinforcement of calcium phosphate materials with silk fibroin (SF) has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/?-tricalcium phosphate composite (Sema3A CMs/SF/?-TCP) in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/?-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction (RT-qPCR) and activity of alkaline phosphatase (ALP) were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/?-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/?-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/?-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/?-TCP and SF/?-TCP composites. In conclusion, Sema3A CMs/SF/?-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.

  15. The interaction of /sup 125/I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method

    SciTech Connect

    Fan, J.Y.; Carpentier, J.L.; Van Obberghen, E.; Blackett, N.M.; Grunfeld, C.; Gorden, P.; Orci, L.

    1983-07-01

    The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when /sup 125/I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, /sup 125/I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.

  16. A commercial formulation of glyphosate inhibits proliferation and differentiation to adipocytes and induces apoptosis in 3T3-L1 fibroblasts.

    PubMed

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

    2012-09-01

    Glyphosate-based herbicides are extensively used for weed control all over the world. Therefore, it is important to investigate the putative toxic effects of these formulations which include not only glyphosate itself but also surfactants that may also be toxic. 3T3-L1 fibroblasts are a useful tool to study adipocyte differentiation, this cell line can be induced to differentiate by addition of a differentiation mixture containing insulin, dexamethasone and 3-isobutyl-1-methylxanthine. We used this cell line to investigate the effect of a commercial formulation of glyphosate (GF) on proliferation, survival and differentiation. It was found that treatment of exponentially growing cells with GF for 48h inhibited proliferation in a dose-dependent manner. In addition, treatment with GF dilution 1:2000 during 24 or 48h inhibited proliferation and increased cell death, as evaluated by trypan blue-exclusion, in a time-dependent manner. We showed that treatment of 3T3-L1 fibroblasts with GF increased caspase-3 like activity and annexin-V positive cells as evaluated by flow cytometric analysis, which are both indicative of induction of apoptosis. It was also found that after the removal of GF, remaining cells were able to restore proliferation. On the other hand, GF treatment severely inhibited the differentiation of 3T3-L1 fibroblasts to adipocytes. According to our results, a glyphosate-based herbicide inhibits proliferation and differentiation in this mammalian cell line and induces apoptosis suggesting GF-mediated cellular damage. Thus, GF is a potential risk factor for human health and the environment. PMID:22546541

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

    SciTech Connect

    Kusunoki, Chisato; Yang, Liu; Yoshizaki, Takeshi; Nakagawa, Fumiyuki; Ishikado, Atsushi; Kondo, Motoyuki; Morino, Katsutaro; Sekine, Osamu; Ugi, Satoshi; Nishio, Yoshihiko; Kashiwagi, Atsunori; Maegawa, Hiroshi

    2013-01-04

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

  18. Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

    PubMed Central

    Kim, Hyun-Jung; Kim, Chang-Hyun; Lee, Do-Hyun; Han, Min-Woo; Kim, Mi-Young; Ju, Jae-Hyun

    2011-01-01

    Eotaxin is an important inflammatory chemokine in eosinophil chemotaxis and activation and, thus, is implicated in asthma. Recently, obesity was associated with an increased prevalence of asthma, but the relationship between obesity and eotaxin expression has only been partially understood in obese mice and human studies. Therefore, we studied the expression patterns of eotaxin in 3T3-L1 preadipocytes/adipocytes to determine whether eotaxin levels are influenced by body weight gain and/or reduction in diet-induced obese mice. First, we investigated eotaxin expression during differentiation in 3T3-L1 adipocytes. Then, we treated 3T3-L1 preadipocytes/adipocytes with tumor necrosis factor-alpha (TNF-?), eotaxin, interleukin (IL)-4, IL-5, or leptin. To examine the effects of weight loss in high-fat diet induced obese mice, we fed C57BL/6 mice a high-fat diet or a normal diet for 26 weeks. Then, half of the high-fat diet group were fed a normal diet until 30 weeks to reduce weight. Epididymal adipose tissue, visceral adipose tissue, serum, and bronchoalveolar fluid of mice were examined for eotaxin expression. The results showed that eotaxin expression levels increased with adipocyte differentiation and that more eotaxin was expressed when the cells were stimulated with TNF-?, eotaxin, IL-4, IL-5, or leptin. An in vivo study showed that eotaxin levels were reduced in visceral adipose tissues when high-fat diet fed mice underwent weight loss. Taken together, these results indicate a close relationship between eotaxin expression and obesity as well as weight loss, thus, they indirectly show a relation to asthma. PMID:21487491

  19. Substance P Activates the Wnt Signal Transduction Pathway and Enhances the Differentiation of Mouse Preosteoblastic MC3T3-E1 Cells

    PubMed Central

    Mei, Gang; Zou, Zhenlv; Fu, Su; Xia, Liheng; Zhou, Jian; Zhang, Yongtao; Tuo, Yonghua; Wang, Zhao; Jin, Dan

    2014-01-01

    Recent experiments have explored the impact of Wnt/?-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10?10 to 10?8 M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/?-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/?-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on ?-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10?9 to 10?8 M) significantly up-regulated the expressions of osteoblastic genes. SP (10?8 M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and ?-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10?8 M) promoted the transfer of ?-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/?-catenin signaling pathway. PMID:24733069

  20. Harmonic considerations for electrical distribution feeders

    NASA Astrophysics Data System (ADS)

    1988-03-01

    Harmonics on the electric power distribution system can cause motor overheating, capacitor failures,watt-hour meter error, and relay malfunctions. The degree of problems caused by harmonics is greatly dependent on the characteristics of the distribution feeder, which can absorb a considerable percentage of its capacity in harmonic currents without ill effects. However, power factor correction capacitors can cause resonances near harmonic voltages that can result in intolerable distortion. Both motor loads and resistive loads can decrease the effect of resonance significantly. This report describes useful techniques to analyze, suppress, and measure harmonics on distribution feeders. Applicable areas for manual analysis and computer analysis are explained. The basic formulae are presented as well as sophisticated computer methods. Emphasis is placed on the fundamental principle. Models of harmonic-producing devices are presented and their limitations discussed. Most distribution feeder harmonics analyses can be performed using simple current source models. Filtering of specific loads and general, dispersed load is discussed. The fundamental principle in filtering distribution feeders is to shorten the harmonic current path. 3-dimensional plots enhance the understanding of the filtering action. Equipment and procedures for making measurements are described.

  1. Abingdon Feeder Cattle Association 234 West Valley Street, Suite B

    E-print Network

    Liskiewicz, Maciej

    Abingdon Feeder Cattle Association 234 West Valley Street, Suite B Abingdon, VA 24210 "Moving Commission, the Abingdon Feeder Cattle Association, and Southwest Virginia Beef Producers. #12;2 BEEF BUILDER improvements to improve production, management and marketing of feeder cattle in tobacco dependent communities

  2. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Feeder circuits. 58.25-65 Section 58.25-65 Shipping... AND RELATED SYSTEMS Steering Gear § 58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear power units must have at least two feeder circuits, which must be...

  3. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Feeder circuits. 58.25-65 Section 58.25-65 Shipping... AND RELATED SYSTEMS Steering Gear § 58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear power units must have at least two feeder circuits, which must be...

  4. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Feeder circuits. 58.25-65 Section 58.25-65 Shipping... AND RELATED SYSTEMS Steering Gear § 58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear power units must have at least two feeder circuits, which must be...

  5. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Feeder circuits. 58.25-65 Section 58.25-65 Shipping... AND RELATED SYSTEMS Steering Gear § 58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear power units must have at least two feeder circuits, which must be...

  6. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Feeder circuits. 58.25-65 Section 58.25-65 Shipping... AND RELATED SYSTEMS Steering Gear § 58.25-65 Feeder circuits. (a) Each vessel with one or more electric-driven steering-gear power units must have at least two feeder circuits, which must be...

  7. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Fruit or syrup feeders. 58.625 Section 58.625 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards....625 Fruit or syrup feeders. Fruit or syrup feeders inject flavoring material into the...

  8. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 3 2011-01-01 2011-01-01 false Fruit or syrup feeders. 58.625 Section 58.625 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards....625 Fruit or syrup feeders. Fruit or syrup feeders inject flavoring material into the...

  9. Arctiin inhibits adipogenesis in 3T3-L1 cells and decreases adiposity and body weight in mice fed a high-fat diet

    PubMed Central

    Min, Byulchorong; Lee, Heejin; Song, Ji Hye; Han, Myung Joo

    2014-01-01

    BACKGROUND/OBJECTIVES The purpose of this study was to examine the effects and associated mechanisms of arctiin, a lignan compound found in burdock, on adipogenesis in 3T3-L1 cells. Also, the effects of arctiin supplementation in obese mice fed a high-fat diet on adiposity were examined. MATERIALS/METHODS 3T3-L1 cells were treated with arctiin (12.5 to 100 µM) during differentiation for 8 days. The accumulation of lipid droplets was determined by Oil Red O staining and intracellular triglyceride contents. The expressions of genes related to adipogenesis were measured by real-time RT-PCR and Western blot analyses. For in vivo study, C57BL/6J mice were first fed either a control diet (CON) or high-fat diet (HF) to induce obesity, and then fed CON, HF, or HF with 500 mg/kg BW arctiin (HF + AC) for four weeks. RESULTS Arctiin treatment to 3T3-L1 pre-adipocytes markedly decreased adipogenesis in a dose-dependent manner. The arctiin treatment significantly decreased the protein levels of the key adipogenic regulators PPAR? and C/EBP?, and also significantly inhibited the expression of SREBP-1c, fatty acid synthase, fatty acid-binding protein and lipoprotein lipase. Also, arctiin greatly increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target phosphorylated-acetyl CoA carboxylase. Furthermore, administration of arctiin significantly decreased the body weight in obese mice fed with the high-fat diet. The epididymal, perirenal or total visceral adipose tissue weights of mice were all significantly lower in the HF + AC than in the HF. Arctiin administration also decreased the sizes of lipid droplets in the epididymal adipose tissue. CONCLUSIONS Arctiin inhibited adipogenesis in 3T3-L1 adipocytes through the inhibition of PPAR? and C/EBP? and the activation of AMPK signaling pathways. These findings suggest that arctiin has a potential benefit in preventing obesity. PMID:25489405

  10. Isoflavonoids from Crotalaria albida Inhibit Adipocyte Differentiation and Lipid Accumulation in 3T3-L1 Cells via Suppression of PPAR-? Pathway

    PubMed Central

    Sun, Qinhu; Chou, Guixin

    2015-01-01

    Two 2?-isopropenyl dihydrofuran isoflavonoids (1 and 3), one 2?-isopropenyl dihydrofuran chromone (2), as well as 13 known compounds were isolated from the herbs of Crotalaria albida. Their structures and relative configurations were elucidated via NMR and HRESIMS analyses. The 2? S absolute configuration of 1 and 2 were deduced by comparing their NOESY spectra with that of 3, which was determined via single crystal X-ray diffraction (CuK?). The 3R absolute configuration of 1 was determined by CD. Compounds 1, 2, and 3 inhibit the adipocyte differentiation and lipid accumulation of 3T3-L1 through down-regulation of PPAR-? activity. PMID:26285147

  11. Isoflavonoids from Crotalaria albida Inhibit Adipocyte Differentiation and Lipid Accumulation in 3T3-L1 Cells via Suppression of PPAR-? Pathway.

    PubMed

    Sun, Qinhu; Chou, Guixin

    2015-01-01

    Two 2?-isopropenyl dihydrofuran isoflavonoids (1 and 3), one 2?-isopropenyl dihydrofuran chromone (2), as well as 13 known compounds were isolated from the herbs of Crotalaria albida. Their structures and relative configurations were elucidated via NMR and HRESIMS analyses. The 2? S absolute configuration of 1 and 2 were deduced by comparing their NOESY spectra with that of 3, which was determined via single crystal X-ray diffraction (CuK?). The 3R absolute configuration of 1 was determined by CD. Compounds 1, 2, and 3 inhibit the adipocyte differentiation and lipid accumulation of 3T3-L1 through down-regulation of PPAR-? activity. PMID:26285147

  12. 4-Hydroxyisoleucine ameliorates an insulin resistant-like state in 3T3-L1 adipocytes by regulating TACE/TIMP3 expression

    PubMed Central

    Gao, Feng; Du, Wen; Zafar, Mohammad Ishraq; Shafqat, Raja Adeel; Jian, Liumeng; Cai, Qin; Lu, Furong

    2015-01-01

    Background Obesity-associated insulin resistance (IR) is highly correlated with soluble tumor necrosis factor-? (sTNF-?), which is released from transmembranous TNF-? by TNF-? converting enzyme (TACE). In vivo, TACE activity is suppressed by tissue inhibitor of metalloproteinase 3 (TIMP3). Agents that can interact with TACE/TIMP3 to improve obesity-related IR would be highly valuable. In the current study, we assessed whether (2S,3R,4S)-4-hydroxyisoleucine (4-HIL) could modulate TACE/TIMP3 and ameliorate an obesity-induced IR-like state in 3T3-L1 adipocytes. Materials and methods 3T3-L1 adipocytes were incubated in the presence of 25 mM glucose and 0.6 nM insulin to induce an IR-like state, and were then treated with different concentrations of 4-HIL or 10 µM pioglitazone (positive control). The glucose uptake rate was determined using the 2-deoxy-[3H]-d-glucose method, and the levels of sTNF-? in the cell supernatant were determined using ELISA. The protein expression of TACE, TIMP3, and insulin signaling-related molecules was measured using western blotting. Results Exposure to high glucose and insulin for 18 hours increased the levels of sTNF-? in the cell supernatant. The phosphorylation of insulin receptor substrate-1 (IRS-1) Ser307 and Akt Ser473 was increased, whereas the protein expression of IRS-1, Akt, and glucose transporter-4 was decreased. The insulin-induced glucose uptake was reduced by 67% in 3T3-L1 adipocytes, which indicated the presence of an IR-like state. The above indexes, which demonstrated the successful induction of an IR-like state, were reversed by 4-HIL in a dose-dependent manner by downregulating and upregulating the protein expression of TACE and TIMP3 proteins, respectively. Conclusion 4-HIL improved an obesity-associated IR-like state in 3T3-L1 adipocytes by targeting TACE/TIMP3 and the insulin signaling pathway. PMID:26527864

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

    PubMed

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

    2015-01-01

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

  14. A growth factor-responsive gene of murine BALB/c 3T3 cells encodes a protein homologous to human tissue factor

    SciTech Connect

    Hartzell, S.; Ryder, K.; Lanahan, A.; Nathans, D.; Lau, L.F.

    1989-06-01

    Polypeptide growth factors rapidly induce the transcription of a set of genes that appear to mediate cell growth. The authors report that one of the genes induced in BALB/c mouse 3T3 cells encodes a transmembrane protein (mTF) homologous to human tissue factor, which is involved in the proteolytic activation of blood clotting. mTF mRNA is present in many murine tissues and cell lines. The authors' results raise the possibility that mTF may also play a role in cell growth.

  15. Antiobesity effects of the water-soluble fraction of the ethanol extract of Smilax china L. leaf in 3T3-L1 adipocytes

    PubMed Central

    Kang, Yun Hwan; Kim, Kyoung Kon; Kim, Dae Jung

    2015-01-01

    BACKGROUND/OBJECTIVES Several medicinal properties of Smilax china L. have been studied including antioxidant, anti-inflammatory, and anti-cancer effects. However, the antiobesity activity and mechanism by which the water-soluble fraction of this plant mediates its effects are not clear. In the present study, we investigated the lipolytic actions of the water-soluble fraction of Smilax china L. leaf ethanol extract (wsSCLE) in 3T3-L1 adipocytes. MATERIALS/METHODS The wsSCLE was identified by measuring the total polyphenol and flavonoid content. The wsSCLE was evaluated for its effects on cell viability, lipid accumulation, glycerol, and cyclic adenosine monophosphate (cAMP) contents. In addition, western blot analysis was used to evaluate the effects on protein kinase A (PKA), PKA substrates (PKAs), and hormone-sensitive lipase (HSL). For the lipid accumulation assay, 3T3-L1 adipocytes were treated with different doses of wsSCLE for 9 days starting 2 days post-confluence. In other cell experiments, mature 3T3-L1 adipocytes were treated for 24 h with wsSCLE. RESULTS Results showed that treatment with wsSCLE at 0.05, 0.1, and 0.25 mg/mL had no effect on cell morphology and viability. Without evidence of toxicity, wsSCLE treatment decreased lipid accumulation compared with the untreated adipocyte controls as shown by the lower absorbance of Oil Red O stain. The wsSCLE significantly induced glycerol release and cAMP production in mature 3T3-L1 cells. Furthermore, protein levels of phosphorylated PKA, PKAs, and HSL significantly increased following wsSCLE treatment. CONCLUSION These results demonstrate that the potential antiobesity activity of wsSCLE is at least in part due to the stimulation of cAMP-PKA-HSL signaling. In addition, the wsSCLE-stimulated lipolysis induced by the signaling is mediated via activation of the ?-adrenergic receptor. PMID:26634049

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

    PubMed

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

    2015-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC-MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells. PMID:25685661

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  18. Effect of methylated tea catechins from Chinese oolong tea on the proliferation and differentiation of 3T3-L1 preadipocyte.

    PubMed

    Yang, Yang; Qiao, Longliang; Zhang, Xin; Wu, Zufang; Weng, Peifang

    2015-07-01

    As the important component of tea catechins in oolong tea, (-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3?Me) has exhibited various beneficial effects, however, little attention about its obesity prevention effect is available. In this study, the inhibitory effects of tea catechin monomers, including their methylated forms on the proliferation and differentiation of 3T3-L1 preadipocyte were studied. The major methylated tea catechins in oolong tea were identified as EGCG3?Me and ECG3?Me. The accumulation of triglyceride was significantly reduced in a concentration-dependent manner in groups treated with EGCG3?Me at concentrations of 20, 40 and 80?g/mL, and the accumulation of lipid was decreased to 89.42±2.66%, 64.36±3.13% and 39.37±2.79%, respectively. Both EGCG3?Me and EGCG treatments showed a significant inhibitory effect on adipogenesis, while EGCG3?Me showed a relatively higher effect than EGCG, which was contrary to the results of cytotoxic activity. For ECG and ECG3?Me, ECG3?Me also showed a relatively higher antiobesity effect and lower cytotoxic activity. The results of activity screening showed that methylated tea catechins, including EGCG3?Me and ECG3?Me inhibited the proliferation and differentiation of 3T3-L1 preadipocyte. The difference of inhibitory effects for tested compounds may be due to their structural difference (the hydroxyl group at C-3 in D ring substituted by methoxy group). PMID:26002426

  19. Effects of nickel-smelting fumes on the regulation of NIH/3T3 cell viability, necrosis, and expression of hMLH1 and RASSF1A.

    PubMed

    Wang, Jun; Yu, Cui-Ping; Hu, Xue-Ying; Wu, Yong-Hui

    2014-01-01

    Nickel is widely used and distributed in various industries. This study investigated the effect of nickel-smelting fumes on the regulation of NIH/3T3 cell viability, apoptosis, and necrosis and the expression of the tumor suppressor genes hMLH1 and RASSF1A. Cell viability was determined using a methylthiazolyl tetrazolium colorimetric assay. NIH/3T3 cell viability was reduced after exposure to different concentrations of nickel-smelting fumes, but cell apoptosis and necrosis were induced. Moreover, cell morphology changed significantly after exposure to different concentrations of nickel-smelting fumes, as determined using an inverted microscope or transmission electron microscope. Real-time RT-PCR and Western blot analyses showed that exposure of cells to concentrations of ?100 µg/mL of nickel-smelting fumes upregulated the expression of hMLH1 and RASSF1A compared to the negative controls. These data suggest that nickel-smelting fumes could be toxic to cells, upregulating the expression of hMLH1 and RASSF1A and in turn inducing cell apoptosis and necrosis. PMID:24579805

  20. Mango (Mangifera indica L.) peel extract fractions from different cultivars differentially affect lipid accumulation in 3T3-L1 adipocyte cells.

    PubMed

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

    2013-02-26

    Plant phytochemicals are increasingly recognised as sources of bioactive molecules which may have potential benefit in many health conditions. In mangoes, peel extracts from different cultivars exhibit varying effects on adipogenesis in the 3T3-L1 adipocyte cell line. In this study, the effects of preparative HPLC fractions of methanol peel extracts from Irwin, Nam Doc Mai and Kensington Pride mangoes were evaluated. Fraction 1 contained the most hydrophilic components while subsequent fractions contained increasingly more hydrophobic components. High content imaging was used to assess mango peel fraction effects on lipid accumulation, nuclei count and nuclear area in differentiating 3T3-L1 cells. For all three mango cultivars, the more hydrophilic peel fractions 1-3 inhibited lipid accumulation with greater potency than the more hydrophobic peel fractions 4. For all three cultivars, the more lipophilic fraction 4 had concentrations that enhanced lipid accumulation greater than fractions 1-3 as assessed by lipid droplet integrated intensity. The potency of this fraction 4 varied significantly between cultivars. Using mass spectrometry, five long chain free fatty acids were detected in fraction 4; these were not present in any other peel extract fractions. Total levels varied between cultivars, with Irwin fraction 4 containing the highest levels of these free fatty acids. Lipophilic components appear to be responsible for the lipid accumulation promoting effects of some mango extracts and are the likely cause of the diverse effects of peel extracts from different mango cultivars on lipid accumulation. PMID:23295454

  1. Selective nuclear translocation of protein kinase C alpha in Swiss 3T3 cells treated with IGF-I, PDGF and EGF.

    PubMed

    Neri, L M; Billi, A M; Manzoli, L; Rubbini, S; Gilmour, R S; Cocco, L; Martelli, A M

    1994-06-20

    To determine the subcellular distribution of PKC after GFs treatment we have employed a combined immunochemical and in situ confocal microscopy analysis. In quiescent Swiss 3T3 cells only a faint PKC positivity was observable in the nucleus while a strong reaction was seen in the cytoplasm. IGF-I and to a lesser extent PDGF and EGF induced, after 45 min of treatment, a nuclear translocation of PKC detected by a pan-anti-PKC antibody and nuclear fluorescence was distributed in the nuclear interior except for the nucleolar regions. Bombesin and FGF did not affect the sub-cellular distribution of the enzyme. To further the understanding of which PKC isoform was involved in the translocation process, we have tested nine isozyme-specific anti-PKC antibodies. Immunoblotting analysis revealed the presence in Swiss 3T3 fibroblasts of alpha, beta I, epsilon and zeta isoforms. In isolated nuclei from GF-exposed cells only the alpha isozyme was detected: immunostaining was very intense after IGF-I treatment and clearly observable after PDGF and EGF stimulation. This result was strongly supported by the in situ confocal microscopy which parallels the Western blot analysis. These data demonstrate that several, but not all, GFs acting through tyrosine kinase receptor induce the intranuclear translocation of PKC alpha and, because of the dramatic effect of IGF-I, strengthen the case for a link between the activation of nuclear inositol lipid cycle and PKC translocation induced by this GF. PMID:8013664

  2. Effect of borate glass composition on its conversion to hydroxyapatite and on the proliferation of MC3T3-E1 cells.

    PubMed

    Brown, Roger F; Rahaman, Mohamed N; Dwilewicz, Agatha B; Huang, Wenhai; Day, Delbert E; Li, Yadong; Bal, B Sonny

    2009-02-01

    Glasses containing varying amounts of B(2)O(3) were prepared by partially or fully replacing the SiO(2) in silicate 45S5 bioactive glass with B(2)O(3). The effects of the B(2)O(3) content of the glass on its conversion to hydroxyapatite (HA) and on the proliferation of MC3T3-E1 cells were investigated in vitro. Conversion of the glasses to HA in dilute (20 mM) K(2)HPO(4) solution was monitored using weight loss and pH measurements. Proliferation of MC3T3-E1 cells was determined qualitatively by assay of cell density at the glass interface after incubation for 1 day and 3 days, and quantitatively by fluorescent measurements of total DNA in cultures incubated for 4 days. Higher B(2)O(3) content of the glass increased the conversion rate to HA, but also resulted in a greater inhibition of cell proliferation under static culture conditions. For a given mass of glass in the culture medium, the inhibition of cell proliferation was alleviated by using glasses with lower B(2)O(3) content, by incubating the cell cultures under dynamic rather than static conditions, or by partially converting the glass to HA prior to cell culture. PMID:18306284

  3. Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

    PubMed

    Hadjicharalambous, Chrystalleni; Kozlova, Diana; Sokolova, Viktoriya; Epple, Matthias; Chatzinikolaidou, Maria

    2015-12-01

    Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3834-3842, 2015. PMID:26097146

  4. Interleukin-1? Increases Angptl4 (FIAF) Expression via the JNK Signaling Pathway in Osteoblastic MC3T3-E1 Cells.

    PubMed

    Noh, J M; Shen, C; Kim, S J; Kim, M R; Kim, S H; Kim, J H; Park, B H; Park, J H

    2015-09-01

    Angiopoietin-like protein 4 (Angptl4), also known as fasting-induced adiopogenic factor (FIAF), has recently been reported to influence bone metabolism. However, there have been few studies on regulatory factors other than hypoxia for Angptl4 in bone, and particularly in osteoblasts. Expression of interleukin-1? (IL-1?), a proinflammatory cytokine, is increased in serum or bone microenvironments in inflammatory bone diseases or estrogen deficient-conditions. The present study was conducted to determine whether Angptl4 expression in osteoblasts is affected by IL-1? and investigate its involvement in MAP kinase signaling pathways. Angptl4 RNA levels were increased by IL-1? treatment in murine MC3T3-E1 osteoblastic cells. Western blotting and immunofluorescent staining showed a corresponding increase in Angptl4 protein. IL-1? treatment of osteoblasts induced phosphorylation of mitogen-activated protein kinases (MAPKs) including extracellular regulated kinases (ERKs), p38, and c-Jun N-terminal kinase (JNK). Furthermore, SP600125, an inhibitor of JNK, significantly blocked the upregulation of Angptl4 by IL-1?. In contrast, treatment with an inhibitor of p38 MAP kinase (SB203580) or an ERK inhibitor (PD98059) produced responses similar to those seen with the DMSO control. Taken together, these results suggest that IL-1? increases Angptl4 expression through a mechanism dependent on the JNK-MAPK signaling pathway in MC3T3-E1 cells. PMID:26069075

  5. Phototoxicity of bituminous tars-correspondence between results of 3T3 NRU PT, 3D skin model and experimental human data.

    PubMed

    Jirová, D; Kejlová, K; Bendová, H; Ditrichová, D; Mezulániková, M

    2005-10-01

    Bituminous tars (Ichthammol and Ichthyol Pale) are widely used in pharmaceutical, veterinary and cosmetic industries for their anti-microbial, anti-inflammatory and anti-pruritic effects. In contrast to coal tar, no phototoxicity of bituminous tars has been reported in man, although both Ichthammol and Ichthyol Pale exhibit UV absorption which is higher and broader for the former. The validated 3T3 NRU phototoxicity test indicated phototoxic potential of both substances. The phototoxicity test in a 3D human skin model (EpiDerm) only confirmed phototoxicity for Ichthammol. Human data on Ichthammol phototoxicity are missing. A photopatch test in human volunteers was performed in order to clarify the discrepancy between the phototoxicity found in the skin model and the absence of reported human phototoxicity. Following 4h exposure to 5% and 10% aqueous solutions of Ichthammol and Ichthyol Pale the test sites were irradiated with a UVA dose of 5 J/cm(2). Early phototoxic reaction (erythema) within 4-6h after irradiation was only elicited by Ichthammol and not by Ichthyol Pale. These data correspond well with those from the 3D skin model test and suggest the necessity to employ several test systems for final phototoxicity assessment. In addition to the results obtained in 3T3 NRU PT, further testing on 3D skin models may better reflect bioavailability of a given chemical in the skin, relevant to the situation in humans. PMID:16061351

  6. Traditional Korean Herbal Formula Samsoeum Attenuates Adipogenesis by Regulating the Phosphorylation of ERK1/2 in 3T3-L1 Cells

    PubMed Central

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

    2015-01-01

    Adipogenesis is the cell differentiation process from preadipocytes into adipocytes and the critical action in the development of obesity. In the present study, we conducted in vitro analyses to investigate the inhibitory effects of Samsoeum (SSE), a traditional herbal decoction. SSE had no significant cytotoxic effect against either the undifferentiated or differentiated 3T3-L1 cells. Oil Red O staining results showed that SSE significantly inhibited fat accumulation in adipocytes. SSE treatment consistently reduced the intracellular triglyceride content in the cells. SSE significantly inactivated glycerol-3-phosphate dehydrogenase (GPDH), a major link between carbohydrate and lipid metabolisms in 3T3-L1 adipocytes, and markedly inhibited the production of leptin, an important adipokine, in differentiated cells. SSE markedly suppressed the mRNA expression of the adipogenesis-related genes peroxisome proliferator-activated receptor-gamma (PPAR-?), CCAAT/enhancer binding protein-alpha (C/EBP-?), fatty acid synthase (FAS), lipoprotein lipase (LPL), and fatty acid binding protein 4 (FABP4). Importantly, SSE increased the phosphorylation of ERK1/2, but not p38 MAPK and JNK, in adipose cells. Overall, our results indicate that SSE exerts antiadipogenic activity and modulates expressions of adipogenesis-related genes and ERK1/2 activation in adipocytes. PMID:26483846

  7. Quercitrin and taxifolin stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells.

    PubMed

    Satué, María; Arriero, Maria del Mar; Monjo, Marta; Ramis, Joana Maria

    2013-11-15

    Flavonoids are natural antioxidants that positively influence bone metabolism. The present study screened among different flavonoids to identify biomolecules for potential use in bone regeneration. For this purpose, we used MC3T3-E1 and RAW264.7 cells to evaluate their effect on cell viability and cell differentiation. First, different doses of chrysin, diosmetin, galangin, quercitrin and taxifolin were analyzed to determine the optimum concentration to induce osteoblast differentiation. After 48h of treatment, doses ?100?M of diosmetin and galangin and also 500?M taxifolin revealed a toxic effect on cells. The same effect was observed in cells treated with doses ?100?M of chrysin after 14 days of treatment. However, the safe doses of quercitrin (200 and 500?M) and taxifolin (100 and 200?M) induced bone sialoprotein and osteocalcin mRNA expression. Also higher osteocalcin secreted levels were determined in 100?M taxifolin osteoblast treated samples when compared with the control ones. On the other hand, quercitrin and taxifolin decreased Rankl gene expression in osteoblasts, suggesting an inhibition of osteoclast formation. Indeed, osteoclastogenesis suppression by quercitrin and taxifolin treatment was observed in RAW264.7 cells. Based on these findings, the present study demonstrates that quercitrin and taxifolin promote osteoblast differentiation in MC3T3-E1 cells and also inhibit osteoclastogenesis in RAW264.7 cells, showing a positive effect of these flavonoids on bone metabolism. PMID:24060614

  8. Sambucus Williamsii Hance Promotes MC3T3-E1 Cells Proliferation and Differentiation via BMP-2/Smad/p38/JNK/Runx2 Signaling Pathway.

    PubMed

    Yang, Bingyou; Lin, Xiaoying; Yang, Chunli; Tan, Jinyan; Li, Wei; Kuang, Haixue

    2015-11-01

    The 50% ethanol elution fractions of root-bark of Sambucus Williamsii Hance (rbSWH) evaluated the effect of proliferation and differentiation on preosteoblast MC3T3-E1 cell, and the mechanism of actions. We found that rbSWH(30, 60, and 90?µg/mL) can enhance cell proliferation by MTT assay and promote alkaline phosphatase (ALP) and bone Gla protein (BGP) activities, type I collagen (Col-I) synthesis, and mineralization nodule formation in primary cultured osteoblasts. The results showed that rbSWH can increase mRNA levels of BMP-2 and Runx2 using real-time reverse transcription-quantitative polymerase chain reaction, whereas the BMP-2 antagonist Noggin attenuated the increase of ALP activity induced by rbSWH, indicating that BMP-2 expression was required for the action of rbSWH in osteoblastic. We also found that rbSWH can enhance the expressions of BMP-2, BMPRIB, BMPRII, phosphorylation of Smad, JNK and p38, and Runx2 proteins by western blotting. In addition, pretreatment of cells with p38 inhibitor (SB203580) or JNK inhibitor (SP600125) can antagonize the elevation of BMP-2 expression, ALP activity, and cell viability induced by rbSWH. Taken together, our results provided an evidence that rbSWH can promote MC3T3-E1 cell proliferation and differentiation via BMP-2/Smad/p38/JNK/Runx2 signaling pathway. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26455827

  9. A novel Mn(2+)-doped core/shell quantum dot-based intracellular probe for fluoride anions sensing in MC3T3-E1 osteoblastic cells.

    PubMed

    Wang, Huan; Hu, Tian-Yu; Zhao, Zhi-Tao; Zhang, Xiu-Yun; Wang, Ying; Duan, Xiao-Qin; Liu, Da-Wei; Jing, Ling; Ma, Qiang

    2016-03-01

    In this paper, 3-aminobenzeneboronic acid functionalized Mn(2+)-doped ZnTe/ZnSe quantum dots (APBA-dQDs) were prepared. The APBA functional groups had strong binding ability with F(-), resulting in the quenchment of dQDs photoluminescence (PL). Under the optimal condition, the fluorescence intensity of APBA-dQDs was related linearly to the concentration of F(-) in the range of 0.25-1.5µmol/L with a detection limit of 0.1µmol/L. The selectivity of fluorescence quenching of APBA-dQDs for F(-) was enhanced. Moreover, the proposed methodology for the sensing of F(-) at EM 560nm in MC3T3-E1 osteoblastic cells was demonstrated and got a satisfactory results. The results indicate that the APBA-dQDs are promising candidates for intracellular in MC3T3-E1 osteoblastic cells. To the best of our knowledge, it was the first report of F(-) sensing by using the quenched fluorescence of APBA-dQDs in non-cancerous cells. PMID:26717843

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC–MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells. PMID:25685661

  12. Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells

    SciTech Connect

    Li, Qiang; Kamemura, Kazuo

    2014-07-18

    Highlights: • The majority of EWS localizes stably in the cytosol in 3T3-L1 preadipocytes. • Adipogenic stimuli induce the nuclear localization of EWS. • Adipogenesis promotes O-GlcNAcylation of EWS. • O-GlcNAcylation stimulates the recruitment of EWS to the nuclear periphery. - Abstract: Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughout adipogenesis. Instead, an adipogenesis-dependent increase in O-linked ?-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.

  13. Cadmium-109 uptake by tumors derived from Balb C/3T3 cell lines with varying degrees of the transformed phenotype

    SciTech Connect

    Morton, K.; Alazraki, N.; Winge, D.; Lynch, R.E.

    1986-05-01

    To determine if tumors are rich in metallothionein, the authors measured the vivo uptake of subcutaneously-injected carrier-free cadmium-109 in tumors and in normal tissues of Balb/C mice. The tumors were grown in the mice from cultured Balb/3T3 cells transformed by the Moloney murine sarcoma virus. Uptake of cadmium-109 per gram of tissue was greatest for liver, kidney, and spleen. However, tumor uptake of cadmium-109 was markedly greater than that in blood, skeletal muscle, bones, intestine or adipose tissue. B Sephadex G-75 chromatography, the radioactivity in tumor and in liver lysates eluted with cytochrome-C, a molecule similar in molecular weight to metal-lothionein. To determine if metallothionein levels are related to the degree of malignancy of tumors, cadmium-109 uptake in the tumors from the virally-transformed cells was compared to that in tumors from non-transformed Balb/3T3 cells and two derivative chemically transformed cell lines. There was strong correlation between the substrate-independent growth in soft agarose of the four cell lines, the rate of growth of the corresponding tumors, and the amount of cadmium-109 uptake in the tumors. The authors conclude that metallothionein levels may be elevated in tumors as a function of the degree of expression of the transformed phenotype.

  14. AKT1/PKB? Kinase Is Frequently Elevated in Human Cancers and Its Constitutive Activation Is Required for Oncogenic Transformation in NIH3T3 Cells

    PubMed Central

    Sun, Mei; Wang, Gen; Paciga, June E.; Feldman, Richard I.; Yuan, Zeng-Qiang; Ma, Xiao-Ling; Shelley, Sue A.; Jove, Richard; Tsichlis, Philip N.; Nicosia, Santo V.; Cheng, Jin Q.

    2001-01-01

    Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention. PMID:11485901

  15. Exposure to p,p'-DDE enhances differentiation of 3T3-L1 preadipocytes in a model of sub-optimal differentiation.

    PubMed

    Mangum, Lauren H; Howell, George E; Chambers, Janice E

    2015-10-14

    The incidence of obesity is increasing worldwide at an alarming rate. Recently, exposure to environmental contaminants, especially organochlorines such as p,p'-dichlorodiphenyldichloroethylene (DDE), has been implicated as a possible causative factor in the increasing obesity epidemic. The objective of this study was to evaluate the ability of DDE to alter adipogenesis in a model of sub-optimal differentiation. 3T3-L1 preadipocytes were induced to differentiate in the presence of DDE (0.01-100?M) using a sub-optimal differentiation cocktail. Eight days after the initiation of differentiation, adipogenesis was assessed through neutral lipid staining, triglyceride accumulation, and expression of markers of terminal differentiation. Exposure to DDE induced a concentration dependent increase in intracellular neutral lipid accumulation as determined by Oil Red O staining and triglyceride assay. Alterations in lipid accumulation were accompanied by upregulation of genetic markers of differentiation. DDE (10?M) enhanced expression of fatty acid binding protein 4 and Sterol regulatory element-binding protein-1c at the 2.5 and 20?M concentrations. DDE (2.5, 10, and 20?M) induced upregulation of leptin and fatty acid synthase, as compared to sub-optimal vehicle control (0.05% ethanol). Our results indicate that DDE is capable of enhancing adipogenesis and intracellular lipid accumulation in 3T3-L1 cells through upregulation of molecular targets responsible for lipid storage. PMID:26200599

  16. Tangeritin inhibits adipogenesis by down-regulating C/EBP?, C/EBP?, and PPAR? expression in 3T3-L1 fat cells.

    PubMed

    He, Y F; Liu, F Y; Zhang, W X

    2015-01-01

    The treatment of obese patients is a topic investigated by an increasing number of researchers. This study aimed to elucidate the possible inhibitory effect of tangeritin on the development and function of fat cells. 3T3-L1 fat cells were grown to confluence and subjected to different concentrations of tangeritin. The most effective tangeritin inhibition concentration was determined by the MTT assay. The treated cells were subjected to real-time reverse transcriptase PCR and western blot analysis, to detect changes in the CCAAT/enhancer binding protein (C/EBP)?, C/EBP?, and peroxisome proliferator activated receptor (PPAR)? expression levels. The MTT assay revealed that the fat cell growth was inhibited at a 20 ng/mL concentration of tangeritin. The results of real-time PCR revealed a significant decrease in the expression of C/EBP?, C/EBP?, and PPAR? mRNA, following the treatment with tangeritin. Western blot analysis also presented similar results at a protein level. Therefore, we concluded that tangeritin inhibits adipogenesis via the down-regulation of C/EBP?, C/EBP?, and PPAR? mRNA and protein expression in 3T3-L1 cells. PMID:26535679

  17. Syzygium aqueum leaf extract and its bioactive compounds enhances pre-adipocyte differentiation and 2-NBDG uptake in 3T3-L1 cells.

    PubMed

    Manaharan, Thamilvaani; Ming, Cheng Hwee; Palanisamy, Uma D

    2013-01-15

    The insulin-like and/or insulin-sensitising effects of Syzygium aqueum leaf extract and its six bioactive compounds; 4-hydroxybenzaldehyde, myricetin-3-O-rhamnoside, europetin-3-O-rhamnoside, phloretin, myrigalone-G and myrigalone-B were investigated in 3T3-L1 adipocytes. We observed that, S. aqueum leaf extract (0.04-5 ?g/ml) and its six bioactive compounds (0.08-10 ?M) at non-cytotoxic concentrations were effectively enhance adipogenesis, stimulate glucose uptake and increase adiponectin secretion in 3T3-L1 adipocytes. Clearly, the compounds myricetin-3-O-rhamnoside and europetin-3-O-rhamnoside showed insulin-like and insulin-sensitising effects on adipocytes from a concentration of 0.08 ?M. These compounds were far better than rosiglitazone and the other isolated compounds in enhancing adipogenesis, stimulating 2-NBDG uptake and increasing adiponectin secretion at all the concentrations tested. These suggest the antidiabetic potential of S. aqueum leaf extract and its six bioactive compounds. However, further molecular interaction studies to explain the mechanisms of action are highly warranted. PMID:23122070

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

    SciTech Connect

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

    2012-07-01

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

  19. Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

    PubMed

    Ghasemi-Dehkordi, Payam; Allahbakhshian-Farsani, Mehdi; Abdian, Narges; Mirzaeian, Amin; Saffari-Chaleshtori, Javad; Heybati, Fatemeh; Mardani, Gashtasb; Karimi-Taghanaki, Alireza; Doosti, Abbas; Jami, Mohammad-Saeid; Abolhasani, Marziyeh; Hashemzadeh-Chaleshtori, Morteza

    2015-09-01

    Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p?feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches. PMID:25820945

  20. Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

    SciTech Connect

    Xue, Peng; School of Public Health, China Medical University, Shenyang 110001 ; Hou, Yongyong; Zhang, Qiang; Woods, Courtney G.; Yarborough, Kathy; Liu, Huiyu; Sun, Guifan; Andersen, Melvin E.; Pi, Jingbo

    2011-04-08

    Highlights: {yields} In 3T3-L1 adipocytes iAs{sup 3+} decreases insulin-stimulated glucose uptake. {yields} iAs{sup 3+} attenuates insulin-induced phosphorylation of AKT S473. {yields} iAs{sup 3+} activates the cellular adaptive oxidative stress response. {yields} iAs{sup 3+} impairs insulin-stimulated ROS signaling. {yields} iAs{sup 3+} decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 {mu}M) inorganic arsenite (iAs{sup 3+}) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs{sup 3+} exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs{sup 3+} exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in adipocytes. Taken together our studies suggest that prolonged low-level iAs{sup 3+} exposure activates the cellular adaptive oxidative stress response, which impairs insulin-stimulated ROS signaling that is involved in ISGU, and thus causes insulin resistance in adipocytes.

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

    PubMed

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

    2015-02-01

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

  2. UV-irradiated 7-dehydrocholesterol coating on polystyrene surfaces is converted to active vitamin D by osteoblastic MC3T3-E1 cells.

    PubMed

    Satué, María; Córdoba, Alba; Ramis, Joana M; Monjo, Marta

    2013-06-01

    The aim of the present study was to determine the effects of UV irradiation on the conversion of 7-dehydrocholesterol (7-DHC), which has been coated onto a polystyrene surface, to cholecalciferol (D3), and the resulting effect on the formation of vitamin D (1,25-D3) by MC3T3-E1 cells. The changes in gene expression of the enzymes regulating its hydroxylation, Cyp27b1 and Cyp27a1, were monitored as well as the net effect of the UV-treated 7-DHC coating on cell viability and osteoblast differentiation. MC3T3-E1 cells were found to express the enzymes required for synthesizing active 1,25-D3, and we found a dose-dependent increase in the production of both 25-D3 and 1,25-D3 levels for UV-activated 7-DHC samples unlike UV-untreated ones. Cell viability revealed no cytotoxic effect for any of the treatments, but only for the highest dose of 7-DHC (20 nmol per well) that was UV-irradiated. Furthermore, osteoblast differentiation was increased in cells treated with some of the higher doses of 7-DHC when UV-irradiated, as shown by collagen-I, osterix and osteocalcin relative mRNA levels. The conversion of 7-DHC to preD3 exogenously by UV irradiation and later to 25-D3 by MC3T3-E1 cells was determined for the optimum 7-DHC dose (0.2 nmol per well), i.e. 8.6 ± 0.7% of UV-activated 7-DHC was converted to preD3 and 6.7 ± 2.8% of preD3 was finally converted to 25-D3 under the conditions studied. In conclusion, we demonstrate that an exogenous coating of 7-DHC, when UV-irradiated, can be used to endogenously produce active vitamin D. We hereby provide the scientific basis for UV-activated 7-DHC coating as a feasible approach for implant therapeutics focused on bone regeneration. PMID:23538933

  3. Catabolism of Branched Chain Amino Acids Contributes Significantly to Synthesis of Odd-Chain and Even-Chain Fatty Acids in 3T3-L1 Adipocytes.

    PubMed

    Crown, Scott B; Marze, Nicholas; Antoniewicz, Maciek R

    2015-01-01

    The branched chain amino acids (BCAA) valine, leucine and isoleucine have been implicated in a number of diseases including obesity, insulin resistance, and type 2 diabetes mellitus, although the mechanisms are still poorly understood. Adipose tissue plays an important role in BCAA homeostasis by actively metabolizing circulating BCAA. In this work, we have investigated the link between BCAA catabolism and fatty acid synthesis in 3T3-L1 adipocytes using parallel 13C-labeling experiments, mass spectrometry and model-based isotopomer data analysis. Specifically, we performed parallel labeling experiments with four fully 13C-labeled tracers, [U-13C]valine, [U-13C]leucine, [U-13C]isoleucine and [U-13C]glutamine. We measured mass isotopomer distributions of fatty acids and intracellular metabolites by GC-MS and analyzed the data using the isotopomer spectral analysis (ISA) framework. We demonstrate that 3T3-L1 adipocytes accumulate significant amounts of even chain length (C14:0, C16:0 and C18:0) and odd chain length (C15:0 and C17:0) fatty acids under standard cell culture conditions. Using a novel GC-MS method, we demonstrate that propionyl-CoA acts as the primer on fatty acid synthase for the production of odd chain fatty acids. BCAA contributed significantly to the production of all fatty acids. Leucine and isoleucine contributed at least 25% to lipogenic acetyl-CoA pool, and valine and isoleucine contributed 100% to lipogenic propionyl-CoA pool. Our results further suggest that low activity of methylmalonyl-CoA mutase and mass action kinetics of propionyl-CoA on fatty acid synthase result in high rates of odd chain fatty acid synthesis in 3T3-L1 cells. Overall, this work provides important new insights into the connection between BCAA catabolism and fatty acid synthesis in adipocytes and underscores the high capacity of adipocytes for metabolizing BCAA. PMID:26710334

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

    PubMed

    Tsuchiya, T; Kato-Masatsuji, E; Tsuzuki, T; Umeda, M

    2000-11-10

    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 microg/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 of sodium ascorbate and ascorbic acid-2-glucoside (AG) were comparable, but weaker than those of APM; L (+)-ascorbic acid-2-sulfate ester disodium 2H(2)O showed little effect. When phorbol 12, 13-didecanoate or tumor necrosis factor alpha (TNF-alpha) were used as promoters, APM also effectively suppressed transformation. PMID:11098084

  5. Vasoactive intestinal peptide synergistically stimulates DNA synthesis in mouse 3T3 cells: Role of cAMP, Ca sup 2+ , and protein kinase C

    SciTech Connect

    Zurier, B.B.; Kozma, M.; Sinnett-Smith, J.; Rozengurt, E. )

    1988-05-01

    Vasoactive intestinal peptide synergistically stimulated initiation of DNA synthesis in Swiss 3T3 cells. The peptide stimulated ({sup 3}H)thymidine incorporation in the presence of insulin and either forskolin or an inhibitor of cAMP phosphodiesterase in a concentration-dependent manner. Half-maximal effect was obtained at 1 nM. At mitogenic concentrations, VIP stimulated a marked accumulation (eightfold) of cAMP. In contrast to other growth-promoting neuropeptides, VIP did not induce an increase in cytosolic free Ca{sup 2+} or the activation of protein kinase C. The authors conclude that neuropeptides can modulate long-term cell proliferation through multiple signaling pathways.

  6. Phosphorylation of the human transferrin receptor by protein kinase C is not required for endocytosis and recycling in mouse 3T3 cells.

    PubMed Central

    Zerial, M; Suomalainen, M; Zanetti-Schneider, M; Schneider, C; Garoff, H

    1987-01-01

    We have investigated the role of phosphorylation in the endocytosis of the human transferrin receptor (TR) by replacing its phosphorylation site, Ser24, with Ala through site-directed mutagenesis of the TR cDNA. The TR Ala24 mutant expressed in mouse 3T3 cells was not phosphorylated, even following stimulation of protein kinase C by phorbol ester. However, in spite of this defect the mutant was efficiently endocytosed and recycled back to the plasma membrane with kinetics similar to those of TR and a control mutant TR Ala63. Thus, these results confirm earlier results by Davis et al. (1986, J. Biol. Chem., 261-9034-9041) that Ser24 of human TR is the phosphorylation site for protein kinase C but do not support a role of this modification as a signal for TR endocytosis and recycling. Images Fig. 1. Fig. 2. Fig. 3. PMID:3479328

  7. Overexpression of tumor necrosis factor receptor-associated protein 1 (TRAP1), leads to mitochondrial aberrations in mouse fibroblast NIH/3T3 cells

    PubMed Central

    Im, Chang-Nim; Seo, Jeong-Sun

    2014-01-01

    Cancer cells undergo uncontrolled proliferation, and aberrant mitochondrial alterations. Tumor necrosis factor receptorassociated protein 1 (TRAP1) is a mitochondrial heat shock protein. TRAP1 mRNA is highly expressed in some cancer cell lines and tumor tissues. However, the effects of its overexpression on mitochondria are unclear. In this study, we assessed mitochondrial changes accompanying TRAP1 overexpression, in a mouse cell line, NIH/3T3. We found that overexpression of TRAP1 leads to a series of mitochondrial aberrations, including increase in basal ROS levels, and decrease in mitochondrial biogenesis, together with a decrease in peroxisome proliferator-activated receptor gamma coactivator-1? (PGC-1?) mRNA levels. We also observed increased extracellular signal-regulated kinase (ERK) phosphorylation, and enhanced proliferation of TRAP1 overexpressing cells. This study suggests that overexpression of TRAP1 might be a critical link between mitochondrial disturbances and carcinogenesis. [BMB Reports 2014; 47(5): 280-285] PMID:24286320

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

    PubMed

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

    2014-01-01

    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

  9. Potentiation of endothelin-1-induced prostaglandin E2 formation by Ni(2+) and Sr(2+) in murine osteoblastic MC3T3-E1 cells.

    PubMed

    Leis, Hans Jörg; Windischhofer, Werner

    2016-01-01

    Cation recognition mechanisms beyond calcium-sensing receptors are still largely unexplored and consequently there is surprisingly little information on linking of this primary event to key metabolic features of different cell systems, such as arachidonic acid metabolism. However, information on the modulatory role of extracellular cations in cellular function is scarce. In this study we have demonstrated, that Ni(2+) and Sr(2+) potentiate endothelin-1 induced prostaglandin E2 formation in the osteoblastic cell line, MC3T3-E1, even in the absence of extracellular calcium. The effect is strictly dependent of receptor-mediated signal transduction processes evoked by endothelin-1 and arachidonate release involves cytosolic phospholipase A2 activity. The ligation sites, at least for Ni(2+) are extracellular. The data suggest a novel activation mechanism for arachidonate release and subsequent prostaglandin formation that does not require calcium. PMID:26653747

  10. Enamel Matrix Derivative Inhibits Adipocyte Differentiation of 3T3-L1 Cells via Activation of TGF-?RI Kinase Activity

    PubMed Central

    Gruber, Reinhard; Bosshardt, Dieter D.; Miron, Richard J.; Gemperli, Anja C.; Buser, Daniel; Sculean, Anton

    2013-01-01

    Enamel matrix derivative (EMD), an extract of fetal porcine enamel, and TGF-? can both suppress adipogenic differentiation. However, there have been no studies that functionally link the role of EMD and TGF-? in vitro. Herein, we examined whether TGF-? signaling contributes to EMD-induced suppression of adipogenic differentiation. Adipogenesis was studied with 3T3-L1 preadipocytes in the presence of SB431542, an inhibitor of TGF-?RI kinase activity. SB431542 reversed the inhibitory effect of EMD on adipogenic differentiation, based on Oil Red O staining and mRNA expression of lipid regulated genes. SB431542 also reduced EMD-stimulated expression of connective tissue growth factor (CTGF), an autocrine inhibitor of adipogenic differentiation. Moreover, short interfering (si)RNAs for CTGF partially reversed the EMD-induced suppression of lipid regulated genes. We conclude that the TGF-?RI - CTGF axis is involved in the anti-adipogenic effects of EMD in vitro. PMID:23951076

  11. Ameliorating effects of fermented rice bran extract on oxidative stress induced by high glucose and hydrogen peroxide in 3T3-L1 adipocytes.

    PubMed

    Kim, Dongyeop; Han, Gi Dong

    2011-09-01

    In this study, we investigated whether fermented rice bran (FRB) can ameliorate the oxidative stress induced by high glucose and hydrogen peroxide (H(2)O(2)) in 3T3-L1 adipocytes by analyzing reactive oxygen species (ROS), oil red O staining, as well as the expression of mRNAs related to glucose homeostasis and adipogenesis. It was first confirmed that rice bran fermented by Issatchenkia orientalis MFST1 extract increased free phenolic content compared to non-fermented rice bran. The FRB extract strongly inhibited ROS generation and upregulated the expression of PPAR-? and adiponectin. Moreover, FRB upregulated GLUT4 related to glucose transportation and insulin sensitivity. Taken together, FRB extract ameliorated oxidative stress-induced insulin resistance by neutralizing free radicals and upregulating adiponectin in adipocytes. Our results provide information toward understanding the beneficial effects of FRB on oxidative stress. PMID:21748436

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

    SciTech Connect

    Kuroyanagi, Kayo; Kang, Min-Sook; Goto, Tsuyoshi; Hirai, Shizuka; Ohyama, Kana; Kusudo, Tatsuya; Yu, Rina; Yano, Masamichi; Sasaki, Takao; Takahashi, Nobuyuki; Kawada, Teruo

    2008-02-01

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

  13. Cytotoxicity of Agaricus sylvaticus in non-tumor cells (NIH/3T3) and tumor (OSCC-3) using tetrazolium (MTT) assay.

    PubMed

    Orsine, Joice Vinhal Costa; Marques Brito, Luíssa; Silva, Renata Carvalho; Santos Almeida, Maria de Fátima Menezes; Novaes, Maria Rita Carvalho Garbi

    2013-01-01

    The purpose of this study was to assess the cytotoxic effect of the non-fractionated aqueous extract of A. sylvaticus mushroom in cultures of non-tumor cells (NIH3T3) and tumor cells (OSCC-3). The cells were maintained in DMEN cell culture medium added of 10% of fetal bovine serum and 1% antibiotic. For the cytotoxicity test we prepared the aqueous mushroom extract at concentrations of 0.01 mg.ml?¹, 0.02 mg.ml?¹, 0.04 mg.ml?¹, 0.08 mg.ml?¹, 0.16 mg.ml?¹, and 0.32 mg.ml?¹. For the culture, 2 x 10? cells/ml was deposited in 96-well microplates during 24 hour incubation with subsequent exchange of medium by another containing the mushroom concentrations. After 24 hour incubation the medium was discarded and 100 ml of tetrazolium blue (MTT) was added at a concentration of 5 mg.ml?¹. The microplates were incubated for 2 h at 37° C. Spectrophotometric analysis was performed using 570 nm wavelength. From the values of the optical densities we determined the drug concentration capable of reducing cell viability by 50%. Therefore, the mushroom A. sylvaticus, at all concentrations tested, did not show cytotoxic effects, once the inhibitory concentration (IC??) obtained for tumor cells OSCC-3 was 0.06194 mg.ml?¹, and the IC?? checked for non-tumor cells NIH3T3 was 0,06468 mg.ml?¹. This test made it possible to determine that A. sylvaticus mushroom has no cytotoxic effects, suggesting its use safe for human consumption. PMID:23889648

  14. Anti-adipogenic activity of the edible brown alga Ecklonia stolonifera and its constituent fucosterol in 3T3-L1 adipocytes.

    PubMed

    Jung, Hyun Ah; Jung, Hee Jin; Jeong, Hyun Young; Kwon, Hyun Ju; Kim, Min-Sun; Choi, Jae Sue

    2014-06-01

    Fucosterol is a sterol metabolite of brown algae and regulates genes involved with cholesterol homeostasis. As a part of our continuous search for anti-obesity agents from natural marine sources, the anti-adipogenic activities of Ecklonia stolonifera and its sterol, fucosterol, were evaluated for the inhibition of adipocyte differentiation and lipid formation. Oil Red O staining was used to evaluate triglyceride contents in 3T3-L1 pre-adipocytes primed by differentiation medium (DM) I and DM II. The methanolic extract of E. stolonifera showed strong anti-adipogenic activity, and was thus fractionated with several solvents. Among the tested fractions, the dichloromethane (CH2Cl2) fraction was found to be the most active fraction, with significant inhibition (40.5 %) of intracellular lipid accumulation at a non-toxic concentration, followed by the ethyl acetate fraction (30.2 %) at the same concentration, while the n-butanol and water fractions did not show inhibitory activity within the tested concentrations. The strong anti-adipogenic CH2Cl2-soluble fraction was further purified by a repeated chromatography to yield fucosterol. Fucosterol reduced lipid contents in a concentration-dependent manner without showing any cytotoxicity. Fucosterol treatment also yielded a decrease in the expression of the adipocyte marker proteins peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer-binding protein ? (C/EBP?) in a concentration-dependent manner. Taken together, these results suggest that fucosterol inhibits expression of PPAR? and C/EBP?, resulting in a decrease of lipid accumulation in 3T3-L1 pre-adipocytes, indicating that the potential use of E. stolonifera and its bioactive fucosterol as an anti-obesity agent. PMID:24014306

  15. Peptide derived from desalinated boiled tuna extract inhibits adipogenesis through the downregulation of C/EBP-? and PPAR-? in 3T3-L1 adipocytes.

    PubMed

    Kim, Young-Min; Kim, Eun-Young; Kim, In-Hye; Nam, Taek-Jeong

    2015-05-01

    Recently, obesity has increased due to a variety of reasons, including the availability of 'fast food' and high-fat diets. Developing anti-obesity functional drugs and foods from natural sources may offer solutions to this global concern. Generally, tuna is a high-protein, low-fat and low-calorie food with various bioactive effects. It may improve memory, reduce cholesterol levels and positively affect the development of brain cells. In this study, we screened the anti-obesity potential of peptides derived from tuna protein. We then observed protein bands by the Coomassie blue staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The protein mixture was concentrated and desalted using in-gel trypsin digestion and a C18 nano column and Poros R2 reversed-phase preparation, prior to quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS). We screened the peptides for their ability to affect adipogenesis in 3T3-L1 adipocytes. We also measured glucose uptake, triglyceride levels and lipid droplets using Oil Red O staining. As a result, we confirmed that one peptide inhibited adipocyte differentiation. We also observed the expression of obesity-related genes by western blot analysis and reverse transcription-polymerase chain reaction. The peptide from the tuna extract significantly reduced the expression levels of CCAAT/enhancer-binding protein ? (C/EBP-?) and peroxisome proliferator-activated receptor-? (PPAR-?) adipocyte marker genes. Thus, our data suggest that this peptide from boiled tuna extract reduces lipid components and adipogenesis in 3T3-L1 cells, and these characteristics may be of value in the development of anti-obesity foods. PMID:25761066

  16. Phlorotannins isolated from the edible brown alga Ecklonia stolonifera exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating C/EBP? and PPAR?.

    PubMed

    Jung, Hyun Ah; Jung, Hee Jin; Jeong, Hyun Young; Kwon, Hyun Ju; Ali, Md Yousof; Choi, Jae Sue

    2014-01-01

    The dramatic increase in obesity-related diseases emphasizes the need to elucidate the cellular and molecular mechanisms underlying fat metabolism. Inhibition of adipocyte differentiation has been suggested to be an important strategy for preventing or treating obesity. In our previous study, we characterized an Ecklonia stolonifera extract and non-polar fractions thereof, including dichloromethane and ethyl acetate fractions. We showed that these fractions inhibited adipocyte differentiation and lipid formation/accumulation in 3T3-L1 preadipocytes, as assessed by Oil Red O staining. As part of our ongoing search for anti-obesity agents derived from E. stolonifera, in this work, we characterized five known phlorotannins, including phloroglucinol, eckol, dieckol, dioxinodehydroeckol, and phlorofucofuroeckol A, all of which were isolated from the active ethyl acetate fraction of E. stolonifera. We determined the chemical structures of these phlorotannins through comparisons of published nuclear magnetic resonance (NMR) spectral data. Furthermore, we screened these phlorotannins for their abilities to inhibit adipogenesis over a range of concentrations (12.5-100 ?M). Of these five phlorotannins, phloroglucinol, eckol, and phlorofucofuroeckol A significantly concentration-dependently inhibited lipid accumulation in 3T3-L1 cells without affecting cell viability. In addition, the five isolated phlorotannins also significantly reduced the expression levels of several adipocyte marker genes, including proliferator activated receptor ? (PPAR?) and CCAAT/enhancer-binding protein ? (C/EBP?), although they did so to different extents. These results suggest that the molecular weight of a phlorotannin is an important factor affecting its ability to inhibit adipocyte differentiation and modulate the expression levels of adipocyte marker genes. PMID:24334103

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

    SciTech Connect

    Wang, Weirong; Lin, Qinqin; Lin, Rong; Zhang, Jiye; Ren, Feng; Zhang, Jianfeng; Ji, Meixi; Li, Yanxiang

    2013-06-10

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

  18. Phillyrin, a natural lignan, attenuates tumor necrosis factor ?-mediated insulin resistance and lipolytic acceleration in 3T3-L1 adipocytes.

    PubMed

    Kong, Poren; Zhang, Linlin; Guo, Yuyu; Lu, Yingli; Lin, Dongping

    2014-07-01

    In obese adipose tissue, tumor necrosis factor-? secreted from macrophages plays an important role in the adipocyte dysfunctions, including insulin resistance, lipolytic acceleration, and changes of adipokines, which promote the development of obesity-related complications. Phillyrin, an active ingredient found in many medicinal plants and certain functional foods, elicits anti-obesity and anti-inflammatory properties in vivo. The aim of the current study was to investigate the role of phillyrin in preventing tumor necrosis factor ?-induced insulin resistance or lipolytic acceleration in 3T3-L1 adipocytes. Our results showed that phillyrin partially restored insulin-stimulated 2-DOG uptake, which was reduced by tumor necrosis factor-?, with concomitant restoration in serine phosphorylation of insulin receptor substrate-1 and insulin-stimulated Glut4 translocation to plasma membrane. Phillyrin also dose-dependently prevented tumor necrosis factor ?-stimulated adipocyte lipolysis with preserved downregulation of perilipin. The mitogen-activated protein kinases and I kappaB kinase activation was promoted in tumor necrosis factor ?-stimulated adipocytes, but pretreatment with 40 µM phillyrin inhibited the phosphorylation of extracellular signal-regulated kinases1/2, stress-activated protein kinase/Jun N-terminal kinase and I kappaB kinase (p<0.05). Moreover, phillyrin could inhibit the expressions of interleukin-6 and monocyte chemoattractant protein-1 induced by tumor necrosis factor-?. Using transwell coculture method with 3T3-L1 adipocytes and RAW 264.7 macrophages, the enhanced productions of tumor necrosis factor-? and free fatty acids in the medium were significantly reduced by phillyrin (p<0.05). These results indicate that phillyrin exerts a beneficial effect on adipocyte dysfunctions induced by tumor necrosis factor-? through suppression of the activation of I kappaB kinase and N-terminal kinase. Phillyrin may have the potential to ameliorate the inflammatory changes and insulin resistance in obese adipose tissue. PMID:24995500

  19. Alpha-Lipoic Acid Promotes Osteoblastic Formation in H2O2 -Treated MC3T3-E1 Cells and Prevents Bone Loss in Ovariectomized Rats.

    PubMed

    Fu, Chao; Xu, Dong; Wang, Chang-Yuan; Jin, Yue; Liu, Qi; Meng, Qiang; Liu, Ke-Xin; Sun, Hui-Jun; Liu, Mo-Zhen

    2015-09-01

    Alpha-lipoic acid (ALA), a naturally occurring compound and dietary supplement, has been established as a potent antioxidant that is a strong scavenger of free radicals. Recently, accumulating evidences has indicated the relationship between oxidative stress and osteoporosis (OP). Some studies have investigated the possible beneficial effects of ALA on OP both in vivo and in vitro; however, the precise mechanism(s) underlying the bone-protective action of ALA remains unclear. Considering this, we focused on the anti-oxidative capacity of ALA to exert bone-protective effects in vitro and in vivo. In the present study, the effects of ALA on osteoblastic formation in H(2)O(2) -treated MC3T3-E1 pre-osteoblasts and ovariectomy (OVX)-induced bone loss in rats were investigated. The results showed that ALA promoted osteoblast differentiation, mineralization and maturation and inhibited osteoblast apoptosis, thus increasing the OPG/receptor activator of nuclear factor-?B (NF-?B) ligand (RANKL) ratio and leading to enhanced bone formation in vitro and inhibited bone loss in vivo. Further study revealed that ALA exerted its bone-protective effects by inhibiting reactive oxygen species (ROS) generation by down-regulating Nox4 gene expression and protein synthesis and attenuating the transcriptional activation of NF-?B. In addition, ALA might exert its bone-protective effects by activating the Wnt/Lrp5/?-catenin signaling pathway. Taken together, the present study indicated that ALA promoted osteoblastic formation in H(2)O(2) -treated MC3T3-E1 cells and prevented OVX-induced bone loss in rats by regulating Nox4/ROS/NF-?B and Wnt/Lrp5/?-catenin signaling pathways, which provided possible mechanisms of bone-protective effects in regulating osteoblastic formation and preventing bone loss. Taken together, the results suggest that ALA may be a candidate for clinical OP treatment. PMID:25655087

  20. 31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

    PubMed

    Civan, M M; Williams, S R; Gadian, D G; Rozengurt, E

    1986-01-01

    Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event. PMID:3543375

  1. Coenzyme Q10 increases the fatty acid oxidation through AMPK-mediated PPAR? induction in 3T3-L1 preadipocytes.

    PubMed

    Lee, Soo Kyung; Lee, Jung Ok; Kim, Ji Hae; Kim, Nami; You, Ga Young; Moon, Ji Wook; Sha, Jie; Kim, Su Jin; Lee, Yong Woo; Kang, Ho Jin; Park, Sun Hwa; Kim, Hyeon Soo

    2012-12-01

    Coenzyme Q10(CoQ10) is a known anti-adipogenic factor. However, the mechanism by which CoQ10 acts is unclear. In this study, we found that CoQ10 increased the phosphorylation of AMP-activated protein kinase (AMPK) in 3T3-L1preadipocytes. CoQ10 induced an increase in cytoplasmic calcium concentrations, which is reflected by increased Fluo-3 intensity under confocal microscopy recording. Either inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) or knock-down CaMKK blocked CoQ10-induced AMPK phosphorylation, suggesting the involvement of calcium in CoQ10-mediated AMPK signaling. CoQ10 also increased the expression of peroxisome proliferator-activated receptor alpha (PPAR?) at both the mRNA and protein levels. Knock down of AMPK with siRNA or inhibition of AMPK using an AMPK inhibitor compound C blocked CoQ10-induced expression of PPAR?, indicating that AMPK plays a critical role in PPAR? induction. In addition, CoQ10 increased fatty acid oxidation in 3T3-L1preadipocytes. The promoter activity of PPAR? was increased by CoQ10 in an AMPK-dependent fashion. Moreover, the induction of acyl-CoA oxidase (ACO), a target gene of PPAR?, was blocked under the PPAR? knock down condition. Furthermore, treatment with CoQ10 blocked differentiation-induced adipogenesis. This blockade was not observed under the PPAR? knock-down condition. Collectively, these results demonstrate that CoQ10 induces PPAR? expression via the calcium-mediated AMPK signal pathway and suppresses differentiation-induced adipogenesis. PMID:22885103

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

    PubMed

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

    2014-11-14

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

  3. Isolation of genes specifically expressed in flat revertant cells derived from activated ras-transformed NIH 3T3 cells by treatment with azatyrosine.

    PubMed Central

    Krzyzosiak, W J; Shindo-Okada, N; Teshima, H; Nakajima, K; Nishimura, S

    1992-01-01

    We previously reported that mouse NIH 3T3 cells transformed by transfection of activated human c-Ha-ras become apparently normal upon treatment with the antibiotic azatyrosine. The revertant cells maintain their normal phenotype during prolonged culture in the absence of azatyrosine, although activated p21ras is still expressed. The normal phenotype induced by azatyrosine could be due to activation of expression of some cellular gene(s) in the cells that results in suppression of ras function. To identify the genes with increased expression in the revertant cells, we adopted differential screening of recombinants from a phage cDNA library made from mRNA of the revertant cells, hybridized with 32P-labeled cDNAs made from mRNAs of the ras-transformed NIH 3T3 cells and the revertant cells. Two clones thus isolated were found to be almost identical to the ras recision gene (rrg), which was identified as a tumor-suppressor gene by Contente et al. [Contente, S., Kenyon, K., Rimoldi, D. & Friedman, R. M. (1990) Science 249, 796-798]. Other genes identified were the collagen type III and rhoB genes. Approximately half the clones were found to contain a sequence corresponding to that of the murine retrovirus-like intracisternal A particle. We speculate that azatyrosine activates several cellular genes in the ras-transformed cells and that some of these genes, including rrg, act cooperatively to counteract ras function, resulting in reversion of the ras-transformed cells to the normal phenotype. Images PMID:1594588

  4. Real-time monitoring of inflammation status in 3T3-L1 adipocytes possessing a secretory Gaussia luciferase gene under the control of nuclear factor-kappa B response element

    SciTech Connect

    Nagasaki, Haruka; Yoshimura, Takeshi; Aoki, Naohito

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Inflammation status in adipocytes can be monitored by the new assay system. Black-Right-Pointing-Pointer Only an aliquot of conditioned medium is required without cell lysis. Black-Right-Pointing-Pointer Inflammation-attenuating compounds can be screened more conveniently. -- Abstract: We have established 3T3-L1 cells possessing a secretory Gaussia luciferase (GLuc) gene under the control of nuclear factor-kappa B (NF-{kappa}B) response element. The 3T3-L1 cells named 3T3-L1-NF-{kappa}B-RE-GLuc could differentiate into adipocyte as comparably as parental 3T3-L1 cells. Inflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1{beta} induced GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes in a concentration- and time-dependent manner. GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes was also induced when cultured with RAW264.7 macrophages and was dramatically enhanced by lipopolysaccharide (LPS)-activated macrophages. An NF-{kappa}B activation inhibitor BAY-11-7085 and an antioxidant N-acetyl cysteine significantly suppressed GLuc secretion induced by macrophages. Finally, we found that rosemary-derived carnosic acid strongly suppressed GLuc secretion induced by macrophages and on the contrary up-regulated adiponectin secretion. Collectively, by using 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes, inflammation status can be monitored in real time and inflammation-attenuating compounds can be screened more conveniently.

  5. ?-Tocotrienol attenuates TNF-?-induced changes in secretion and gene expression of MCP-1, IL-6 and adiponectin in 3T3-L1 adipocytes.

    PubMed

    Matsunaga, Tetsuro; Shoji, Ayae; Gu, Ning; Joo, Erina; Li, Shiho; Adachi, Tetsuya; Yamazaki, Hanae; Yasuda, Koichiro; Kondoh, Takashi; Tsuda, Kinsuke

    2012-04-01

    Tocotrienols, members of the vitamin E family, have been shown to possess anti-inflammatory properties and display activity against a variety of chronic diseases, such as cancer, cardiovascular and neurological diseases. However, whether tocotrienols contribute to the prevention of inflammatory responses in adipose tissue remains to be elucidated. In this study, we examined the effects of ?-tocotrienol, the most common tocotrienol isomer, on tumor necrosis factor-? (TNF-?)-induced inflammatory responses by measuring the expression of the adipokines, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and adiponectin in 3T3-L1 adipocytes. Exposure to TNF-? (10 ng/ml) for 24 h increased MCP-1 and IL-6 secretion, and decreased adiponectin secretion and peroxisome proliferator-activated receptor-? (PPAR?) mRNA expression. ?-tocotrienol effectively improved the TNF-?-induced adverse changes in MCP-1, IL-6 and adiponectin secretion, and in MCP-1, IL-6, adiponectin and PPAR? mRNA expression. Furthermore, TNF-?-mediated I?B-? phosphorylation and nuclear factor-?B (NF-?B) activation were significantly suppressed by the ?-tocotrienol treatment. Our results suggest that ?-tocotrienol may improve obesity-related functional abnormalities in adipocytes by attenuating NF-?B activation and the expression of inflammatory adipokines. PMID:22293775

  6. ?-tocotrienol attenuates TNF-?-induced changes in secretion and gene expression of MCP-1, IL-6 and adiponectin in 3T3-L1 adipocytes

    PubMed Central

    MATSUNAGA, TETSURO; SHOJI, AYAE; GU, NING; JOO, ERINA; LI, SHIHO; ADACHI, TETSUYA; YAMAZAKI, HANAE; YASUDA, KOICHIRO; KONDOH, TAKASHI; TSUDA, KINSUKE

    2012-01-01

    Tocotrienols, members of the vitamin E family, have been shown to possess anti-inflammatory properties and display activity against a variety of chronic diseases, such as cancer, cardiovascular and neurological diseases. However, whether tocotrienols contribute to the prevention of inflammatory responses in adipose tissue remains to be elucidated. In this study, we examined the effects of ?-tocotrienol, the most common tocotrienol isomer, on tumor necrosis factor-? (TNF-?)-induced inflammatory responses by measuring the expression of the adipokines, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and adiponectin in 3T3-L1 adipocytes. Exposure to TNF-? (10 ng/ml) for 24 h increased MCP-1 and IL-6 secretion, and decreased adiponectin secretion and peroxisome proliferator-activated receptor-? (PPAR?) mRNA expression. ?-tocotrienol effectively improved the TNF-?-induced adverse changes in MCP-1, IL-6 and adiponectin secretion, and in MCP-1, IL-6, adiponectin and PPAR? mRNA expression. Furthermore, TNF-?-mediated I?B-? phosphorylation and nuclear factor-?B (NF-?B) activation were significantly suppressed by the ?-tocotrienol treatment. Our results suggest that ?-tocotrienol may improve obesity-related functional abnormalities in adipocytes by attenuating NF-?B activation and the expression of inflammatory adipokines. PMID:22293775

  7. Purification and Characterization of Aporphine Alkaloids from Leaves of Nelumbo nucifera Gaertn and Their Effects on Glucose Consumption in 3T3-L1 Adipocytes

    PubMed Central

    Ma, Chengjun; Wang, Jinjun; Chu, Hongmei; Zhang, Xiaoxiao; Wang, Zhenhua; Wang, Honglun; Li, Gang

    2014-01-01

    Aporphine alkaloids from the leaves of Nelumbo nucifera Gaertn are substances of great interest because of their important pharmacological activities, particularly anti-diabetic, anti-obesity, anti-hyperlipidemic, anti-oxidant, and anti-HIV’s activities. In order to produce large amounts of pure alkaloid for research purposes, a novel method using high-speed counter-current chromatography (HSCCC) was developed. Without any initial cleanup steps, four main aporphine alkaloids, including 2-hydroxy-1-methoxyaporphine, pronuciferine, nuciferine and roemerine were successfully purified from the crude extract by HSCCC in one step. The separation was performed with a simple two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (5:3:3:2.5:5, v/v/v/v/v). In each operation, 100 mg crude extracts was separated and yielded 6.3 mg of 2-hydroxy-1-methoxyaporphine (95.1% purity), 1.1 mg of pronuciferine (96.8% purity), 8.5 mg of nuciferine (98.9% purity), and 2.7 mg of roemerine (97.4%) respectively. The chemical structure of four aporphine alkaloids are identified by means of electrospray ionization MS (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Moreover, the effects of four separated aporphine alkaloids on insulin-stimulated glucose consumption were examined in 3T3-L1 adipocytes. The results showed that 2-hydroxy-1-methoxyaporphine and pronuciferine increased the glucose consumption significantly as rosiglitazone did. PMID:24577311

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

    PubMed Central

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

    2014-01-01

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

  9. Neoplastic transformation and tumorigenesis associated with overexpression of imup-1 and imup-2 genes in cultured NIH/3T3 mouse fibroblasts

    SciTech Connect

    Ryoo, Zae Young . E-mail: jaewoong64@hanmail.net; Jung, Boo Kyoung; Lee, Sang Ryeul; Kim, Myoung Ok; Kim, Sung Hyun; Kim, Hyo Jin; Ahn, Jung Yong; Lee, Tae-Hoon; Cho, Youl Hee; Park, Jae Hak; Kim, Jin Kyeoung

    2006-10-27

    Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation.

  10. Extract from Edible Red Seaweed (Gelidium amansii) Inhibits Lipid Accumulation and ROS Production during Differentiation in 3T3-L1 Cells

    PubMed Central

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

    2012-01-01

    Gelidium (G.) amansii is a red alga widely distributed in the shallow waters around East Asian countries. We investigated the effect of G. amansii on lipid accumulation and ROS (Reactive Oxygen Species) production in 3T3-L1 cells. G. amansii extracts dose-dependently inhibited lipid formation and ROS generation in cultured cells. Our results showed that anti-adipogenic effect of G. amansii was due to the reduction in mRNA expressions of PPAR? peroxisome proliferator-activated receptor-? and aP2 (adipocyte protein 2). G. amansii extracts significantly decreased mRNA levels of a ROS-generator, NOX4 (nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4), and increased the protein levels of antioxidant enzymes including SOD1/2 (superoxide dis-mutases), Gpx (glutathione peroxidase), and GR (glutathione reductase), which can lead to the reduction of ROS in the cell. In addition, the G. amansii extract enhanced mRNA levels of adiponectin, one of the adipokines secreted from adipocytes, and GLUT4, glucose uptake protein. Taken together, our study shows that G. amansii extract inhibited lipid accumulation and ROS production by controlling adipogenic signals and ROS regulating genes. PMID:24471074

  11. Dieckol, a phlorotannin isolated from a brown seaweed, Ecklonia cava, inhibits adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1 preadipocytes.

    PubMed

    Ko, Seok-Chun; Lee, Myoungsook; Lee, Ji-Hyeok; Lee, Seung-Hong; Lim, Yunsook; Jeon, You-Jin

    2013-11-01

    In this study, we assessed the potential inhibitory effect of 5 species of brown seaweeds on adipogenesis the differentiation of 3T3-L1 preadipocytes into mature adipocytes by measuring Oil-Red O staining. The Ecklonia cava extract tested herein evidenced profound adipogenesis inhibitory effect, compared to that exhibited by the other four brown seaweed extracts. Thus, E. cava was selected for isolation of active compounds and finally the three polyphenol compounds of phlorotannins were obtained and their inhibitory effect on adipogenesis was observed. Among the phlorotannins, dieckol exhibited greatest potential adipogenesis inhibition and down-regulated the expression of peroxisome proliferator-activated receptor-? (PPAR?), CCAAT/enhancer-binding proteins (C/EBP?), sterol regulatory element-binding protein 1 (SREBP1) and fatty acid binding protein 4 (FABP4) in a dose-dependent manner. The specific mechanism mediating the effects of dieckol was confirmed by AMP-activated protein kinase (AMPK) activation. These results demonstrate inhibitory effect of dieckol compound on adipogenesis through the activation of the AMPK signal pathway. PMID:24211593

  12. Isomeric C12-alkamides from the roots of Echinacea purpurea improve basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes.

    PubMed

    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

    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

  13. Mechanism of transforming growth factor-beta1-induced expression of vascular endothelial growth factor in murine osteoblastic MC3T3-E1 cells.

    PubMed

    Chua, C C; Hamdy, R C; Chua, B H

    2000-06-01

    Transforming growth factor-beta1 (TGF-beta1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-beta1 is not clearly understood. In this study, we found that the addition of TGF-beta1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-beta1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-kappaB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-beta1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-beta1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-beta1 may be attributed to the production of VEGF. PMID:10838160

  14. ?-Mangostin Improves Glucose Uptake and Inhibits Adipocytes Differentiation in 3T3-L1 Cells via PPAR?, GLUT4, and Leptin Expressions

    PubMed Central

    Taher, Muhammad; Mohamed Amiroudine, Mohamed Zaffar Ali; Tengku Zakaria, Tengku Muhamad Faris Syafiq; Ichwan, Solachuddin J. A.; Kaderi, Mohd Arifin; Ahmed, Qamar Uddin; Zakaria, Zainul Amiruddin

    2015-01-01

    Obesity has been often associated with the occurrence of cardiovascular diseases, type 2 diabetes, and cancer. The development of obesity is also accompanied by significant differentiation of preadipocytes into adipocytes. In this study, we investigated the activity of ?-mangostin, a major xanthone component isolated from the stem bark of G. malaccensis, on glucose uptake and adipocyte differentiation of 3T3-L1 cells focusing on PPAR?, GLUT4, and leptin expressions. ?-Mangostin was found to inhibit cytoplasmic lipid accumulation and adipogenic differentiation. Cells treated with 50??M of ?-mangostin reduced intracellular fat accumulation dose-dependently up to 44.4% relative to MDI-treated cells. Analyses of 2-deoxy-D-[3H] glucose uptake activity showed that ?-mangostin significantly improved the glucose uptake (P < 0.05) with highest activity found at 25??M. In addition, ?-mangostin increased the amount of free fatty acids (FFA) released. The highest glycerol release level was observed at 50??M of ?-mangostin. qRT-PCR analysis showed reduced lipid accumulation via inhibition of PPAR? gene expression. Induction of glucose uptake and free fatty acid release by ?-mangostin were accompanied by increasing mRNA expression of GLUT4 and leptin. These evidences propose that ?-mangostin might be possible candidate for the effective management of obesity in future. PMID:25873982

  15. Active compounds from Lagerstroemia speciosa, insulin-like glucose uptake-stimulatory/inhibitory and adipocyte differentiation-inhibitory activities in 3T3-L1 cells.

    PubMed

    Bai, Naisheng; He, Kan; Roller, Marc; Zheng, Bolin; Chen, Xiaozhuo; Shao, Zhongguang; Peng, Tangsheng; Zheng, Qunyi

    2008-12-24

    Seven ellagitannins, lagerstroemin (1), flosin B (2), stachyurin (3), casuarinin (4), casuariin (5), epipunicacortein A (6), and 2, 3-(S)-hexahydroxydiphenoyl-alpha/beta-D-glucose (7), together with one ellagic acid sulfate, 3-O-methyl-ellagic acid 4'-sulfate (8), ellagic acid (9), and four methyl ellagic acid derivatives, 3-O-methylellagic acid (10), 3,3'-di-O-methylellagic acid (11), 3,4,3'-tri-O-methylellagic acid (12), and 3,4,8,9,10-pentahydroxydibenzo[b,d]pyran-6-one (13), were identified by the bioassay-directed isolation from the leaves of Lagerstroemia speciosa (L.) Pers. The chemical structures of these components were established on the basis of one- and two-dimensional NMR and high-resolution mass spectroscopic analyses. Other known compounds, including corosolic acid, gallic acid, 4-hydroxybenzoic acid, 3-O-methylprotocatechuic acid, caffeic acid, p-coumaric acid, kaempferol, quercetin, and isoquercitrin, were also isolated from the same plant. The obtained ellagitannins exhibited strong activities in both stimulating insulin-like glucose uptake (1-5 and 7) and inhibiting adipocyte differentiation (1 and 4) in 3T3-L1 cells. Meanwhile, ellagic acid derivatives (10-13) showed an inhibitory effect on glucose transport assay. This study is the first to report an inhibitory effect for methyl ellagic acid derivatives. PMID:19053366

  16. Inhibitory effects of harpagoside on TNF-?-induced pro-inflammatory adipokine expression through PPAR-? activation in 3T3-L1 adipocytes.

    PubMed

    Kim, Tae Kon; Park, Kyoung Sik

    2015-12-01

    Obesity is closely associated with increased production of pro-inflammatory adipokines, including interleukin (IL)-6, plasminogen activator inhibitor (PAI)-1, and adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, which contribute to chronic and low-grade inflammation in adipose tissue. Harpagoside, a major iridoid glycoside present in devil's claw, has been reported to show anti-inflammatory activities by suppression of lipopolysaccharide (LPS)-induced production of inflammatory cytokines in murine macrophages. The present study is aimed to investigate the effects of harpagoside on both tumor necrosis factor (TNF)-?-induced inflammatory adipokine expression and its underlying signaling pathways in differentiated 3T3-L1 cells. Harpagoside significantly inhibited TNF-?-induced mRNA synthesis and protein production of the atherogenic adipokines including IL-6, PAI-1, and MCP-1. Further investigation of the molecular mechanism revealed that pretreatment with harpagoside activated peroxisome proliferator-activated receptor (PPAR)-?. These findings suggest that the clinical application of medicinal plants which contain harpagoside may lead to a partial prevention of obesity-induced atherosclerosis by attenuating inflammatory responses. PMID:26049170

  17. Metabolic fate of glucose on 3T3-L1 adipocytes treated with grape seed-derived procyanidin extract (GSPE). Comparison with the effects of insulin.

    PubMed

    Pinent, M; Bladé, M C; Salvadó, M J; Arola, L; Ardévol, A

    2005-07-27

    In this paper we investigate the effects of a grape seed procyanidin extract (GSPE) on the metabolic fate of glucose in adipocytes. Differentiated 3T3-L1 cells were treated with 140 mg/L GSPE or 100 nM insulin for a short period (1 h, acute treatment) or for a long period (15 h, chronic treatment). 2-Deoxy-[1-(3)H]glucose uptake and [1-(14)C]glucose incorporation into cells, glycogen, and lipid were measured. We found that GSPE mimicked the anabolic effects of insulin but there were several important differences. GSPE stimulated glycogen synthesis less than insulin. After chronic exposure, GSPE induced a higher incorporation of glucose into lipid, mainly due to the increase in glucose directed to glycerol synthesis. Our main conclusions, therefore, are that GSPE has insulinomimetic properties and activates glycogen and lipid synthesis. However, the differences between the effects of GSPE and the effects of insulin indicate that GSPE uses mechanisms complementary to those of insulin signaling pathways to bring about these effects. PMID:16028976

  18. Suppressing the activity of ERR? in 3T3-L1 adipocytes reduces mitochondrial biogenesis but enhances glycolysis and basal glucose uptake

    PubMed Central

    Nie, Yaohui; Wong, Chiwai

    2009-01-01

    Estrogen-related receptor ? (ERR?) is thought to primarily regulate lipid oxidation and control the transcription of genes in the oxidative phosphorylation pathway in skeletal and cardiac muscles. However, its role in white adipose tissue is not well studied. In this study, we aimed to establish a role for ERR? in adipocytes by down-regulating its activity through its inverse agonist XCT-790 in differentiated 3T3-L1 adipocytes. We found that XCT-790 differentially reduced the expression of ERR? target genes. Specifically, XCT-790 reduced the expressions of peroxisome proliferator-activated receptor ? co-activator-1? (PGC-1?), resulting in reductions of mitochondrial biogenesis, adiogenesis and lipogeneis. Through suppressing the expression of another ERR? target gene pyruvate dehydrogenase kinase 2 (PDK2), we found that XCT-790 not only enhanced the conversion of pyruvate to acetyl-CoA and hyper-activated the tricarboxylic acid (TCA) cycle, but also led to higher levels of mitochondrial membrane potential and reactive oxidant species (ROS) production. Additionally, XCT-790 treatment also resulted in enhanced rates of glycolysis and basal glucose uptake. Therefore, ERR? stands at the crossroad of glucose and fatty acid utilization and acts as a homeostatic switch to regulate the flux of TCA cycle, mitochondrial membrane potential and glycolysis to maintain a steady level of ATP production, particularly, when mitochondrial biogenesis is reduced. PMID:18544047

  19. Effects of an AMP-activated protein kinase inhibitor, compound C, on adipogenic differentiation of 3T3-L1 cells.

    PubMed

    Gao, Ye; Zhou, Yi; Xu, Aimin; Wu, Donghai

    2008-09-01

    The role of AMP-activated protein kinase (AMPK) in adipocyte differentiation is not completely understood. Here we reported that an AMPK inhibitor, compound C, significantly inhibited adipogenic differentiation of 3T3-L1 cells in a dose dependent manner, and this inhibitory effect was primarily effective in the initial stage of differentiation. Compound C prevented the mitotic clonal expansion (MCE) of preadipocytes, probably by inhibiting expression of CCAAT/enhancer-binding protein (C/EBP)beta and delta, and subsequently blocked the expression of C/EBPalpha and peroxisome proliferator-activated receptor (PPAR)gamma and transcriptional activation of genes that produce the adipocyte phenotype. AMPK activity was also suppressed by compound C treatment during the early phase of adipogenic differentiation, which indicated that suppressed activation of AMPK by compound C may inhibit the MCE process of preadipocytes. Our results suggest that compound C might serve as a useful molecule in both basic and clinical research on adipogenesis and as a potential lead compound for the treatment of obesity. PMID:18758065

  20. Toddaculin, Isolated from of Toddalia asiatica (L.) Lam., Inhibited Osteoclastogenesis in RAW 264 Cells and Enhanced Osteoblastogenesis in MC3T3-E1 Cells

    PubMed Central

    Watanabe, Akio; Kumagai, Momochika; Mishima, Takashi; Ito, Junya; Otoki, Yurika; Harada, Teppei; Kato, Tsuyoshi; Yoshida, Mikihiko; Suzuki, Misora; Yoshida, Izumi; Fujita, Kazuhiro; Watai, Masatoshi; Nakagawa, Kiyotaka; Miyazawa, Teruo

    2015-01-01

    Osteoporosis with bone loss is widely recognized as a major health problem. Bone homeostasis is maintained by balancing bone formation and bone resorption. The imbalance caused by increased bone resorption over bone formation can lead to various bone-related diseases such as osteoporosis and rheumatoid arthritis. Osteoclasts are the principal cells responsible for bone resorption and the main targets of anti-resorptive therapies. However, excessive inhibition of osteoclast differentiation may lead to inhibition of osteoblast differentiation. Therefore, it is important to screen for new compounds capable of inhibiting bone resorption and enhancing bone formation. Toddalia asiatica (L.) Lam. has been utilized traditionally for medicinal purposes such as the treatment of rheumatism. Currently, the extract is considered to be a good source of pharmacological agents for the treatment of bone-related diseases, but the active compounds have yet to be identified. We investigated whether toddaculin, derived from Toddalia asiatica (L.) Lam., affects both processes by inhibiting bone resorption and enhancing bone formation. Towards this end, we used pre-osteoclastic RAW 264 cells and pre-osteoblastic MC3T3-E1 cells. We found that toddaculin not only inhibited the differentiation of osteoclasts via activation of the NF-?B, ERK 1/2, and p38 MAPK signaling pathways, but it also induced differentiation and mineralization of osteoblasts by regulating differentiation factors. Thus, toddaculin might be beneficial for the prevention and treatment of osteoporosis. PMID:25993011

  1. Ezrin, radixin, and moesin phosphorylation in NIH3T3 cells revealed angiotensin II type 1 receptor cell-type-dependent biased signaling.

    PubMed

    Ibrahim, Islam A A E-H; Nakaya, Michio; Kurose, Hitoshi

    2013-01-01

    ?-Arrestin-biased agonists are a new class of drugs with promising therapeutic effects. The molecular mechanisms of ?-arrestin-biased agonists are still not completely identified. Here, we investigated the effect of angiotensin II (AngII) and [Sar1,Ile4,Ile8] AngII (SII), a ?-arrestin-biased agonist, on ezrin-radixin-moesin (ERM) phosphorylation in NIH3T3 cells (a fibroblast cell line) stably expressing AngII type 1A receptor. ERM proteins are cross-linkers between the plasma membrane and the actin cytoskeleton and control a number of signaling pathways. We also investigated the role of G?q protein and ?-arrestins in mediating ERM phosphorylation. We found that AngII stimulates ERM phosphorylation by acting as a ?-arrestin-biased agonist and AngII-stimulated ERM phosphorylation is mediated by ?-arrestin2 not ?-arrestin1. We also found that SII inhibits ERM phosphorylation by acting as a G?q protein-biased agonist. We concluded that ERM phosphorylation is a unique ?-arrestin-biased agonism signal. Both AngII and SII can activate either G?q protein or ?-arrestin-mediated signaling as functional biased agonists according to the type of the cell on which they act. PMID:23575451

  2. ?-Mangostin Improves Glucose Uptake and Inhibits Adipocytes Differentiation in 3T3-L1 Cells via PPAR?, GLUT4, and Leptin Expressions.

    PubMed

    Taher, Muhammad; Mohamed Amiroudine, Mohamed Zaffar Ali; Tengku Zakaria, Tengku Muhamad Faris Syafiq; Susanti, Deny; Ichwan, Solachuddin J A; Kaderi, Mohd Arifin; Ahmed, Qamar Uddin; Zakaria, Zainul Amiruddin

    2015-01-01

    Obesity has been often associated with the occurrence of cardiovascular diseases, type 2 diabetes, and cancer. The development of obesity is also accompanied by significant differentiation of preadipocytes into adipocytes. In this study, we investigated the activity of ?-mangostin, a major xanthone component isolated from the stem bark of G. malaccensis, on glucose uptake and adipocyte differentiation of 3T3-L1 cells focusing on PPAR?, GLUT4, and leptin expressions. ?-Mangostin was found to inhibit cytoplasmic lipid accumulation and adipogenic differentiation. Cells treated with 50??M of ?-mangostin reduced intracellular fat accumulation dose-dependently up to 44.4% relative to MDI-treated cells. Analyses of 2-deoxy-D-[(3)H] glucose uptake activity showed that ?-mangostin significantly improved the glucose uptake (P < 0.05) with highest activity found at 25??M. In addition, ?-mangostin increased the amount of free fatty acids (FFA) released. The highest glycerol release level was observed at 50??M of ?-mangostin. qRT-PCR analysis showed reduced lipid accumulation via inhibition of PPAR? gene expression. Induction of glucose uptake and free fatty acid release by ?-mangostin were accompanied by increasing mRNA expression of GLUT4 and leptin. These evidences propose that ?-mangostin might be possible candidate for the effective management of obesity in future. PMID:25873982

  3. mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

    PubMed Central

    Watanabe, Sadanori; Ando, Yoshikazu; Yasuda, Shingo; Hosoya, Hiroshi; Watanabe, Naoki; Ishizaki, Toshimasa

    2008-01-01

    mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell. PMID:18287523

  4. Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

    SciTech Connect

    Kodama, H.; Iizuka, M.; Tomiyama, T.; Yoshida, K.; Seki, M.; Suda, T.; Nishikawa, S. )

    1991-01-01

    Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

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

    SciTech Connect

    Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

    2010-01-01

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

  6. Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

    PubMed Central

    Axelrod, J H; Reich, R; Miskin, R

    1989-01-01

    The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities. Images PMID:2747645

  7. Toddaculin, Isolated from of Toddalia asiatica (L.) Lam., Inhibited Osteoclastogenesis in RAW 264 Cells and Enhanced Osteoblastogenesis in MC3T3-E1 Cells.

    PubMed

    Watanabe, Akio; Kumagai, Momochika; Mishima, Takashi; Ito, Junya; Otoki, Yurika; Harada, Teppei; Kato, Tsuyoshi; Yoshida, Mikihiko; Suzuki, Misora; Yoshida, Izumi; Fujita, Kazuhiro; Watai, Masatoshi; Nakagawa, Kiyotaka; Miyazawa, Teruo

    2015-01-01

    Osteoporosis with bone loss is widely recognized as a major health problem. Bone homeostasis is maintained by balancing bone formation and bone resorption. The imbalance caused by increased bone resorption over bone formation can lead to various bone-related diseases such as osteoporosis and rheumatoid arthritis. Osteoclasts are the principal cells responsible for bone resorption and the main targets of anti-resorptive therapies. However, excessive inhibition of osteoclast differentiation may lead to inhibition of osteoblast differentiation. Therefore, it is important to screen for new compounds capable of inhibiting bone resorption and enhancing bone formation. Toddalia asiatica (L.) Lam. has been utilized traditionally for medicinal purposes such as the treatment of rheumatism. Currently, the extract is considered to be a good source of pharmacological agents for the treatment of bone-related diseases, but the active compounds have yet to be identified. We investigated whether toddaculin, derived from Toddalia asiatica (L.) Lam., affects both processes by inhibiting bone resorption and enhancing bone formation. Towards this end, we used pre-osteoclastic RAW 264 cells and pre-osteoblastic MC3T3-E1 cells. We found that toddaculin not only inhibited the differentiation of osteoclasts via activation of the NF-?B, ERK 1/2, and p38 MAPK signaling pathways, but it also induced differentiation and mineralization of osteoblasts by regulating differentiation factors. Thus, toddaculin might be beneficial for the prevention and treatment of osteoporosis. PMID:25993011

  8. 19. DETAIL OF STAMP BATTERY AUTOMATIC FEEDER, LOOKING EAST. THIS ...

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

    19. DETAIL OF STAMP BATTERY AUTOMATIC FEEDER, LOOKING EAST. THIS IS THE MIDDLE OF THREE FEEDERS, ONE FOR EACH STAMP BATTERY. THE CHUTE (UPPER RIGHT) INTRODUCED THE CRUSHED ORE FROM THE ORE BIN. FLOW WAS CONTROLLED BY A SLIDING DOOR ON THE UPPER LEVEL. - Skidoo Mine, Park Route 38 (Skidoo Road), Death Valley Junction, Inyo County, CA

  9. Faculty Articulation with Feeder High Schools and Local Employers.

    ERIC Educational Resources Information Center

    Parrott, Marietta

    As a first step in developing an articulation plan with feeder high schools, a College of the Sequoias (COS) task force developed and distributed a survey to all full-time faculty members to determine if individual faculty members were articulating with feeder high schools and local businesses, and if they would be willing to participate in an…

  10. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING... fire bulkheads forming fire zones, at least one emergency lighting feeder must supply only the emergency lights between two adjacent main vertical fire zone bulkheads. The emergency lighting feeder...

  11. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency lighting feeders. For a vessel with fire bulkheads forming fire zones,...

  12. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING... fire bulkheads forming fire zones, at least one emergency lighting feeder must supply only the emergency lights between two adjacent main vertical fire zone bulkheads. The emergency lighting feeder...

  13. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING... fire bulkheads forming fire zones, at least one emergency lighting feeder must supply only the emergency lights between two adjacent main vertical fire zone bulkheads. The emergency lighting feeder...

  14. 46 CFR 111.75-1 - Lighting feeders.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Lighting feeders. 111.75-1 Section 111.75-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 Lighting feeders. (a) Passenger vessels. On a passenger vessel with fire bulkheads...

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

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING... fire bulkheads forming fire zones, at least one emergency lighting feeder must supply only the emergency lights between two adjacent main vertical fire zone bulkheads. The emergency lighting feeder...

  16. 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

  17. Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.

    PubMed

    Yu, G; Okawa, H; Okita, K; Kamano, Y; Wang, F; Saeki, M; Yatani, H; Egusa, H

    2016-01-01

    Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin ?5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of autologous hGF-iPSCs, thus providing an important step toward the future therapeutic application of hGF-iPSCs. PMID:26467419

  18. Heat or radiofrequency plasma glow discharge treatment of a titanium alloy stimulates osteoblast gene expression in the MC3T3 osteoprogenitor cell line

    PubMed Central

    Rapuano, Bruce E.; Hackshaw, Kyle

    2012-01-01

    Purpose The purpose of this study was to determine whether increasing the Ti6Al4V surface oxide negative charge through heat (600?) or radiofrequency plasma glow discharge (RFGD) pretreatment, with or without a subsequent coating with fibronectin, stimulated osteoblast gene marker expression in the MC3T3 osteoprogenitor cell line. Methods Quantitative real-time polymerase chain reaction was used to measure changes over time in the mRNA levels for osteoblast gene markers, including alkaline phosphatase, bone sialoprotein, collagen type I (?1), osteocalcin, osteopontin and parathyroid hormone-related peptide (PTH-rP), and the osteoblast precursor genes Runx2 and osterix. Results Osteoprogenitors began to differentiate earlier on disks that were pretreated with heat or RFGD. The pretreatments increased gene marker expression in the absence of a fibronectin coating. However, pretreatments increased osteoblast gene expression for fibronectin-coated disks more than uncoated disks, suggesting a surface oxide-mediated specific enhancement of fibronectin's bioactivity. Heat pretreatment had greater effects on the mRNA expression of genes for PTH-rP, alkaline phosphatase and osteocalcin while RFGD pretreatment had greater effects on osteopontin and bone sialoprotein gene expression. Conclusions The results suggest that heat and RFGD pretreatments of the Ti6Al4V surface oxide stimulated osteoblast differentiation through an enhancement of (a) coated fibronectin's bioactivity and (b) the bioactivities of other serum or matrix proteins. The quantitative differences in the effects of the two pretreatments on osteoblast gene marker expression may have arisen from the unique physico-chemical characteristics of each resultant oxide surface. Therefore, engineering the Ti6Al4V surface oxide to become more negatively charged can be used to accelerate osteoblast differentiation through fibronectin-dependent and independent mechanisms. PMID:22803011

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

    PubMed Central

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

    2013-01-01

    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

  20. mTORC1 inhibition via rapamycin promotes triacylglycerol lipolysis and release of free fatty acids in 3T3-L1 adipocytes.

    PubMed

    Soliman, Ghada A; Acosta-Jaquez, Hugo A; Fingar, Diane C

    2010-12-01

    Signaling by mTOR complex 1 (mTORC1) promotes anabolic cellular processes in response to growth factors, nutrients, and hormonal cues. Numerous clinical trials employing the mTORC1 inhibitor rapamycin (aka sirolimus) to immuno-suppress patients following organ transplantation have documented the development of hypertriglyceridemia and elevated serum free fatty acids (FFA). We therefore investigated the cellular role of mTORC1 in control of triacylglycerol (TAG) metabolism using cultured murine 3T3-L1 adipocytes. We found that treatment of adipocytes with rapamycin reduced insulin-stimulated TAG storage ~50%. To determine whether rapamycin reduces TAG storage by upregulating lipolytic rate, we treated adipocytes in the absence and presence of rapamycin and isoproterenol, a ?2-adrenergic agonist that activates the cAMP/protein kinase A (PKA) pathway to promote lipolysis. We found that rapamycin augmented isoproterenol-induced lipolysis without altering cAMP levels. Rapamycin enhanced the isoproterenol-stimulated phosphorylation of hormone sensitive lipase (HSL) on Ser-563 (a PKA site), but had no effect on the phosphorylation of HSL S565 (an AMPK site). Additionally, rapamycin did not affect the isoproterenol-mediated phosphorylation of perilipin, a protein that coats the lipid droplet to initiate lipolysis upon phosphorylation by PKA. These data demonstrate that inhibition of mTORC1 signaling synergizes with the ?-adrenergic-cAMP/PKA pathway to augment phosphorylation of HSL to promote hormone-induced lipolysis. Moreover, they reveal a novel metabolic function for mTORC1; mTORC1 signaling suppresses lipolysis, thus augmenting TAG storage. PMID:21042876

  1. Glycerol Production from Glucose and Fructose by 3T3-L1 Cells: A Mechanism of Adipocyte Defense from Excess Substrate

    PubMed Central

    Romero, María del Mar; Sabater, David; Fernández-López, José Antonio; Remesar, Xavier; Alemany, Marià

    2015-01-01

    Cultured adipocytes (3T3-L1) produce large amounts of 3C fragments; largely lactate, depending on medium glucose levels. Increased glycolysis has been observed also in vivo in different sites of rat white adipose tissue. We investigated whether fructose can substitute glucose as source of lactate, and, especially whether the glycerol released to the medium was of lipolytic or glycolytic origin. Fructose conversion to lactate and glycerol was lower than that of glucose. The fast exhaustion of medium glucose was unrelated to significant changes in lipid storage. Fructose inhibited to a higher degree than glucose the expression of lipogenic enzymes. When both hexoses were present, the effects of fructose on gene expression prevailed over those of glucose. Adipocytes expressed fructokinase, but not aldolase b. Substantive release of glycerol accompanied lactate when fructose was the substrate. The mass of cell triacylglycerol (and its lack of change) could not justify the comparatively higher amount of glycerol released. Consequently, most of this glycerol should be derived from the glycolytic pathway, since its lipolytic origin could not be (quantitatively) sustained. Proportionally (with respect to lactate plus glycerol), more glycerol was produced from fructose than from glucose, which suggests that part of fructose was catabolized by the alternate (hepatic) fructose pathway. Earlier described adipose glycerophophatase activity may help explain the glycolytic origin of most of the glycerol. However, no gene is known for this enzyme in mammals, which suggests that this function may be carried out by one of the known phosphatases in the tissue. Break up of glycerol-3P to yield glycerol, may be a limiting factor for the synthesis of triacylglycerols through control of glycerol-3P availability. A phosphatase pathway such as that described may have a potential regulatory function, and explain the production of glycerol by adipocytes in the absence of lipolytic stimulation. PMID:26426115

  2. Tension Force Downregulates Matrix Metalloproteinase Expression and Upregulates the Expression of Their Inhibitors through MAPK Signaling Pathways in MC3T3-E1 cells

    PubMed Central

    Karasawa, Yoko; Tanaka, Hideki; Nakai, Kumiko; Tanabe, Natsuko; Kawato, Takayuki; Maeno, Masao; Shimizu, Noriyoshi

    2015-01-01

    Objective: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways. Design: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting. Results: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3. Conclusions: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts. PMID:26640410

  3. Effects of Varying Degrees of Intermittent Hypoxia on Proinflammatory Cytokines and Adipokines in Rats and 3T3-L1 Adipocytes

    PubMed Central

    Zhou, Qin; Zhu, Hui; Niu, Wen-yan; Feng, Jing; Wang, Yan; Cao, Jie; Chen, Bao-yuan

    2014-01-01

    Objectives Intermittent hypoxia (IH), resulted from recurring episodes of upper airway obstruction, is the hallmark feature and the most important pathophysiologic pathway of obstructive sleep apnea (OSA). IH is believed to be the most important factor causing systemic inflammation. Studies suggest that insulin resistance (IR) is positively associated with OSA. In this study, we hypothesized that the recurrence of IH might result in cellular and systemic inflammation, which was manifested through the levels of proinflammatory cytokines and adipokines after IH exposure, and because IR is linked with inflammation tightly, this inflammatory situation may implicate an IR status. Methods We developed an IH 3T3-L1 adipocyte and rat model respectively, recapitulating the nocturnal oxygen profile in OSA. In IH cells, nuclear factor kappa B (NF-?B) DNA binding reactions, hypoxia-inducible factor-1? (HIF-1?), glucose transporter-1 (Glut-1), necrosis factor alpha (TNF-?), interleukin (IL) -6, leptin, adiponectin mRNA transcriptional activities and protein expressions were measured. In IH rats, blood glucose, insulin, TNF-?, IL-6, leptin and adiponectin levels were analyzed. Results The insulin and blood glucose levels in rats and NF-?B DNA binding activities in cells had significantly statistical results described as severe IH>moderate IH>mild IH>sustained hypoxia>control. The mRNA and protein levels of HIF-1? and Glut-1 in severe IH group were the highest. In cellular and animal models, both the mRNA and protein levels of TNF-?, IL-6 and leptin were the highest in severe IH group, when the lowest in severe IH group for adiponectin. Conclusions Oxidative stress and the release of pro-inflammatory cytokines/adipokines, which are the systemic inflammatory markers, are associated with IH closely and are proportional to the severity of IH. Because IR and glucose intolerance are linked with inflammation tightly, our results may implicate the clinical relationships between OSA and IR. PMID:24466027

  4. Proliferation and osteogenic response of MC3T3-E1 pre-osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria.

    PubMed

    Hadjicharalambous, Chrystalleni; Mygdali, Evdokia; Prymak, Oleg; Buyakov, Ales; Kulkov, Sergei; Chatzinikolaidou, Maria

    2015-11-01

    Dense zirconia ceramics are used in bone applications due to their mechanical strength and biocompatibility, but lack osseointegration. A porous interface in contact with bone tissue may lead to better bone bonding but the biological properties of porous zirconia are not widely explored. The present study focuses on the manufacturing of an yttria- (YSZ) and a magnesia-stabilized (MgSZ) porous zirconia, and on their in vitro biological investigation. The sintered ceramics had similar characteristics of porosity, pore size and interconnectivity. Their elastic moduli and compressive strength values were within the range of the values of human cortical bone. MC3T3-E1 pre-osteoblasts were used to investigate the proliferation, alkaline phosphatase (ALP) activity, collagen deposition and expression profile of four genes involved in bone metabolism of cells on porous ceramics. Scanning electron and fluorescence microscopy were employed to visualize cell morphology and growth. Pre-osteoblasts adhered well on both ceramics but cell numbers on YSZ were higher. Cells exhibited an increase in ALP activity and collagen deposition after 14 days on both MgSZ and YSZ, with higher levels on YSZ. Real-time quantitative polymerase chain reaction (qPCR) showed that the expression of bone sialoprotein (Bsp) and collagen type I (col1aI) were significantly higher on YSZ. No significant differences were found in their ability to regulate the early gene expression of Runx2 and Alp. Nevertheless, the biomineralized calcium content was similar on both ceramics after 21 days, indicating that despite chemical differences, both scaffolds direct the pre-osteoblasts toward a mature state capable of mineralizing the extracellular matrix. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3612-3624, 2015. PMID:25847599

  5. Absence of an adipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes: increase of lipid catabolism and reduction of adipokine expression

    PubMed Central

    Wang, P.; Renes, J.; Bouwman, F.; Bunschoten, A.; Mariman, E.

    2007-01-01

    Aims/hypothesis The thiazolidinedione (TZD) rosiglitazone is a peroxisome proliferator-activated receptor-? agonist that induces adipocyte differentiation and, hence, lipid accumulation. This is in apparent contrast to the long-term glucose-lowering, insulin-sensitising effect of rosiglitazone. We tested whether the action of rosiglitazone involves specific effects on mature adipocytes, which are different from those on preadipocytes. Materials and methods Differentiated mature 3T3-L1 adipocytes were used as an in vitro model. Transcriptomics, proteomics and assays of metabolism were applied to assess the effect of rosiglitazone in different insulin and glucose conditions. Results Rosiglitazone does not induce an increase, but rather a decrease in the lipid content of mature adipocytes. Analysis of transcriptome data, confirmed by quantitative RT-PCR and measurements of lipolysis, indicates that an altered energy metabolism may underlie this change. The pathway analysis shows a consistent picture dominated by lipid catabolism. In addition, we confirmed at both mRNA level and protein level that rosiglitazone represses adipokine expression and production, except for genes encoding adiponectin and apolipoprotein E. Moreover, transcriptome changes indicate that a general repression of genes encoding secreted proteins occurs. Conclusions/interpretation Our findings suggest that the change of adiposity as seen in vivo reflects a shift in balance between the different effects of TZDs on preadipocytes and on mature adipocytes, while the changes in circulating adipokine levels primarily result from an effect on mature adipocytes. Electronic supplementary material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00125-006-0565-0 and is accessible to authorised users only PMID:17245590

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

    PubMed Central

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

    2015-01-01

    Our previous studies showed that nitric oxide (NO) could induce osteoblast apoptosis. MicroRNA-1 (miR-1), a skeletal- and cardiac muscle-specific small non-coding RNA, contributes to the regulation of multiple cell activities. In this study, we evaluated the roles of miR-1 in NO-induced insults to osteoblasts and the possible mechanisms. Exposure of mouse MC3T3-E1 cells to sodium nitroprusside (SNP) increased amounts of cellular NO and intracellular reactive oxygen species. Sequentially, SNP decreased cell survival but induced caspase-3 activation, DNA fragmentation, and cell apoptosis. In parallel, treatment with SNP induced miR-1 expression in a time-dependent manner. Application of miR-1 antisense inhibitors to osteoblasts caused significant inhibition of SNP-induced miR-1 expression. Knocking down miR-1 concurrently attenuated SNP-induced alterations in cell morphology and survival. Consecutively, SNP time-dependently inhibited heat-shock protein (HSP)-70 messenger (m)RNA and protein expressions. A bioinformatic search predicted the existence of miR-1-specific binding elements in the 3'-untranslational region of HSP-70 mRNA. Downregulation of miR-1 expression simultaneously lessened SNP-induced inhibition of HSP-70 mRNA and protein expressions. Consequently, SNP-induced modifications in the mitochondrial membrane potential, caspase-3 activation, DNA fragmentation, and apoptotic insults were significantly alleviated by miR-1 antisense inhibitors. Therefore, this study showed that miR-1 participates in NO-induced apoptotic insults through targeting HSP-70 gene expression. PMID:25678843

  7. Facilitation of adipocyte differentiation of 3T3-L1 cells by debrominated tetrabromobisphenol A compounds detected in Japanese breast milk.

    PubMed

    Akiyama, Ema; Kakutani, Hideki; Nakao, Teruyuki; Motomura, Yuka; Takano, Yuki; Sorakubo, Ryota; Mizuno, Ayami; Aozasa, Osamu; Tachibana, Keisuke; Doi, Takefumi; Ohta, Souichi

    2015-07-01

    Tetrabromobisphenol A (TeBBPA) is widely used type of brominated flame retardant. In this study, we newly synthesized materials for the debrominated congeners, 2,2',6-tribromobisphenol A (TriBBPA), 2,2'-dibromobisphenol A (2,2'-DiBBPA), 2,6-dibromobisphenol A (2,6-DiBBPA), and 2-monobromobisphenol A (MoBBPA) and evaluated the actual extent of contamination with bisphenol A (BPA), TeBBPA and debrominated congeners in Japanese breast milk samples. TriBBPA was detected at higher levels than that of TeBBPA, while DiBBPA and MoBBPA were detected at lower levels than that of TeBBPA. This observation suggested that humans are exposed to debrominated congeners, which might cause adverse effects. Contamination of the congeners in breast milk was concern about risk infant health, having vulnerable defense system. As pilot study by in vitro experiment, we assessed the toxic potency of debrominated congeners by studying their effect on adipocyte differentiation in 3T3-L1 cells. We observed 2,6-DiBBPA, TriBBPA and TeBBPA elevated the lipid accumulation and adipocyte-specific protein 2 expression in a manner dependent on the number of substituted bromines. Moreover, PPAR? transcriptional activities increased in a dose-dependent manner in the presence of 2,6-DiBBPA and TriBBPA as well as TeBBPA. Our study clarified that TeBBPA and its debrominated congeners accumulated in breast milk and the debrominated congeners promoted adipocyte differentiation, showing that a comprehensive evaluation of the influences of these compounds including the debrominated congeners of TeBBPA on health in infants is necessary. PMID:25863188

  8. Regulation of Apelin and Its Receptor Expression in Adipose Tissues of Obesity Rats with Hypertension and Cultured 3T3-L1 Adipocytes

    PubMed Central

    Wu, Hongxian; Cheng, Xian Wu; Hao, Changning; Zhang, Zhi; Yao, Huali; Murohara, Toyoaki; Dai, Qiuyan

    2014-01-01

    The apelin/APJ system has been implicated in obesity-related hypertension. We investigated the mechanism responsible for the pathogenesis of obesity-related hypertension with a special focus on the crosstalk between AngII/its type 1 receptor (AT1R) signaling and apelin/APJ expression. Sprague-Dawley rats fed a high-fat (obesity-related hypertension, OH) or normal-fat diet (NF) for 15 weeks were randomly assigned to one of two groups and administered vehicle or perindopril for 4 weeks. Compared to the NF rats, the OH rats showed lower levels of plasma apelin and apelin/APJ mRNAs of perirenal adipose tissues, and these changes were restored by perindopril. Administration of the AT1R antagonist olmesartan resulted in the restoration of the reduction of apelin and APJ expressions induced by AngII for 48 h in 3T3-L1 adipocytes. Among several inhibitors for extracellular signal-regulated kinases 1/2 (ERK1/2) PD98059, p38 mitogen-activated protein kinase (p38MAPK) SB203580 and phosphatidylinositol 3-kinase (PI3K) LY294002, the latter showed an additive effect on AngII-mediated inhibitory effects. In addition, the levels of p-Akt, p-ERK and p38MAPK proteins were decreased by long-term treatment with AngII (120 min), and these changes were restored by Olmesartan. Apelin/APJ appears to be impaired in obesity-related hypertension. The AngII inhibition-mediated beneficial effects are likely attributable, at least in part, to restoration of p38/ERK-dependent apelin/APJ expression in diet-induced obesity-related hypertension. PMID:24770651

  9. Protein kinase C activation potently down-regulates the expression of its major substrate, 80K, in Swiss 3T3 cells.

    PubMed

    Brooks, S F; Herget, T; Erusalimsky, J D; Rozengurt, E

    1991-09-01

    The amino acid sequence of 80K, the major acidic protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts, was deduced from a cDNA nucleotide sequence. Overall, 25% of the predicted amino acid sequence is supported by direct protein sequence data. Southern blot analysis suggests that the mouse genome contains a single copy of this gene. Two 80K mRNA species, a major band of 2.25 kb and a minor band of 3.9 kb, were detected by Northern blot analysis. Stimulation of PKC by biologically active phorbol esters, including phorbol-12, 13-dibutyrate (PDB), reduced the steady state level of 80K mRNA to 8.8% of control within 5-7 h. This effect was dose-dependent, and was abolished by prior depletion of PKC. The PDB-induced down-regulation of 80K mRNA levels was transient, and recovery coincided with the disappearance of PKC activity. A similar transient decrease in 80K mRNA levels was also demonstrated in tertiary cultures of mouse embryo fibroblasts. The down-regulation of 80K mRNA levels was completely abolished by actinomycin D, cycloheximide or anisomycin if added up to 30 min after PDB addition. Since the rate of transcription of the 80K gene was unaltered by PDB treatment, we concluded that the PKC-induced down-regulation of 80K mRNA is mediated by a post-transcriptional mechanism. In addition, PDB transiently decreased the level of 80K protein within 14-18 h, thus reflecting the effects of this phorbol ester on mRNA expression. PMID:1868832

  10. Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors.

    PubMed

    Borgatti, P; Martelli, A M; Bellacosa, A; Casto, R; Massari, L; Capitani, S; Neri, L M

    2000-07-14

    An active phosphatidylinositol 3-kinase (PI3K) has been shown in nuclei of different cell types. The products of this enzyme, i.e. inositides phosphorylated in the D3 position of the inositol ring, may act as second messengers themselves. Nuclear PI3K translocation has been demonstrated to be related to an analogous translocation of a PtdIns(3,4,5)P(3) activated PKC, the zeta isozyme. We have examined the issue of whether or not in the osteoblast-like clonal cell line MC3T3-E1 there may be observed an insulin-like growth factor-I- (IGF-I) and platelet-derived growth factor- (PDGF) dependent nuclear translocation of an active Akt/PKB. Western blot analysis showed a maximal nuclear translocation after 20 min of IGF-I stimulation or after 30 min of PDGF treatment. Both growth factors increased rapidly and transiently the enzyme activity of immunoprecipitable nuclear Akt/PKB on a similar time scale and after 60 min the values were slightly higher than the basal levels. Enzyme translocation was blocked by the specific PI3K inhibitor, LY294002, as well as cell entry into S-phase. Confocal microscopy showed an evident increase in immunostaining intensity in the nuclear interior after growth factor treatment but no changes in the subcellular distribution of Akt/PKB when a LY294002 pre-treatment was administered to the cells. These findings strongly suggest that the intranuclear translocation of Akt/PKB is an important step in signalling pathways that mediate cell proliferation. PMID:10899305

  11. Effects of long-term 50Hz power-line frequency electromagnetic field on cell behavior in Balb/c 3T3 cells.

    PubMed

    An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

    2015-01-01

    Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn't change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein. PMID:25695503

  12. TSPY potentiates cell proliferation and tumorigenesis by promoting cell cycle progression in HeLa and NIH3T3 cells

    PubMed Central

    Oram, Shane W; Liu, Xing Xing; Lee, Tin-Lap; Chan, Wai-Yee; Lau, Yun-Fai Chris

    2006-01-01

    Background TSPY is a repeated gene mapped to the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Elevated levels of TSPY have been observed in gonadoblastoma specimens and a variety of other tumor tissues, including testicular germ cell tumors, prostate cancer, melanoma, and liver cancer. TSPY contains a SET/NAP domain that is present in a family of cyclin B and/or histone binding proteins represented by the oncoprotein SET and the nucleosome assembly protein 1 (NAP1), involved in cell cycle regulation and replication. Methods To determine a possible cellular function for TSPY, we manipulated the TSPY expression in HeLa and NIH3T3 cells using the Tet-off system. Cell proliferation, colony formation assays and tumor growth in nude mice were utilized to determine the TSPY effects on cell growth and tumorigenesis. Cell cycle analysis and cell synchronization techniques were used to determine cell cycle profiles. Microarray and RT-PCR were used to investigate gene expression in TSPY expressing cells. Results Our findings suggest that TSPY expression increases cell proliferation in vitro and tumorigenesis in vivo. Ectopic expression of TSPY results in a smaller population of the host cells in the G2/M phase of the cell cycle. Using cell synchronization techniques, we show that TSPY is capable of mediating a rapid transition of the cells through the G2/M phase. Microarray analysis demonstrates that numerous genes involved in the cell cycle and apoptosis are affected by TSPY expression in the HeLa cells. Conclusion These data, taken together, have provided important insights on the probable functions of TSPY in cell cycle progression, cell proliferation, and tumorigenesis. PMID:16762081

  13. Transformation of BALB/c-3T3 cells: II. Investigation of experimental parameters that influence detection of benzo[a]pyrene-induced transformation.

    PubMed Central

    Matthews, E J

    1993-01-01

    Benzo[a]pyrene (BaP) induced significant morphological transformation of clone A31-1-13 BALB/c-3T3 cells without exogenous activation. Therefore, BaP was selected as a model to determine the internal consistency of detection of chemical-induced transformation. BaP induced a continuum of type I-III foci of different sizes, and the ratio of type I-III to type III foci/vessel was usually about 2-fold. The major finding was that BaP induced highly significant transformation responses, and the magnitude of these responses were inversely correlated with the cytotoxicity of the treatment doses. Thus, the induction of BaP-induced transformation behaved as though it was caused by a mutational event. Variability among responses were shown to depend on the serum lot and the cryopreserved ampule of cells. In addition, experiments with low spontaneous transformation responses had an impaired ability to detect BaP; however, experiments with high or normal spontaneous responses had a normal ability to detect BaP. Because the expression of BaP-induced transformation depended on both the cytotoxicity of the treatment and the cumulative number of mitoses, the frequency of BaP-induced transformation should be reported as the number of foci/vessel, but not expressed as the number of foci/viable cell surviving the chemical treatment. These conclusions are important because the same 110 experiments described in this report were also used to evaluate the transformation responses of many different carcinogenic and noncarcinogenic chemicals. These data are being reported separately. PMID:8243399

  14. Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples

    PubMed Central

    Dessaux, Sophie; Besse, Anthony; Brahimi-Adouane, Sabrina; Emile, Jean-François; Blay, Jean-Yves; Alberti, Laurent

    2013-01-01

    Objective Most gain of function mutations of tyrosine kinase receptors in human tumours are hemizygous. Gastrointestinal stromal tumours (GIST) with homozygous mutations have a worse prognosis. We aimed to identify genes differentially regulated by hemizygous and heterozygous KIT mutations. Materials and Methods Expression of 94 genes and 384 miRNA was analysed with low density arrays in five NIH3T3 cell lines expressing the full-length human KIT cDNA wild-type (WT), hemizygous KIT mutation with del557-558 (D6) or del564-581 (D54) and heterozygous WT/D6 or WT/D54. Expression of 5 of these genes and 384 miRNA was then analysed in GISTs samples. Results Unsupervised and supervised hierarchical clustering of the mRNA and miRNA profiles showed that heterozygous mutants clustered with KIT WT expressing cells while hemizygous mutants were distinct. Among hemizygous cells, D6 and D54 expressing cells clustered separately. Most deregulated genes have been reported as potentially implicated in cancer and severals, as ANXA8 and FBN1, are highlighted by both, mRNA and miRNA analyses. MiRNA and mRNA analyses in GISTs samples confirmed that their expressions varied according to the mutation of the alleles. Interestingly, RGS16, a membrane protein of the regulator of G protein family, correlate with the subcellular localization of KIT mutants and might be responsible for regulation of the PI3K/AKT signalling pathway. Conclusion Patterns of mRNA and miRNA expression in cells and tumours depend on heterozygous/hemizygous status of KIT mutations, and deletion/presence of TYR568 & TYR570 residues. Thus each mutation of KIT may drive specific oncogenic pathways. PMID:23593401

  15. Protein kinase C activation potently down-regulates the expression of its major substrate, 80K, in Swiss 3T3 cells.

    PubMed Central

    Brooks, S F; Herget, T; Erusalimsky, J D; Rozengurt, E

    1991-01-01

    The amino acid sequence of 80K, the major acidic protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts, was deduced from a cDNA nucleotide sequence. Overall, 25% of the predicted amino acid sequence is supported by direct protein sequence data. Southern blot analysis suggests that the mouse genome contains a single copy of this gene. Two 80K mRNA species, a major band of 2.25 kb and a minor band of 3.9 kb, were detected by Northern blot analysis. Stimulation of PKC by biologically active phorbol esters, including phorbol-12, 13-dibutyrate (PDB), reduced the steady state level of 80K mRNA to 8.8% of control within 5-7 h. This effect was dose-dependent, and was abolished by prior depletion of PKC. The PDB-induced down-regulation of 80K mRNA levels was transient, and recovery coincided with the disappearance of PKC activity. A similar transient decrease in 80K mRNA levels was also demonstrated in tertiary cultures of mouse embryo fibroblasts. The down-regulation of 80K mRNA levels was completely abolished by actinomycin D, cycloheximide or anisomycin if added up to 30 min after PDB addition. Since the rate of transcription of the 80K gene was unaltered by PDB treatment, we concluded that the PKC-induced down-regulation of 80K mRNA is mediated by a post-transcriptional mechanism. In addition, PDB transiently decreased the level of 80K protein within 14-18 h, thus reflecting the effects of this phorbol ester on mRNA expression. Images PMID:1868832

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

    PubMed

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

    2014-01-01

    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

  17. Saikosaponin a, an active compound of Radix Bupleuri, attenuates inflammation in hypertrophied 3T3-L1 adipocytes via ERK/NF-?B signaling pathways.

    PubMed

    Kim, Sung Ok; Park, Ji Yeoung; Jeon, Seo Young; Yang, Chea Ha; Kim, Mi Ryeo

    2015-04-01

    Bupleurum falcatum L. is employed in oriental medicine in Korea. This root has been used for anti-inflammatory, anti-pyretic, and anti-hepatotoxic effects in the treatments of common cold, fever, and hepatitis. One of major bioactive compounds of Radix Bupleuri is the saikosaponin a (SSNa). However, little is known concerning the effects of SSNa on obesity associated with a state of low-grade inflammation. Consequently, this study was conducted to determine the inhibition of the inflammation pathway of SSNa in obesity. MTT assay was conducted for cytotoxicity and viability; nuclear and cytoplasmic fractions were extracted from adipocytes for translocation of nuclear factor-?B cells (NF-?B); nitric oxide (NO) production and secretion using Griess reagent; reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting for mRNA and protein levels associated with inflammation in the hypertrophied adipocytes. The results revealed that SSNa significantly decreased the expression of tumor necrosis factor-? (TNF?), interleukin (IL)-1? and IL-6 as proinflammatory cytokines, compared to that of non-treated control cells. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as inflammatory factors were reduced by treatment of these cells with SSNa and also suppressed NO production. Phosphorylation of I?B? was inhibited and translocation of NF-?B was suppressed via the ERK pathway in response to SSNa treatment. In conclusion, the results demonstrated that SSNa can inhibit the expression of inflammatory-associated genes in hypertrophied 3T3-L1 adipocytes and is a potent inhibitor of NF-?B activation. Thus these results suggest that SSNa is a novel therapeutic agent against that can be used against obesity-associated inflammation. PMID:25672367

  18. Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke.

    PubMed

    Knörr-Wittmann, Constanze; Hengstermann, Arnd; Gebel, Stephan; Alam, Jawed; Müller, Thomas

    2005-12-01

    Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up to 5000 different chemicals, many of which are prooxidant. Here we show that, at least in vitro, the cellular response designed to combat oxidative stress resulting from CS exposure is primarily controlled by the transcription factor Nrf2, a principal inducer of antioxidant and phase II-related genes. The prominent role of Nrf2 in the cellular response to CS is substantiated by the following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i) Nrf2 is strongly stabilized and becomes detectable in nuclear extracts. (ii) Nuclear localization of Nrf2 coincides with increased DNA binding of a putative Nrf2/MafK heterodimer to its cognate cis-regulatory site, i.e., the antioxidant-responsive element (ARE). (iii) Studies on the regulatory elements of the oxidative stress-inducible gene heme oxygenase-1 (hmox1) using various hmox1 promoter/luciferase reporter constructs revealed that the strong CS-dependent expression of this gene is primarily governed by the distal enhancers 1 ("E1") and 2 ("E2"), which both contain three canonical ARE-like stress-responsive elements (StREs). Notably, depletion of Nrf2 levels caused by RNA interference significantly compromised CS-induced hmox1 promoter activation, based on the distinct Nrf2 sensitivity exhibited by E1 and E2. Finally, (iv) siRNA-dependent knock-down of Nrf2 completely abrogated CS-induced expression of phase II-related genes. Taken together, these results confirm the outstanding role of Nrf2 both in sensing (oxidant) stress and in orchestrating an efficient transcriptional response aimed at resolving the stressing conditions. PMID:16274879

  19. Isolation of the molecular species of monogalactosyldiacylglycerols from brown edible seaweed Sargassum horneri and their inhibitory effects on triglyceride accumulation in 3T3-L1 adipocytes.

    PubMed

    Ma, Ai-Cui; Chen, Zhen; Wang, Tao; Song, Ni; Yan, Qian; Fang, Yu-Chun; Guan, Hua-Shi; Liu, Hong-Bing

    2014-11-19

    The chemical composition of monogalactosyldiacylglycerols (MGDGs) from brown alga Sargassum horneri and their inhibitory effects on lipid accumulation were investigated in this study. A total of 10 molecular species of MGDGs were identified using nuclear magnetic resonance, alkaline hydrolysis, gas chromatography-flame ionization detector, and high-performance liquid chromatography-tandem mass spectrometry methods. Individual molecular species of MGDGs, including (2S)-1-O-myristoyl-2-O-palmitoleoyl-3-O-?-D-galactopyranosyl-sn-glycerol (1), (2S)-1-O-myristoyl-2-O-linoleyl-3-O-?-D-galactopyranosyl-sn-glycerol (3), (2S)-1-O-palmitoyl-2-O-linolenoyl-3-O-?-D-galactopyranosyl-sn-glycerol (5), (2S)-1-O-myristoyl-2-O-oleyl-3-O-?-D-galactopyranosyl-sn-glycerol (7), (2S)-1-O-palmitoyl-2-O-palmitoleoyl-3-O-?-D-galactopyranosyl-sn-glycerol (8), (2S)-1-O-palmitoyl-2-O-linoleyl-3-O-?-D-galactopyranosyl-sn-glycerol (9), and (2S)-1-O-palmitoyl-2-O-oleyl-3-O-?-D-galactopyranosyl-sn-glycerol (10), were then furnished using semi-preparative high-performance liquid chromatography, and their inhibitory effects on triglyceride (TG) accumulation and free fatty acid (FFA) levels in 3T3-L1 adipocytes were evaluated. Compounds 3 and 9 showed inhibitory effects on TG and FFA accumulation, with TG levels of 1.568 ± 0.2808 and 1.701 ± 0.1460 ?mol/L and FFA levels of 0.149 ± 0.0258 and 0.198 ± 0.0229 mequiv/L, respectively, which were more effective than other compounds. The primary structure-activity relationship suggested that linoleyl [18:2(?-6)] in the sn-2 position played an important role on triglyceride accumulation inhibition. PMID:25363514

  20. Evaluation of antioxidant properties of major dietary polyphenols and their protective effect on 3T3-L1 preadipocytes and red blood cells exposed to oxidative stress.

    PubMed

    Hatia, S; Septembre-Malaterre, A; Le Sage, F; Badiou-Bénéteau, A; Baret, P; Payet, B; Lefebvre d'hellencourt, C; Gonthier, M P

    2014-04-01

    Obesity has been associated with a marked risk of metabolic diseases and requires therapeutic strategies. Changes in redox status with increased oxidative stress in adipose tissue have been linked with obesity-related disorders. Thus, the biological effect of antioxidants such as polyphenols is of high interest. We aimed to measure antioxidant capacities of 28 polyphenols representative of main dietary phenolic acids, flavonoids, stilbenes and curcuminoids. Then, 14 molecules were selected for the evaluation of their effect on 3T3-L1 preadipocytes and human red blood cells exposed to oxidative stress. Analysis of reducing and free radical-scavenging capacities of compounds revealed antioxidant properties related to their structure, with higher activities for flavonoids such as quercetin and epicatechin. Their effects on preadipocytes' viability also depended on their structure, dose and time of exposure. Interestingly, most of the compounds exhibited a protective effect on preadipocytes exposed to oxidative stress, by reversing H?O?-induced anti-proliferative action and reactive oxygen species production. Polyphenols also exerted an anti-inflammatory effect on preadipocytes exposed to H?O? by reducing IL-6 secretion. Importantly, such antioxidant and anti-inflammatory effects were observed in co-exposition (polyphenol and prooxidant during 24 h) or pretreatment (polyphenol during 24 h, then prooxidant for 24 h) conditions. Moreover, compounds protected erythrocytes from AAPH radical-induced lysis. Finally, these results led to demonstrate that antioxidant and anti-inflammatory properties of polyphenols may depend on structure, dose, time of exposure and cell conditioning with oxidative stress. Such findings should be considered for a better understanding of polyphenols' benefits in strategies aiming to prevent obesity-related diseases. PMID:24393006

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

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

    PubMed Central

    2014-01-01

    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?

  3. Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes.

    PubMed Central

    Plée-Gautier, E; Grober, J; Duplus, E; Langin, D; Forest, C

    1996-01-01

    Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 microM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 microM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols. PMID:8836156

  4. A novel coal feeder for production of low sulfur fuel

    SciTech Connect

    Khang, S.J.; Keener, T.; Lin, L.

    1992-01-01

    In this project, a dual-screw feeder was designed and constructed for desulfurization of coal before combustion. The key parts of this reactor are two screw tubes which are used to feed coal and calcined lime particles separately, the inner tube acting as a coal pyrolyzer and the outer tube acting as a desulfurizer with hot calcined lime pellets or other renewable sorbent pellets. The objective of this project is to study the feasibility of an advanced concept of desulfurization using the coal feeder. In this quarter, the following tasks have been performed: (1) Continued measurements of the organic sulfur content in the char which was produced from the pyrolysis process in the coal feeder, (2) Study of the combustion characteristics of the pyrolyzed products by using a combustor which is physically attached to the feeder system. Some preliminary conclusions have been obtained: (1) Under the experimental conditions, the organic sulfur in Ohio No.8 coal is released in the form of H{sub 2}S and 73.1% of the organic sulfur was removed in the dual-screw feeder reactor at a temperature of 475{degrees}C with the residence time of 6 min. (2) The SO{sub 2} emission from the combustor stack reduces as the coal feeder temperature rises, corresponding to the level of H{sub 2}S release from the dual-screw feeder.

  5. Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells

    PubMed Central

    Cho, Young-Eun; Lomeda, Ria-Ann R.; Ryu, Sang-Hoon; Lee, Jong-Hwa; Beattie, John H.

    2007-01-01

    Trace mineral studies involving metal ion chelators have been conducted in investigating the response of gene and protein expressions of certain cell lines but a few had really focused on how these metal ion chelators could affect the availability of important trace minerals such as Zn, Mn, Fe and Cu. The aim of the present study was to investigate the availability of Zn for the treatment of MC3T3-E1 osteoblast-like cells and the availability of some trace minerals in the cell culture media components after using chelexing resin in the FBS and the addition of N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN, membrane-permeable chelator) and diethylenetriaminepentaacetic acid (DTPA, membrane-impermeable chelator) in the treatment medium. Components for the preparation of cell culture medium and Zn-treated medium have been tested for Zn, Mn, Fe and Cu contents by atomic absorption spectrophotometer or inductively coupled plasma spectrophotometer. Also, the expression of bone-related genes (ALP, Runx2, PTH-R, ProCOL I, OPN and OC) was measured on the cellular Zn depletion such as chelexing or TPEN treatment. Results have shown that using the chelexing resin in FBS would significantly decrease the available Zn (p<0.05) (39.4 ± 1.5 µM vs 0.61 ± 10.15 µM) and Mn (p<0.05) (0.74 ± 0.01 µM vs 0.12 ± 0.04 µM). However, levels of Fe and Cu in FBS were not changed by chelexing FBS. The use of TPEN and DTPA as Zn-chelators did not show significant difference on the final concentration of Zn in the treatment medium (0, 3, 6, 9, 12 µM) except for in the addition of higher 15 µM ZnCl2 which showed a significant increase of Zn level in DTPA-chelated treatment medium. Results have shown that both chelators gave the same pattern for the expression of the five bone-related genes between Zn- and Zn+, and TPEN-treated experiments, compared to chelex-treated experiment, showed lower bone-related gene expression, which may imply that TPEN would be a stronger chelator than chelex resin. This study showed that TPEN would be a stronger chelator compared to DTPA or chelex resin and TPEN and chelex resin exerted cellular zinc depletion to be enough for cell study for Zn depletion. PMID:20535382

  6. Transformation of BALB/c-3T3 cells: IV. Rank-ordered potency of 24 chemical responses detected in a sensitive new assay procedure.

    PubMed

    Matthews, E J; Spalding, J W; Tennant, R W

    1993-07-01

    This report introduces an improved method of detecting chemical-induced morphological transformation of A-31-1-13 BALB/c-3T3 cells. The new procedure uses an increased target cell population to assess chemical-induced damage by increasing the initial seeding density and by delaying the initiation time of chemical treatment. Furthermore, a newly developed co-culture clonal survival assay was used to select chemical doses for the transformation assay. This assay measured the relative cloning efficiency (RCE) of chemical treatments in high-density cell cultures. In addition, transformation assay sensitivity was enhanced through the use of improved methods to solubilize many chemicals. From a group of 24 chemicals tested in at least two trials, clear evidence of chemical-induced transformation was detected for 12 chemicals (aphidicolin, barium chloride-2H2O, 5-bromo-2'-deoxyuridine, C.I. direct blue 15, trans-cinnamaldehyde, cytosine arabinoside, diphenylnitrosamine, manganese sulfate-H2O, 2-mercaptobenzimidazole, mezerein, riddelliine, and 2,6-xylidine); 2 chemicals had equivocal activity [C.I. direct blue 218 and mono(2-ethylhexyl)phthalate], 9 chemicals were inactive [carisoprodol, chloramphenicol sodium succinate, 4-chloro-2-nitroaniline, C.I. acid red 114, isobutyraldehyde, mono(2-ethylhexyl)adipate, sodium fluoride, and 12-O-tetradecanoylphorbol-13-acetate), and 1 chemical had an indeterminate response (2,6-dinitrotoluene). All positive responses were detected in the absence of an exogenous activation system and exhibited significant activity at two or more consecutive doses. This report also presents a mathematical method that uses t-statistics for rank-ordering the potency of chemical-induced transformation responses. This model detects sensitivity differences in experiments used to evaluate chemical-induced transformation. Furthermore, it provides a method to estimate a chemical's transformation response in terms of the historical behavior of the assay, as well as its future activity. The most active of the 24 chemicals was mezerein, and the least active chemical was diphenylnitrosamine. PMID:8243401

  7. Transformation of BALB/c-3T3 cells: IV. Rank-ordered potency of 24 chemical responses detected in a sensitive new assay procedure.

    PubMed Central

    Matthews, E J; Spalding, J W; Tennant, R W

    1993-01-01

    This report introduces an improved method of detecting chemical-induced morphological transformation of A-31-1-13 BALB/c-3T3 cells. The new procedure uses an increased target cell population to assess chemical-induced damage by increasing the initial seeding density and by delaying the initiation time of chemical treatment. Furthermore, a newly developed co-culture clonal survival assay was used to select chemical doses for the transformation assay. This assay measured the relative cloning efficiency (RCE) of chemical treatments in high-density cell cultures. In addition, transformation assay sensitivity was enhanced through the use of improved methods to solubilize many chemicals. From a group of 24 chemicals tested in at least two trials, clear evidence of chemical-induced transformation was detected for 12 chemicals (aphidicolin, barium chloride-2H2O, 5-bromo-2'-deoxyuridine, C.I. direct blue 15, trans-cinnamaldehyde, cytosine arabinoside, diphenylnitrosamine, manganese sulfate-H2O, 2-mercaptobenzimidazole, mezerein, riddelliine, and 2,6-xylidine); 2 chemicals had equivocal activity [C.I. direct blue 218 and mono(2-ethylhexyl)phthalate], 9 chemicals were inactive [carisoprodol, chloramphenicol sodium succinate, 4-chloro-2-nitroaniline, C.I. acid red 114, isobutyraldehyde, mono(2-ethylhexyl)adipate, sodium fluoride, and 12-O-tetradecanoylphorbol-13-acetate), and 1 chemical had an indeterminate response (2,6-dinitrotoluene). All positive responses were detected in the absence of an exogenous activation system and exhibited significant activity at two or more consecutive doses. This report also presents a mathematical method that uses t-statistics for rank-ordering the potency of chemical-induced transformation responses. This model detects sensitivity differences in experiments used to evaluate chemical-induced transformation. Furthermore, it provides a method to estimate a chemical's transformation response in terms of the historical behavior of the assay, as well as its future activity. The most active of the 24 chemicals was mezerein, and the least active chemical was diphenylnitrosamine. PMID:8243401

  8. The anti-obesity effects of a tuna peptide on 3T3-L1 adipocytes are mediated by the inhibition of the expression of lipogenic and adipogenic genes and by the activation of the Wnt/?-catenin signaling pathway

    PubMed Central

    KIM, YOUNG-MIN; KIM, IN-HYE; CHOI, JEONG-WOOK; LEE, MIN-KYEONG; NAM, TAEK-JEONG

    2015-01-01

    The differentiation of 3T3-L1 cells into adipocytes involves the activation of an organized system of obesity-related genes, of which those encoding CCAAT/enhancer-binding proteins (C/EBPs) and the Wnt-10b protein may play integral roles. In a previous study of ours, we found that a specific peptide found in tuna (sequence D-I-V-D-K-I-E-I; termed TP-D) inhibited 3T3-L1 cell differentiation. In the present study, we observed that the expression of expression of C/EBPs and Wnt-10b was associated with obesity. The initial step of 3T3-L1 cell differentiation involved the upregulation of C/EBP-? expression, which in turn activated various subfactors. An upstream effector of glycogen synthase kinase-3? (GSK-3?) inhibited Wnt-10b expression in 3T3-L1 adipocytes. In a previous study of ours, we sequenced the tuna peptide via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS) and confirmed the anti-obesity effects thereof in 3T3-L1 adipocytes. In the present study, we demonstrate that TP-D inhibits C/EBP and promotes Wnt-10b mRNA expression, thus activating the Wnt pathway. The inhibition of lipid accumulation was measured using a glucose and triglyceride (TG) assay. Our results confirmed that TP-D altered the expression levels of C/EBP-related genes in a dose-dependent manner and activated the Wnt signaling pathway. In addition, we confirmed that total adiponectin and high-molecular weight (HMW) adiponectin levels were reduced by treatment with TP-D. These data indicate that TP-D inhibits adipocyte differentiation through the inhibition of C/EBP genes and the subsequent activation of the Wnt/?-catenin signaling pathway. PMID:26046125

  9. VIEW OF FEEDER TABLE WITH THE BOOM OF THE UNLOADER ...

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

    VIEW OF FEEDER TABLE WITH THE BOOM OF THE UNLOADER IN BACKGROUND. THE CASE HYDRAULIC BOOM HOIST IS TO THE RIGHT. VIEW FROM THE SOUTHWEST - Kekaha Sugar Company, Sugar Mill Building, 8315 Kekaha Road, Kekaha, Kauai County, HI

  10. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...2014-10-01 2014-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15...CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency...

  11. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...2012-10-01 2012-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15...CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency...

  12. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...2013-10-01 2013-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15...CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency...

  13. Analysis of Feeder Bus Network Design and Scheduling Problems

    PubMed Central

    Almasi, Mohammad Hadi; Karim, Mohamed Rehan

    2014-01-01

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

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

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...2011-10-01 2011-10-01 false Emergency lighting feeders. 112.43-15 Section 112.43-15...CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency...

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

    Code of Federal Regulations, 2010 CFR

    2010-10-01

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

  16. 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

  17. A New Angle on Microscopic Suspension Feeders near Boundaries

    PubMed Central

    Pepper, Rachel E.; Roper, Marcus; Ryu, Sangjin; Matsumoto, Nobuyoshi; Nagai, Moeto; Stone, Howard A.

    2013-01-01

    Microscopic sessile suspension feeders are a critical component in aquatic ecosystems, acting as an intermediate trophic stage between bacteria and higher eukaryotic taxa. Because they live attached to boundaries, it has long been thought that recirculation of the feeding currents produced by sessile suspension feeders inhibits their ability to access fresh fluid. However, previous models for the feeding flows of these organisms assume that they feed by pushing fluid perpendicular to surfaces they live upon, whereas we observe that sessile suspension feeders often feed at an angle to these boundaries. Using experiments and calculations, we show that living suspension feeders (Vorticella) likely actively regulate the angle that they feed relative to a substratum. We then use theory and simulations to show that angled feeding increases nutrient and particle uptake by reducing the reprocessing of depleted water. This work resolves an open question of how a key class of suspension-feeding organisms escapes physical limitations associated with their sessile lifestyle. PMID:24138855

  18. Analysis of feeder bus network design and scheduling problems.

    PubMed

    Almasi, Mohammad Hadi; Mirzapour Mounes, Sina; Koting, Suhana; Karim, Mohamed Rehan

    2014-01-01

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

  19. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...feeders inject flavoring material into the semi-frozen product. Product contact surfaces shall be constructed of stainless steel or equally corrosion resistant metal and all pumps shall be in accordance to 3-A Sanitary Standards for dairy...

  20. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...feeders inject flavoring material into the semi-frozen product. Product contact surfaces shall be constructed of stainless steel or equally corrosion resistant metal and all pumps shall be in accordance to 3-A Sanitary Standards for dairy...

  1. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...feeders inject flavoring material into the semi-frozen product. Product contact surfaces shall be constructed of stainless steel or equally corrosion resistant metal and all pumps shall be in accordance to 3-A Sanitary Standards for dairy...

  2. A novel coal feeder for production of low sulfur fuel

    SciTech Connect

    Lin, L.; Khang, S.J.; Keener, T.C.

    1991-01-01

    In this project, a dual-screw feeder was designed for desulfurization of coal. The key parts of this reactor are two screw tubes which are used to feed coal and calcined lime particles separately, the inner tube acting as a coal pyrolyzer and the outer tube acting as a desulfurizer with hot calcined lime pellets or other renewable sorbent pellets. The objective of this project is to study the feasibility of an advanced concept of desulfurization in the coal feeder. (VC)

  3. Functional and aesthetic approach to design of bird feeders

    NASA Astrophysics Data System (ADS)

    Kukhta, A.; Kukhta, M.

    2015-10-01

    Anthropogenic objects which load the urban environment negatively affects the human psyche. The alternative is attracting elements of the natural environment into urban environment, of which some of the most frequently identified are birds. Attracting birds in the city is possible by means of feeders and artificial nests, however, both must be harmonious. The aim of this study is to analyze the essential functions of the feeders, and their integration into the environmental design and development of the city. On this basis an original feeder which is convenient for use by birds and attracts people's attention is developed. In this paper we apply comparative analysis of different types of feeders encountered in Tomsk, bird watching, and evaluate usability of different types of feeders from the position of their convenience both for birds and human beings. Historical-cultural analysis for determining features of the architectural and environmental design of Tomsk is carried out, the method allows us to solve engineering problems. In this study the feeder convenient for bird use is designed which blends harmoniously with the architectural design of Tomsk.

  4. Cleavage of vesicle-associated membrane protein (VAMP)-2 and cellubrevin on GLUT4-containing vesicles inhibits the translocation of GLUT4 in 3T3-L1 adipocytes.

    PubMed

    Tamori, Y; Hashiramoto, M; Araki, S; Kamata, Y; Takahashi, M; Kozaki, S; Kasuga, M

    1996-03-27

    We have identified VAMP isoforms, VAMP-2 and cellubrevin, on GLUT4-containing vesicle membranes isolated from 3T3-Ll adipocytes. These proteins translocate from a low density microsomal fraction to the plasma membrane upon insulin stimulation in a fashion similar to GLUT4. VAMP-1 was not detected in this low density microsomal fraction nor on purified GLUT4-containing vesicles. In streptolysin-O permeabilized 3T3-L1 adipocytes, both VAMP-2 and cellubrevin were cleaved with botulinum neurotoxin isoform B, BoNTx/B. In addition, BoNTx/B partially inhibited insulin-stimulated GLUT4 translocation and glucose transport activity. We conclude that the synaptobrevin isoforms are important components of the insulin-dependent translocation of GLUT4 to the cell surface in adipocytes. PMID:8607835

  5. Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells.

    PubMed

    Liu, Jingli; Yang, Jianhong

    2016-01-01

    Uncarboxylated osteocalcin, an osteoblast-derived protein, plays an important role in the regulation of glucose metabolism. It has previously been demonstrated that high glucose levels inhibit osteoblast proliferation and differentiation. However, the mechanisms through which uncarboxylated osteocalcin regulates osteoblast proliferation and differentiation under high glucose conditions remain unclear. Thus, in the present study, we aimed to examine the effects of uncarboxylated osteocalcin on the proliferation and differentiation of MC3T3-E1 cells under high glucose conditions. We demonstrated that high glucose levels induced the production of reactive oxygen species (ROS) in MC3T3-E1 cells, and this production was inhibited by treatment with uncarboxylated osteocalcin and N-acetyl-L-cysteine (NAC), a ROS scavenger. In addition, we found that uncarboxylated osteocalcin reduced high glucose?induced oxidative stress and increased the mRNA expression of the osteogenic markers, runt-related transcription factor 2 (Runx2), osterix and osteocalcin, as well as the formation of mineralized nodules; it also inhibited adipogenic differentiation, as shown by a decrease in the mRNA expression of the adipogenic markers, peroxisome proliferator?activated receptor ? (PPAR?), adipocyte fatty acid-binding protein (adipocyte protein 2; aP2) and fatty acid synthase (FAS), and reduced lipid drop accumulation. Furthermore, we found that uncarboxylated osteocalcin inhibited PI3K/Akt signaling which was induced by ROS and facilitated the osteogenic differentiation of MC3T3-E1 cells under high glucose conditions. Taken together and to the best of ou knowledge, our results demonstrate for the first time that uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by inhibiting the activation of PI3K/Akt in MC3T3-E1 cells. Therefore, we suggest that uncarboxylated osteocalcin may be a potential therapeutic agent for diabetes-related osteoporosis. PMID:26719856

  6. A Small Molecule Swertisin from Enicostemma littorale Differentiates NIH3T3 Cells into Islet-Like Clusters and Restores Normoglycemia upon Transplantation in Diabetic Balb/c Mice.

    PubMed

    Dadheech, Nidheesh; Soni, Sanket; Srivastava, Abhay; Dadheech, Sucheta; Gupta, Shivika; Gopurappilly, Renjitha; Bhonde, Ramesh R; Gupta, Sarita

    2013-01-01

    Aim. Stem cell therapy is one of the upcoming therapies for the treatment of diabetes. Discovery of potent differentiating agents is a prerequisite for increasing islet mass. The present study is an attempt to screen the potential of novel small biomolecules for their differentiating property into pancreatic islet cells using NIH3T3, as representative of extra pancreatic stem cells/progenitors. Methods. To identify new agents that stimulate islet differentiation, we screened various compounds isolated from Enicostemma littorale using NIH3T3 cells and morphological changes were observed. Characterization was performed by semiquantitative RT-PCR, Q-PCR, immunocytochemistry, immunoblotting, and insulin secretion assay for functional response in newly generated islet-like cell clusters (ILCC). Reversal of hyperglycemia was monitored after transplanting ILCC in STZ-induced diabetic mice. Results. Among various compounds tested, swertisin, an isolated flavonoid, was the most effective in differentiating NIH3T3 into endocrine cells. Swertisin efficiently changed the morphology of NIH3T3 cells from fibroblastic to round aggregate cell cluster in huge numbers. Dithizone (DTZ) stain primarily confirmed differentiation and gene expression studies signified rapid onset of differentiation signaling cascade in swertisin-induced ILCC. Molecular imaging and immunoblotting further confirmed presence of islet specific proteins. Moreover, glucose induced insulin release (in vitro) and decreased fasting blood glucose (FBG) (in vivo) in transplanted diabetic BALB/c mice depicted functional maturity of ILCC. Insulin and glucagon expression in excised islet grafts illustrated survival and functional integrity. Conclusions. Rapid induction for islet differentiation by swertisin, a novel herbal biomolecule, provides low cost and readily available differentiating agent that can be translated as a therapeutic tool for effective treatment in diabetes. PMID:23662125

  7. Inhibitory Effects of Hwangryunhaedok-Tang in 3T3-L1 Adipogenesis by Regulation of Raf/MEK1/ERK1/2 Pathway and PDK1/Akt Phosphorylation

    PubMed Central

    Lee, Ji-Hye; Kim, Dong-Gun; Kim, Taesoo; Lee, Kwang Jin; Ma, Jin Yeul

    2013-01-01

    Hwangryunhaedok-tang (HRT) has been long used as traditional medicine in Asia. However, inhibitory role of HRT is unclear in early stage of 3T3-L1 adipocyte differentiation related to signaling. In the present study, we investigated the inhibitory effects of HRT on upstream signaling of peroxisome proliferation-activity receptor-? (PPAR-?) and CCAAT/enhancer binding protein-? (C/EBP-?) expression in differentiation of 3T3-L1 preadipocytes. We found that HRT significantly inhibited the adipocyte differentiation by downregulating several adipocyte-specific transcription factors including PPAR-?, C/EBP-?, and C/EBP-? in 3T3-L1 preadipocytes. Furthermore, we observed that HRT markedly inhibited the differentiation media-mediated phosphorylation of Raf/extracellular mitogen-activated protein kinase 1 (MEK1)/signal-regulated protein kinase 1/2 (ERK1/2) and phosphorylation of phosphoinositide-dependent kinase 1 (PDK1)/Akt. These results indicate that anti-adipogenesis mechanism involves the downregulation of the major transcription factors of adipogenesis including PPAR-? and C/EBP-? through inhibition of Raf/MEK1/ERK1/2 phosphorylation and PDK1/Akt phosphorylation by HRT. Furthermore, high performance liquid chromatography (HPLC) analysis showed HRT contains active antiobesity constituents such as palmatine, berberine, geniposide, baicalin, baicalein, and wogonin. Taken together, this study suggested that anti-adipogenesis effects of HRT were accounted by downregulation of Raf/MEK1/ERK1/2 pathway and PDK1/Akt pathway during 3T3-L1 adipocyte differentiation. PMID:23762131

  8. Temperature-dependent alteration of 1,25-dihydroxy-vitamin D3 receptor macromolecules in MC3T3-E1 cells: affinity of hexafluoro analog of 1,25-dihydroxyvitamin D3, ST-630, for these forms.

    PubMed

    Tanaka, M; Muramatsu, M; Higuchi, S; Otomo, S

    1994-07-01

    The binding properties of [3H]1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to 1,25(OH)2D3 receptor in mouse osteoblastic MC3T3-E1 cells and the affinity of 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (ST-630) were examined. Sucrose density gradient experiments demonstrated that [3H]1,25(OH)2D3 bound to the 6S macromolecule in the cytosol of MC3T3-E1 cells at 4 degrees C. The inhibitory effect of 1,25(OH)2D3 was compared with that of ST-630. However, at 30 degrees C, only the 3.7S macromolecule was labeled, and 1,25(OH)2D3 and ST-630 demonstrated similar affinity for the 3.7S macromolecule. Under the lower temperature condition, the cytosol from MC3T3-E1 cells showed one binding site for [3H]1,25(OH)2D3 with Kd of 0.31 nM and Bmax of 13.0 fmol/mg protein, respectively. Furthermore, from the competitive binding experiments at 4 degrees C, the affinity of ST-630 was 6.4-fold lower than that of 1,25(OH)2D3. These results suggest that the 6S macromolecule formed at the lower temperature in MC3T3-E1 cells has a property of 1,25(OH)2D3 receptor, but ST-630 has a higher affinity for 3.7S macromolecule and it may be comparable to 1,25(OH)2D3. PMID:7953192

  9. Rat bone marrow-derived mast cells co-cultured with 3T3 fibroblasts in the absence of T-cell derived cytokines require stem cell factor for their survival and maintain their mucosal mast cell-like phenotype.

    PubMed

    MacDonald, A J; Thornton, E M; Newlands, G F; Galli, S J; Moqbel, R; Miller, H R

    1996-07-01

    When cultured without fibroblasts, rat bone marrow-derived mast cells (BMMC) contain abundant rat mast cell proteinase type II (RMCP-II), and exhibit survival and proliferation when maintained in mesenteric lymph node conditioned medium (CM). When BMMC were co-cultured with 3T3 fibroblasts in the absence of CM, BMMC numbers increased for 7 days and the BMMC survived for up to 23 days. There was a gradual loss of stored RMCP-II in BMMC that were co-cultured with 3T3 cells, but the fibroblast microenvironment did not induce a detectable increase in the low levels of the connective tissue mast cell (CTMC)-associated proteinase, RMCP-I, in the BMMC. Nor did 3T3 cell co-culture induce significant heparin synthesis in BMMC as judged by the cells' reactivity with the fluorescent heparin-binding dye, berberine sulphate. These results suggest that rat BMMC, unlike murine BMMC, do not have the potential to develop multiple CTMC-like characteristics upon co-culture with 3T3 cells. However, when BMMC and fibroblast co-cultures were treated with an antibody to recombinant rat stem cell factor (rrSCF), mast cell survival was completely abrogated. This result suggests that endogenous, fibroblast-derived SCF is essential for the maintenance of rat BMMC viability in the absence of CM. On the other hand, prior treatment of the fibroblasts with the anti-rrSCF antibody did not affect the adherence of BMMC to the monolayer, implying that (an) other molecule(s) is(are) involved in the attachment process. The demonstration that rat BMMC survival on fibroblasts in vitro is dependent upon SCF may indicate an important mechanism by which tissue mucosal cells can be maintained in vivo in the absence of T-cell derived factors. PMID:8774353

  10. A novel coal feeder for production of low sulfur fuel

    SciTech Connect

    Khang, S.J.; Keener, T.C.; Line, L.

    1992-01-01

    The following tasks have been performed (1) Change of the single-ended driving mechanism of the inner screw shaft to a double-ended driving mechanism, (2) Study of the total sulfur balance on the feeder reactor system, (3) Analytical measurements of the organic sulfur contents in the feed coal and the product char, (4) Study of the organic sulfur removal efficiency in the feeder reactor, (5) Designing and setting up a combustor which is physically attached to the feeder system, (6) Study of the combustion characteristics of char and volatiles produced from the pyrolysis process in the feeder system, and (7) Modeling the pyrolysis process in the inner screw tube to estimate the heat transfer coefficient. Some preliminary conclusions have been obtained: (1) Double-ended driving mechanism of the inner screw feeder is useful for a uniform stress on the shaft and to raise the reaction temperature without an additional metal torque on the shaft. (2) The total sulfur balance shows that the sulfur removal efficiency of CaO in this reactor is higher than 90%. (3) The organic sulfur in Ohio [number sign]8 coal is 1.72% which amounts to 45.4% of the total sulfur content. (4) A 73.1% of the organic sulfur is removed in the dual-screw feeder reactor at a temperature of 475[degree]C with the residence time of 6 min., (5) The S[sub 2]O emission from the combustor stack reduces as the coal feeder temperature rises, corresponding to the level of H S release from the dual-screw feeder. (6) The total heating value of the coal does not significantly decrease during the pyrolysis and desulfurization processes in the coal feeder. The decrease is mainly due to the loss of sulfur from coal. (7) The heat transfer step is the main resistance to the pyrolysis process in this system. The heat transfer coefficient is estimated to be about 331.7 W/m-K.

  11. Cellular regulation of poly ADP-ribosylation of proteins: II. Augmentation of poly(ADP-ribose) polymerase in SV40 3T3 cells following methotrexate-induced G1/S inhibition of cell cycle progression

    SciTech Connect

    Sooki-Toth, A.; Asghari, F.; Kirsten, E.; Kun, E. )

    1987-05-01

    SV40-3T3 cells were exposed in monolayer cultures to 5{times}10{sup {minus}7} M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5{times}10{sup {minus}7} MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory site of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5{times}10{sup {minus}7} M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.

  12. Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

    PubMed

    Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio; Iwasa, Koichi; Kitawaki, Jo; Ohnishi, Katsunori; Tsujikawa, Muneo; Obayashi, Hiroshi

    2015-07-01

    4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes. PMID:26077869

  13. Tauroursodeoxycholic acid attenuates inorganic phosphate-induced osteoblastic differentiation and mineralization in NIH3T3 fibroblasts by inhibiting the ER stress response PERK-eIF2?-ATF4 pathway.

    PubMed

    Liu, Fang; Cui, Yazhou; Ge, Pinglan; Luan, Jing; Zhou, Xiaoyan; Han, Jinxiang

    2015-02-01

    Ectopic ossification occurs in a wide range of common and genetic diseases, but its molecular mechanisms and effective therapeutic targets remain to be clarified. The aim of the study is to investigate whether endoplasmic reticulum (ER) stress is involved in ectopic ossification and ER stress inhibitor tauroursodeoxycholic acid (TUDCA) has potential to treat the pathological conditions. In this study, inorganic phosphate (Pi)-induced NIH3T3 fibroblasts induced osteogenesis and mineralization was used as an in vitro model for ectopic ossification. Various concentrations of TUDCA (0.1, 1, 5, 10 ?M) were added during osteogenic induction. Osteoblast differentiation and minerlization were determined by RT-qPCR, alkaline phosphatase (ALP) activity assay, Alizarin Red-S (AR-S) staining, and calcium deposition. ER stress signalling components were detected by Western-blot analysis. We found ER stress was activated by inorganic phosphate in NIH3T3 cells. During osteogenic induction, TUDCA inhibited NIH3T3 cells ALP activity and mineral nodule formation. In addition, TUDCA caused decreased expression of osteoblastic markers Runx2, Col1a1, ALP, OCN. Mechanistically, TUDCA inhibited the ER stress response PERK-eIF2?-ATF4 pathway during osteogenesis. In conclusion, TUDCA could inhibit fibroblasts mineralization via supressing the ER stress response PERK-eIF2?-ATF4 pathway, and has potential pharmacologic and therapeutic applications for treating ectopic ossification associated diseases. PMID:25788050

  14. A magnetic flux leakage NDE system for CANDU feeder pipes

    NASA Astrophysics Data System (ADS)

    Mak, Thomas Don

    This work examines the application of different magnetic flux leakage (MFL) inspection concepts to the non destructive evaluation (NDE) of residual (elastic) stresses in CANDURTM reactor feeder pipes. The stress sensitivity of three MFL inspection techniques was examined with flat plate samples, with stress-induced magnetic anisotropy (SMA) demonstrating the greatest stress sensitivity. A prototype SMA testing system was developed to apply magnetic NDE to feeders. The system consists of a flux controller that incorporates feedback from a wire coil and a Hall sensor (FCV2), and a magnetic anisotropy prototype (MAP) probe. The combination of FCV2 and the MAP probe was shown to provide SMA measurements on feeder pipe samples and predict stresses from SMA measurements with a mean accuracy of +/-38MPa.

  15. 20. VIEW OF CATENARY, FEEDER TOWERS AND COS COB POWER ...

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

    20. VIEW OF CATENARY, FEEDER TOWERS AND COS COB POWER PLANT (CENTER BACKGROUND) LOOKING WEST FROM THE NORTH SIDE OF THE MIANUS RIVER BASCULE BRIDGE. NOTE THAT THE CATENARY ENDS AT BRIDGE 313. TRAINS ARE CARRIED ACROSS THE BASCULE BRIDGE BY THEIR OWN MOMENTUM AND PICK UP THE CATENARY ON THE EAST SIDE OF THE RIVER. THE FEEDER LINES CARRY POWER ACROSS THE RIVER ABOVE THE LEVEL OF THE OPENED BASCULE ON THE TOWERS TO THE LEFT AND RIGHT. - New York, New Haven & Hartford Railroad, Cos Cob Power Plant, Sound Shore Drive, Greenwich, Fairfield County, CT

  16. An automatic 14-day paste diet feeder for animals

    NASA Technical Reports Server (NTRS)

    Vasques, Marilyn; Mulenburg, Jerry; Gundo, Dan; Griffith, Jon

    1994-01-01

    During a centrifuge experiment, any interruption that requires stopping the centrifuge may influence the results. Centrifuges often must be stopped for animal maintenance (food, water and waste removal), especially in cases of timed feedings. To eliminate the need for stopping the centrifuge while still providing timed feeding, an automatic paste diet feeder was developed. The feeder is based on a constant volume concept and can deliver a predetermined amount of paste diet at specified time intervals. This unit was supported by water delivery and waste collection systems. The entire system performed reliably and maintained the animals well for a continuous centrifugation experiment of 14 days.

  17. Layers

    NASA Astrophysics Data System (ADS)

    Hong, K. J.; Jeong, T. S.; Youn, C. J.

    2014-09-01

    The temperature-dependent photoresponse characteristics of MnAl2S4 layers have been investigated, for the first time, by use of photocurrent (PC) spectroscopy. Three peaks were observed at all temperatures. The electronic origin of these peaks was associated with band-to-band transitions from the valence-band states ?4( z), ?5( x), and ?5( y) to the conduction-band state ?1( s). On the basis of the relationship between PC-peak energy and temperature, the optical band gap could be well expressed by the expression E g( T) = E g(0) - 2.80 × 10-4 T 2/(287 + T), where E g(0) was estimated to be 3.7920 eV, 3.7955 eV, and 3.8354 eV for the valence-band states ?4( z), ?5( x), and ?5( y), respectively. Results from PC spectroscopy revealed the crystal-field and spin-orbit splitting were 3.5 meV and 39.9 meV. The gradual decrease of PC intensity with decreasing temperature can be explained on the basis of trapping centers associated with native defects in the MnAl2S4 layers. Plots of log J ph, the PC current density, against 1/ T, revealed a dominant trap level in the high-temperature region. By comparing PC and the Hall effect results, we confirmed that this trap level is a shallow donor 18.9 meV below the conduction band.

  18. 34. INTERIOR VIEW, SAME AS ABOVE WITH THE FEEDER ROD ...

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

    34. INTERIOR VIEW, SAME AS ABOVE WITH THE FEEDER ROD BEING OFFERED INTO THE MACHINE; UNLIKE THE OTHER NAIL CUTTING MACHINES, THE NAIL PLATE MUST BE HAND FLIPPED AND HAND FED WITH THIS MACHINE - LaBelle Iron Works, Thirtieth & Wood Streets, Wheeling, Ohio County, WV

  19. A novel coal feeder for production of low sulfur fuel

    SciTech Connect

    Khang, S.J.; Keener, T.; Lin, L.

    1992-01-01

    A dual-screw feeder was designed and tested for desulfurization of coal. The key parts of this reactor are two screw tubes which independently feed coal and calcined lime, the inner tube acting as a coal pyrolizer and the outer tube acting as a desulfurizer with hot calcined lime pellets or other renewable sorbent pellets. The objective of this project is to study the feasibility of this advanced concept of desulfurization using the coal feeder. In this quarter, the following tasks have been performed: (1) Analytical measurements of the organic sulfur content in the feed coal and the product char, (2) setting up the combustor which is physically attached to the feeder system and will be used to study the combustion characteristics of char and volatiles produced from the pyrolysis process. The following preliminary conclusions have been obtained: (1) A 73.1% of the organic sulfur in Ohio {number sign}8 coal was removed in the dual-screw feeder reactor at a temperature of 475{degrees}C with the residence time of 6 min. (2) The combustor works stably and the operating temperature of the combustor can be adjusted by controlling the char feed rate and the air flowrate. 2 tabs.

  20. DYNAMIC RESPONSES OF FEEDER CATTLE TO SIMULATED HEAT WAVES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat stress in feedlot cattle causes reduced performance, and in the most severe cases, death of the animals, thus causing the loss of millions of dollars in revenue to the cattle industry. A study was designed to evaluate the dynamics of thermoregulation and feeding activities when feeder cattle w...

  1. Nearby boundaries create eddies near microscopic filter feeders

    PubMed Central

    Roper, Marcus; Ryu, Sangjin; Matsudaira, Paul; Stone, Howard A.

    2010-01-01

    We show through calculations, simulations and experiments that the eddies often observed near sessile filter feeders are frequently due to the presence of nearby boundaries. We model the common filter feeder Vorticella, which is approximately 50 µm across and which feeds by removing bacteria from ocean or pond water that it draws towards itself. We use both an analytical stokeslet model and a Brinkman flow approximation that exploits the narrow-gap geometry to predict the size of the eddy caused by two parallel no-slip boundaries that represent the slides between which experimental observations are often made. We also use three-dimensional finite-element simulations to fully solve for the flow around a model Vorticella and analyse the influence of multiple nearby boundaries. Additionally, we track particles around live feeding Vorticella in order to determine the experimental flow field. Our models are in good agreement both with each other and with experiments. We also provide approximate equations to predict the experimental eddy sizes owing to boundaries both for the case of a filter feeder between two slides and for the case of a filter feeder attached to a perpendicular surface between two slides. PMID:19942677

  2. TEXAS FEEDER SCHOOLS SUPPLEMENT TO SB 175 REPORT FOR 2013

    E-print Network

    Pillow, Jonathan

    TEXAS FEEDER SCHOOLS SUPPLEMENT TO SB 175 REPORT FOR 2013 HS Code High School Name HS City 2011 TO SB 175 REPORT FOR 2013 HS Code High School Name HS City 2011 Admitted 2011 Enrolled 2012 Admitted Austin 1 1 2440069 Liberal Arts & Science Acad Hs Austin 43 19 40 17 46 24 2440294 L C Anderson High

  3. TEXAS FEEDER SCHOOLS SUPPLEMENT TO SB 175 REPORT FOR 2014

    E-print Network

    Pillow, Jonathan

    TEXAS FEEDER SCHOOLS SUPPLEMENT TO SB 175 REPORT FOR 2014 HS Code High School Name HS City 2012 REPORT FOR 2014 HS Code High School Name HS City 2012 Admitted 2012 Enrolled 2013 Admitted 2013 Enrolled 1 1 1079587 Ace Academy Austin 2 2 2440069 Liberal Arts & Science Acad Hs Austin 40 17 46 24 56 28

  4. Predicting Electricity Distribution Feeder Failures Using Machine Learning Susceptibility Analysis

    E-print Network

    Cortes, Corinna

    into the city from upstate New York, New Jersey and Long Island, as well as from in-city generation facilities feeder cables that supply electricity to the boroughs of New York City. In Manhattan, rankings the force of water, steam, or hot gasses on a turbine, spins an electromagnet, generating large amounts

  5. Development of a Feeder for Uninterrupted Centrifugation Studies

    NASA Technical Reports Server (NTRS)

    Mulenburg, Gerald M.; Vasques, Marilyn F.; Gundo, Daniel P.; Griffith, Jon B.; Wade, Charles E. (Technical Monitor)

    1994-01-01

    A specialized paste diet feeder was developed in support of a hypergravity (2G) centrifuge study. The centrifuge study was to be compared to a previously flown Russian Cosmos spaceflight so experimental parameters of the 14 day spaceflight had to be duplicated. In order to duplicate at hyper G an experiment that took place in weightlessness, all other conditions must be as identical as possible. Stopping the centrifuge to provide maintenance for the animals causes unacceptable changes in experimental research results. Thus the experimental protocol required the delivery of a designated amount of paste diet at regular intervals for a two week period without stopping the centrifuge. A centrifuge and a stationary control cage, each containing 10 laboratory rats, were fitted with feeders that were calibrated to provide 140 plus or minus 2g of paste diet every 6 hours. This paper describes development of the feeder design and results of its operation over the two week experiment. The design philosophy and details of the feeder system are provided with recommendations for future such devices.

  6. PV Interconnection Risk Analysis through Distribution System Impact Signatures and Feeder Zones

    E-print Network

    PV Interconnection Risk Analysis through Distribution System Impact Signatures and Feeder Zones reliability problems. In order to improve the interconnection study process, the use of feeder zones and PV, and location and size of PV. PV impact signatures, hosting capacity, and feeder risk zones are demonstrated

  7. VECTOR-BORNE DISEASES, SURVEILLANCE, PREVENTION The Effects of Bird Feeders on Lyme Disease Prevalence and

    E-print Network

    VECTOR-BORNE DISEASES, SURVEILLANCE, PREVENTION The Effects of Bird Feeders on Lyme Disease, Millbrook, NY 12545 J. Med. Entomol. 40(4): 540Ð546 (2003) ABSTRACT The effects of bird feeders on local in 2001Ð2002 on residential properties with and without bird feeders. Tick densities and infection

  8. Dry coal feeder development program at Ingersoll-Rand Research, Incorporated. [for coal gasification systems

    NASA Technical Reports Server (NTRS)

    Mistry, D. K.; Chen, T. N.

    1977-01-01

    A dry coal screw feeder for feeding coal into coal gasification reactors operating at pressures up to 1500 psig is described. Results on the feeder under several different modes of operation are presented. In addition, three piston feeder concepts and their technical and economical merits are discussed.

  9. Comparison of two approaches to automated PI controller tuning for an industrial weigh belt feeder

    E-print Network

    Collins, Emmanuel

    Comparison of two approaches to automated PI controller tuning for an industrial weigh belt feeder, are applied to an industrial weigh belt feeder that has significant nonlinearities. Both methods do; Weigh belt feeder 1. Introduction Automated controller tuning is of significant in- terest to control

  10. Effect of modified Chung-Sim-Youn-Ja-Tang on anti-inflammatory and anti-lipogenesis in RAW 264.7 and 3T3-L1 cells

    PubMed Central

    Shim, Eun Hyoung; Lee, Hoyoung; Lee, Myeong Soo

    2015-01-01

    Jea-Ma-Chung-Sim-Youn-Ja-Tang (JCST), one of herbal formulae, is modified prescription base on Chung-Sim-Youn-Ja-Tang (CST) which treats cerebrovascular disease for the Tae-Eum-In (TE). This study was designed to determine the anti-inflammatory and anti-adipogenesis effect of CST and JCST in vitro. CST and JCST of various concentrations were added in RAW 264.7 and 3T3-L1 cell. To determine the anti-inflammatory and anti-adipogenesis effects of CST and JCST, the PGE2 production was measured by lipopolysaccharide (LPS) treated RAW 264.7 cell. The adipocytes was determined by Oil red O staining, triglyceride (TG) production, leptin level and the protein expressions of peroxisome proliferator-activated receptor gamma (PPAR?), CCAAT/enhancer binding proteins alpha (C/EBP?) and fatty acid binding protein 4 (FABP4) in 3T3-L1 adipocytes. Our results showed that treatment with JCST significantly decreased PGE2 production on RAW 264.7 cell and suppressed adipocyte differentiation, lipid accumulation, TG production, leptin contents and the protein expressions of PPAR?, C/EBP? and FABP4 on 3T3-L1 adipocytes compared to CST without affecting cell viability. In conclusions, our results suggest that JCST may be useful to inhibit the effect on lipid metabolism compared to CST, and regulates lipogenesis effectively. Therefore, our data provides scientific evidence to support the clinical use of JCST in the treatment of cerebrovascular diseases such as stroke in the TE. PMID:25755734

  11. The Effects of Propionate and Valerate on Insulin Responsiveness for Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myotubes via G Protein-Coupled Receptor 41

    PubMed Central

    Han, Joo-Hui; Kim, In-Su; Jung, Sang-Hyuk; Lee, Sang-Gil; Son, Hwa-Young; Myung, Chang-Seon

    2014-01-01

    Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the G?i/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes. PMID:24748202

  12. Extracellular calcium-sensing-receptor (CaR)-mediated opening of an outward K(+) channel in murine MC3T3-E1 osteoblastic cells: evidence for expression of a functional CaR

    NASA Technical Reports Server (NTRS)

    Ye, C. P.; Yamaguchi, T.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.

  13. Large round bale feeder design affects hay utilization and beef cow behavior.

    PubMed

    Buskirk, D D; Zanella, A J; Harrigan, T M; Van Lente, J L; Gnagey, L M; Kaercher, M J

    2003-01-01

    One hundred sixty beef cows (631 +/- 78 kg) were used to evaluate the quantity of hay loss and feeding behaviors from different round bale feeders. Twenty cows were allotted by weight and body condition score to one of eight pens with four feeder designs: cone, ring, trailer, or cradle. All feeder types provided approximately 37 cm of linear feeder space per animal. Alfalfa and orchardgrass round bales were weighed and sampled before feeding. Hay that fell onto the concrete surrounding the feeder was considered waste and was collected and sampled daily. At the end of a 7-d period, each feeder type was assigned to a different pen for a second 7-d period. On four consecutive days in each period, animal behavior was recorded using a time-lapse video system. Data were collected from 5-min observational intervals from the video tapes every 0.5 h each day. Feeder access, occupancy rate, and occurrence of agonistic interactions were recorded. Dry matter hay waste was 3.5, 6.1, 11.4, and 14.6% for the cone, ring, trailer, and cradle feeders, respectively. Calculated dry matter intake of hay ranged from 1.8 to 2.0% of body weight and was not different among feeder type (P < 0.05). Percentage of organic matter, neutral detergent fiber, acid detergent fiber, and crude protein were all lower and acid detergent lignin was higher in the recovered waste compared to the hay fed (P < 0.05). Cows feeding from the cradle feeder had nearly three times the agonistic interactions and four times the frequency of entrances compared to cows feeding from the other feeder types (P < 0.05). Feed losses were positively correlated with agonistic interactions, frequency of regular and irregular entrances, and feeder occupancy rate (P < 0.05). Agonistic interactions by cows and frequency of feeder entrances differed among feeders and were correlated to feeder design induced feed losses. PMID:12597379

  14. Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition.

    PubMed

    Liu, Xiaokun; Wei, Qiang; Zhang, Junhong; Yang, Wanli; Zhao, Xiaoe; Ma, Baohua

    2015-06-01

    Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 ?M BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers. PMID:25592083

  15. Optimized mixture of hops rho iso-alpha acids-rich extract and acacia proanthocyanidins-rich extract reduces insulin resistance in 3T3-L1 adipocytes and improves glucose and insulin control in db/db mice.

    PubMed

    Tripp, Matthew L; Darland, Gary; Konda, Veera Reddy; Pacioretty, Linda M; Chang, Jyh-Lurn; Bland, Jeffrey S; Babish, John G

    2012-10-01

    Rho iso-alpha acids-rich extract (RIAA) from Humulus lupulus (hops) and proanthocyanidins-rich extracts (PAC) from Acacia nilotica exert anti-inflammatory and anti-diabetic activity in vitro and in vivo. We hypothesized that a combination of these two extracts would exert enhanced effects in vitro on inflammatory markers and insulin signaling, and on nonfasting glucose and insulin in db/db mice. Over 49 tested combinations, RIAA:PAC at 5:1 (6.25 µg/mL) exhibited the greatest reductions in TNF?-stimulated lipolysis and IL-6 release in 3T3-L1 adipocytes, comparable to 5 µg/mL troglitazone. Pretreatment of 3T3-L1 adipocytes with this combination (5 µg/mL) also led to a 3-fold increase in insulin-stimulated glucose uptake that was comparable to 5 µg/mL pioglitazone or 901 µg/mL aspirin. Finally, db/db mice fed with RIAA:PAC at 5:1 (100 mg/kg) for 7 days resulted in 22% decrease in nonfasting glucose and 19% decrease in insulin that was comparable to 0.5 mg/kg rosiglitazone and better than 100 mg/kg metformin. RIAA:PAC mixture may have the potential to be an alternative when conventional therapy is undesirable or ineffective, and future research exploring its long-term clinical application is warranted. PMID:23198019

  16. Optimized mixture of hops rho iso-alpha acids-rich extract and acacia proanthocyanidins-rich extract reduces insulin resistance in 3T3-L1 adipocytes and improves glucose and insulin control in db/db mice

    PubMed Central

    Darland, Gary; Konda, Veera Reddy; Pacioretty, Linda M.; Chang, Jyh-Lurn; Bland, Jeffrey S.; Babish, John G.

    2012-01-01

    Rho iso-alpha acids-rich extract (RIAA) from Humulus lupulus (hops) and proanthocyanidins-rich extracts (PAC) from Acacia nilotica exert anti-inflammatory and anti-diabetic activity in vitro and in vivo. We hypothesized that a combination of these two extracts would exert enhanced effects in vitro on inflammatory markers and insulin signaling, and on nonfasting glucose and insulin in db/db mice. Over 49 tested combinations, RIAA:PAC at 5:1 (6.25 µg/mL) exhibited the greatest reductions in TNF?-stimulated lipolysis and IL-6 release in 3T3-L1 adipocytes, comparable to 5 µg/mL troglitazone. Pretreatment of 3T3-L1 adipocytes with this combination (5 µg/mL) also led to a 3-fold increase in insulin-stimulated glucose uptake that was comparable to 5 µg/mL pioglitazone or 901 µg/mL aspirin. Finally, db/db mice fed with RIAA:PAC at 5:1 (100 mg/kg) for 7 days resulted in 22% decrease in nonfasting glucose and 19% decrease in insulin that was comparable to 0.5 mg/kg rosiglitazone and better than 100 mg/kg metformin. RIAA:PAC mixture may have the potential to be an alternative when conventional therapy is undesirable or ineffective, and future research exploring its long-term clinical application is warranted. PMID:23198019

  17. Scaffold-free formation of a millimeter-scale multicellular spheroid with an internal cavity from magnetically levitated 3T3 cells that ingested iron oxide-containing microspheres.

    PubMed

    Lee, Joon Ho; Hur, Won

    2014-05-01

    This report describes fabrication of a millimeter-scale three-dimensional (3D) multicellular structure with a central cavity based on magnetic levitation of 3T3 cells that had ingested Fe3 O4 -containing microcapsules. Magnetically levitated cells initially formed a disc-shaped cell cluster at the air-medium interface and transformed into a spheroid (up to 2.8?mm in diameter) after 10-day incubation under a magnet. Hematoxylin-and-eosin-stained section revealed that an eosinophilic shell of cells enclosed a pale-staining core of the spheroid. Mitotic or elongated and aligned cells were found at the outer periphery of the shell, while Fe3 O4 deposits were distributed in the inner part of the shell. Surgical dissection indicated that the spheroid had a hollow interior filled with a fluid-state cell suspension. Accordingly, it was demonstrated that magnetically levitated 3T3 cells organized themselves into a tissue-like spheroid, resulting in core cell death. The spheroid can be used as a 3D tissue model and as building blocks that fused to form a more complicated structure. PMID:24254251

  18. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802... Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and...

  19. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802... Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and...

  20. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802... Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and...

  1. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802... Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and...

  2. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802... Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and...

  3. The design and planning of feeder links to broadcasting satellites

    NASA Technical Reports Server (NTRS)

    Kiebler, J. W.

    1985-01-01

    Design and planning guidelines being followed to define feeder link parameters for the U.S. ITU Region 2 DBS system are outlined. The carrier/noise (C/N) ratio determines the signal quality acceptable to the consumer and sets the standards which the link must meet. A 10 dB margin is required to meet a 99 percent availability criterion for the times of heaviest rainfall attenuation. The receiver gain is limited by the bandwidth, system noise temperature, and the antenna size. Three mechanisms have been identified for ensuring receiver discrimination between desired and interfering signals. Test points must be selected to examine the effectiveness of the chosen feeder link configuration in maintaining signal reception and channel separation, of the antennas installed, and of the orbital positions of the satellites.

  4. Effect of force feeder on tablet strength during compression.

    PubMed

    Narang, Ajit S; Rao, Venkatramana M; Guo, Hang; Lu, Jian; Desai, Divyakant S

    2010-11-30

    Mechanical strength of tablets is an important quality attribute, which depends on both formulation and process. In this study, the effect of process variables during compression on tablet tensile strength and tabletability (the ratio of tensile strength to compression pressure) was investigated using a model formulation. Increase in turret and force feeder speeds reduced tablet tensile strength and tabletability. Turret speed affected tabletability through changes in dwell time under the compression cam and the kinetics of consolidation of granules in the die cavity. The effect of force feeder was attributed to the shearing of the granulation, leading to its over-lubrication. A dimensionless equation was derived to estimate total shear imparted by the force feeder on the granulation in terms of a shear number. Scale-independence of the relationship of tabletability with the shear number was explored on a 6-station Korsch press, a 16-station Betapress, and a 35-station Korsch XL-400 press. The use of this relationship, the exact nature of which may be formulation dependent, during tablet development is expected to provide guidance to the scale-up and interchangeability of tablet presses. PMID:20816733

  5. A novel coal feeder for production of low sulfur fuel

    SciTech Connect

    Khang, S.J.; Lin, L.; Keener, T.C.; Yeh, P.

    1991-01-01

    A dual-screw feeder was designed for desulfurization of coal. This reactor contains two screw tubes, the inner tube acting as a coal pyrolizer and the outer tube acting as a desulfurizer with hot calcined lime pellets or other renewable sorbent pellets. The objectives of this project is to study the feasibility of an advanced concept of desulfurization and possibly some denitrification in this coal feeder. In this year, two basic studies have been performed: (1) the desulfurization and (2) the denitrification due to mild pyrolysis. Specifically, the following tasks have been performed: (1) Setting up the Dual-Screw reactor, (2) Determination of the pyrolysis product and the sulfur distribution in char, tar and gas based on experimental data, (3) Study of the devolatilization, the desulfurization kinetics and the denitrification kinetics and obtaining the basic kinetic parameters, (4) Study of the sulfur removal efficiency of lime pellets fed into the outer tube of the dual-feeder reactor, (5) Study of the effect of the coal particle size on pyrolysis and desulfurization, (6) Study of the coal pyrolysis and desulfurization using a TGA(Thermal Gravimetric Analyzer).

  6. The Herbal Medicine KBH-1 Inhibits Fat Accumulation in 3T3-L1 Adipocytes and Reduces High Fat Diet-Induced Obesity through Regulation of the AMPK Pathway

    PubMed Central

    Lee, Ji-Hye; Kim, Taesoo; Lee, Jung-Jin; Lee, Kwang Jin; Kim, Hyun-Kyu; Yun, Bora; Jeon, Jongwook; Kim, Sang Kyum; Ma, Jin Yeul

    2015-01-01

    The aim of this study was to investigate whether a novel formulation of an herbal extract, KBH-1, has an inhibitory effect on obesity. To determine its anti-obesity effects and its underlying mechanism, we performed anti-obesity-related experiments in vitro and in vivo. 3T3-L1 preadipocytes were analyzed for lipid accumulation as well as the protein and gene expression of molecular targets involved in fatty acid synthesis. To determine whether KBH-1 oral administration results in a reduction in high-fat diet (HFD)-induced obesity, we examined five groups (n = 9) of C57BL/6 mice as follows: 10% kcal fat diet-fed mice (ND), 60% kcal fat diet-fed mice (HFD), HFD-fed mice treated with orlistat (tetrahydrolipstatin, marketed under the trade name Xenical), HFD-fed mice treated with 150 mg/kg KBH-1 (KBH-1 150) and HFD-fed mice treated with 300 mg/kg KBH-1 (KBH-1 300). During adipogenesis of 3T3-L1 cells in vitro, KBH-1 significantly reduced lipid accumulation and down-regulated the expression of master adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP) ?, C/EBP ? and peroxisome proliferation-activity receptor (PPAR) ?, which led to the suppression of the expression of several adipocyte-specific genes and proteins. KBH-1 also markedly phosphorylated the adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). In addition, KBH-1-induced the inhibition effect on lipid accumulation and AMPK-mediated signal activation were decreased by blocking AMPK phosphorylation using AMPK siRNA. Furthermore, daily oral administration of KBH-1 resulted in dose-dependent decreases in body weight, fat pad mass and fat tissue size without systemic toxicity. These results suggest that KBH-1 inhibits lipid accumulation by down-regulating the major transcription factors of the adipogenesis pathway by regulating the AMPK pathway in 3T3-L1 adipocytes and in mice with HFD-induced obesity. These results implicate KBH-1, a safe herbal extract, as a potential anti-obesity therapeutic agent. PMID:26649747

  7. Clustering method and representative feeder selection for the California solar initiative

    SciTech Connect

    Broderick, Robert Joseph; Williams, Joseph R.; Munoz-Ramos, Karina

    2014-02-01

    The screening process for DG interconnection procedures needs to be improved in order to increase the PV deployment level on the distribution grid. A significant improvement in the current screening process could be achieved by finding a method to classify the feeders in a utility service territory and determine the sensitivity of particular groups of distribution feeders to the impacts of high PV deployment levels. This report describes the utility distribution feeder characteristics in California for a large dataset of 8,163 feeders and summarizes the California feeder population including the range of characteristics identified and most important to hosting capacity. The report describes the set of feeders that are identified for modeling and analysis as well as feeders identified for the control group. The report presents a method for separating a utilitys distribution feeders into unique clusters using the k-means clustering algorithm. An approach for determining the feeder variables of interest for use in a clustering algorithm is also described. The report presents an approach for choosing the feeder variables to be utilized in the clustering process and a method is identified for determining the optimal number of representative clusters.

  8. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

    PubMed

    Kathirvel, Poonkodi; Ravi, Subban

    2012-01-01

    This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), ?-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3?µg?mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity. PMID:21939371

  9. 2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

    SciTech Connect

    Jeong, Jin Boo; Jeong, Hyung Jin

    2010-10-01

    Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

  10. Ghrelin protects against depleted uranium-induced apoptosis of MC3T3-E1 cells through oxidative stress-mediated p38-mitogen-activated protein kinase pathway.

    PubMed

    Hao, Yuhui; Liu, Cong; Huang, Jiawei; Gu, Ying; Li, Hong; Yang, Zhangyou; Liu, Jing; Wang, Weidong; Li, Rong

    2016-01-01

    Depleted uranium (DU) mainly accumulates in the bone over the long term. Osteoblast cells are responsible for the formation of bone, and they are sensitive to DU damage. However, studies investigating methods of reducing DU damage in osteoblasts are rarely reported. Ghrelin is a stomach hormone that stimulates growth hormones released from the hypothalamic-pituitary axis, and it is believed to play an important physiological role in bone metabolism. This study evaluates the impact of ghrelin on DU-induced apoptosis of the osteoblast MC3T3-E1 and investigates its underlying mechanisms. The results show that ghrelin relieved the intracellular oxidative stress induced by DU, eliminated reactive oxygen species (ROS) and reduced lipid peroxidation by increasing intracellular GSH levels; in addition, ghrelin effectively suppressed apoptosis, enhanced mitochondrial membrane potential, and inhibited cytochrome c release and caspase-3 activation after DU exposure. Moreover, ghrelin significantly reduced the expression of DU-induced phosphorylated p38-mitogen-activated protein kinase (MAPK). A specific inhibitor (SB203580) or specific siRNA of p38-MAPK could significantly suppress DU-induced apoptosis and related signals, whereas ROS production was not affected. In addition, ghrelin receptor inhibition could reduce the anti-apoptosis effect of ghrelin on DU and reverse the effect of ghrelin on intracellular ROS and p38-MAPK after DU exposure. These results suggest that ghrelin can suppress DU-induced apoptosis of MC3T3-E1 cells, reduce DU-induced oxidative stress by interacting with its receptor, and inhibit downstream p38-MAPK activation, thereby suppressing the mitochondrial-dependent apoptosis pathway. PMID:26529667

  11. In vitro biokinetics of chlorpromazine and the influence of different dose metrics on effect concentrations for cytotoxicity in Balb/c 3T3, Caco-2 and HepaRG cell cultures.

    PubMed

    Broeders, Jessica J W; Blaauboer, Bas J; Hermens, Joop L M

    2013-04-01

    The extrapolation of in vitro to in vivo toxicity data is a challenge. Differences in sensitivity between cell systems may be due to intrinsic properties of the cell but also because of differences in exposure. In this study, the cytotoxicity and biokinetics of the antipsychotic chlorpromazine (CPZ) were studied in in vitro assays using different cell types and exposure conditions. Different dose metrics were assessed to express the sensitivity to CPZ. The biokinetics of CPZ were measured in cell cultures of Balb/c 3T3, Caco-2 and HepaRG cells. Cytotoxicity was measured by Alamar Blue and expressed using different dose metrics, including the nominal, measured total and measured free CPZ medium concentrations. CPZ was taken up by the cells; the highest amounts in the cell compartments were found in the Caco-2 and HepaRG cells. CPZ was highly protein-bound in the Caco-2 cell medium containing 10% fetal bovine serum, resulting in lower bioavailable exposure concentrations. Moreover, also uptake into the cells strongly influenced the concentration in the medium. The Balb/c 3T3 cells were the most sensitive to the toxic effect of CPZ. The use of different dose metrics influenced the cytotoxicity results found in the three cell types. The data show that in comparing the sensitivity of the tested cell systems, the freely dissolved concentration is a more appropriate dose metric than total concentration in the medium. The ranking in sensitivity of the three cell types for CPZ was dependent on the dose metric used. PMID:23376437

  12. Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization

    PubMed Central

    Parimala, Mabel; Debjani, M.; Vasanthi, Hannah Rachel; Shoba, Francis Gricilda

    2015-01-01

    Nymphaea nouchali Burm. f. (Family – Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR?) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPAR? activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPAR? target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPAR? activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues. PMID:26605160

  13. Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization.

    PubMed

    Parimala, Mabel; Debjani, M; Vasanthi, Hannah Rachel; Shoba, Francis Gricilda

    2015-01-01

    Nymphaea nouchali Burm. f. (Family - Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR?) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPAR? activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPAR? target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPAR? activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues. PMID:26605160

  14. Transformation of BALB/c-3T3 cells: III. Development of a co-culture clonal survival assay for quantification of chemical cytotoxicity in high-density cell cultures.

    PubMed Central

    Matthews, E J

    1993-01-01

    A co-culture clonal survival assay was developed to measure the cytotoxicity of test chemical treatments to BALB/c-3T3 cells because the standard clonal survival assay using 200 wild type (WT) cells frequently overestimates chemical cytotoxicity when compared with identical treatment doses in high-density cultures. The assay used co-cultures of 3.2 x 10(4) WT cells, the same seeding density used in the transformation assay, and 200 ouabain resistant (OUAr) cells. After a 48-hr test chemical treatment, co-cultured cells were fed with culture medium containing 4 mM ouabain. The test chemical was cytotoxic to an equal percentage of WT and OUAr cells. Ouabain treatments killed the remaining WT cells. Thus, OUAr cells surviving the test chemical treatment measured the relative cloning efficiency (RCE) of all treated cells in the high-density cell co-culture. The co-culture assay succeeded because metabolic cooperation at the OUAr locus was not detected in BALB/c-3T3 cells. Five chemicals induced comparable cytotoxic responses in both assays, including actinomycin D, 5-bromo-2'-deoxyuridine, N'-methyl-N-nitro-N'-nitrosoguanidine, dimethyl sulfoxide and sodium chloride. In contrast, chemical cytotoxic responses detected in the standard and co-culture assays differed by > or = 10-fold for 11-aminoundecanoic acid, benzo[a]pyrene, cytosine arabinoside, and 3-methyl-cholanthrene and differed by > 2-fold for 2-acetylaminofluorene and dimethylnitrosamine. Detection of 11-aminoundecanoic acid-induced transformation was shown to be dependent on selecting treatment doses from the co-culture assay data. Thus, this method permits more accurate assessment of both chemical-induced cytotoxicity and transformation. PMID:8243400

  15. Drynariae Rhizoma promotes osteoblast differentiation and mineralization in MC3T3-E1 cells through regulation of bone morphogenetic protein-2, alkaline phosphatase, type I collagen and collagenase-1.

    PubMed

    Jeong, Ji-Cheon; Lee, Jae-Wook; Yoon, Cheol-Ho; Kim, Hyung-Min; Kim, Cheorl-Ho

    2004-12-01

    In a previous study (Jeong et al., 2003, Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts. International Immunopharmacology 3, 1685-1697), treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extract prevented the intracellular maturation of cathepsin K and thus, it was considered that DR is a pro-drug of a potent bone resorption inhibitor. To further clarify the role of DR in ossification, we investigated the effects of DR on the proliferation and differentiation of osteoblastic cell lines in vitro. In this study, the bone effect of DR is studied. We assessed the effects of DR on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. DR enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the DR was observed at relatively low doses (significant at 50-150 microg/ml and maximal at 150 microg/ml). Northern blot analysis showed that the DR (100 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. DR (60 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that DR has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis. PMID:15465649

  16. Recombinant human cachectin/tumor necrosis factor but not interleukin-1 alpha downregulates lipoprotein lipase gene expression at the transcriptional level in mouse 3T3-L1 adipocytes.

    PubMed Central

    Zechner, R; Newman, T C; Sherry, B; Cerami, A; Breslow, J L

    1988-01-01

    Lipoprotein lipase (LPL) is synthesized primarily in muscle and adipose tissue and by hydrolyzing triglycerides in chylomicrons and very low density lipoprotein allows uptake of the resultant free fatty acids by these tissues. This report describes the cloning of the mouse LPL gene from which probes were derived to study the regulation of LPL synthesis in the 3T3-L1 adipocyte cell culture system. Preconfluent 3T3-L1 preadipocytes had very small amounts of LPL mRNA (less than 1 pg/micrograms of RNA). At confluency, LPL mRNA levels increased to 5 to 15 pg/micrograms of RNA. After insulin and dexamethasone were added, LPL activity and mRNA levels rose in parallel. Peak mRNA levels were reached within 4 to 10 days, achieving LPL mRNA concentrations of 150 to 500 pg/micrograms of RNA. This represents a 15- to 50-fold increase over confluent cells. Two cytokines known to diminish adipose tissue LPL activity were studied to see how their effects were regulated. Recombinant human cachectin/tumor necrosis factor diminished both LPL activity and LPL mRNA levels. The effect on LPL activity compared with mRNA levels was quicker, at a lower dose, and more complete (95 versus 75% maximum effect). The effect of recombinant human cachectin tumor necrosis factor on LPL mRNA levels was shown by nuclear run-on experiments to be exerted transcriptionally. It was also independent of new protein synthesis. Recombinant human interleukin-1 alpha diminished only LPL activity but not mRNA levels. This study suggests that during times of stress, cytokines secreted by activated macrophages can alter energy balance by affecting transcriptional and posttranscriptional processes in adipocytes. Images PMID:3261386

  17. Insulin-like synergistic stimulation of DNA synthesis in Swiss 3T3 cells by the BSC-1 cell-derived growth inhibitor related to transforming growth factor type. beta

    SciTech Connect

    Brown, K.D.; Holley, R.W.

    1987-06-01

    A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type ..beta.. (TGF-..beta..), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. /sup 125/I-EGF binding was measured and the efflux of /sup 45/Ca/sup 2 +/ was measured in response to mitogen stimulation. A similar synergistic response has been demonstrated for TGF-..beta.. purified from human platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-..beta.. in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and the authors have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-..beta.. and insulin converge at a post-receptor stage.

  18. Prostaglandin E2-induced up-regulation of c-fos messenger ribonucleic acid is primarily mediated by 3',5'-cyclic adenosine monophosphate in MC3T3-E1 osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Dietz, T. J.; Hughes-Fulford, M.

    2000-01-01

    The mechanism by which the proto-oncogene, c-fos, is up-regulated in response to PGE2 in the mouse osteoblastic (MC3T3-E1) cell line was investigated using RT-PCR. c-fos messenger RNA up-regulation by dmPGE2 is rapid, starting 10 min post stimulation, and transient. The specific protein kinase A (PKA) inhibitor, H89, inhibited c-fos induction. Moreover, down-regulation of protein kinase C (PKC) activity by chronic TPA treatment had no effect on the induction of c-fos by dmPGE2. We conclude that up-regulation of c-fos by dmPGE2 is primarily dependent on PKA in MC3T3-E1 osteoblasts. In S49 lymphoma wild-type but not S49 cyc- cells, which are deficient in cAMP signaling, dmPGE2 up-regulates c-fos and increases cell growth compared with unstimulated cells. Thus in S49 lymphoma cells, c-fos induction by PGE2 is also dependent on cAMP signaling. The minimal c-fos promoter region required for dmPGE2-induced expression was identified by transfecting c-fos promoter deletion constructs coupled to the chloramphenicol acetyltransferase (CAT) reporter gene into Vero cells. Transfection of a plasmid containing 99 bp c-fos proximal promoter was sufficient to direct c-fos/CAT expression following stimulation with dmPGE2. Because induction of c-fos is mediated by cAMP, these data are consistent with activation of c-fos via the CRE/ATF cis element.

  19. Coal-feeder development. Second quarterly technical progress report, January-March 1980

    SciTech Connect

    Mistry, D.K.

    1981-04-01

    The pilot-scale piston-feeder development is progressing satisfactorily and should proceed as planned. The bench scale testing of components, sub-system and critical areas continued to provide very useful information in support of the development of the complete feeder. The K30M seals and polyurethane scrapers are showing very promising results. The components development facility is being upgraded and testing at the bench scale level should be vigorously perused. The upgrading of the pilot scale feeder and its system will be emphasized during the next quarter to perform feeder capabilities and limitations testing. No progress on the 5.5-in. diameter pilot scale screw feeder has been made because IRRI is waiting decision from METC as to when the feeder can be installed on the 42-in. gas producer.

  20. Semiautomatic cold wire feeder systems increase GTA productivity

    SciTech Connect

    Richardson, M. )

    1995-01-01

    Often, the focus of attempts to increase GTA welding productivity is on studies to determine if justification exists for additional workstations, or for the investment in new fully automated dedicated welding fixtures. Often less costly and simpler solutions can bring about the necessary means to increase production rates and reduce operating costs. For short-run production applications, it is almost impossible to justify the substantial investment in a dedicated automatic fixture. Now, low cost GTA cold wire feeder systems are within the reach of even small shops. The paper views how cold wire equipment has been applied in several GTAW applications to improve results.

  1. Vitamin A Studies in Fattening Feeder Calves and Yearlings. 

    E-print Network

    Marion, Paul T.; Ellis, N. R.; Black, W. H.; Howe, P. E.; Kemmerer, A. R.; Riggs, J. K.; Jones, J. M.; Fraps, G. S.; Dickson, R. E.; Schmidt, H.; Jones, John H.

    1943-01-01

    the vitamin A had been destroyed. D"T TvnTN NO. 630, TEXAS AGRIqULTUhfirr finrnnL.vln~\\ L ~LAL LW.> 179th fected ---A line ca conditi basal I day of th steers to -> ..----- e experiment. This fact, together with the failure of af- improve when fed... STATION A. B. CONNER, DIRECTOR College Station. Texas BULLETIPI' NO. 630 APRIL 1943 VITAMIN A STUDIES IN FATTENING FEEDER CALVES AND YEARLINGS . H.,JONES, H. SCHMIDT, R. E. DIICKSON, G. S. FRAPS, J. M. JONE 3'. K. RIGGS, A. R. ICEMMERER, P. E. HOWE...

  2. The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance.

    PubMed

    Siti-Ismail, Norhayati; Bishop, Anne E; Polak, Julia M; Mantalaris, Athanasios

    2008-10-01

    The majority of methodologies for maintaining human embryonic stem cell (hESC) pluripotency require the use of human or animal feeder cell layers, the most common being murine embryonic fibroblasts. In this study, we applied a protocol aimed at maintaining hESCs in culture without exposure to animal cells or proteins. hESCs were encapsulated in 1.1% (w/v) calcium alginate hydrogels and grown in basic maintenance medium for a period of up to 260 days. Investigation of the cell aggregates formed within the hydrogels yielded no evidence of the formation of any of the three germ layers, although the hESCs retained their pluripotency and could differentiate when they were subsequently cultured in a conditioned environment. Immunohistochemistry and RT-PCR showed that the hESC aggregates expressed protein and gene markers characteristic of pluripotency including Oct-4, Nanog, SSEA-4, TRA-1-60 and TRA-1-81. At the ultrastructural level, the cells were arranged in closely packed clusters and showed no cytoplasmic organelles, suggesting an undifferentiated state. These data show that it is possible to maintain hESCs in an undifferentiated state, without passaging or embryoid body formation, and without animal contamination. PMID:18639332

  3. Clustering Methodology for Classifying Distribution Feeders Robert J. Broderick and Joseph R. Williams

    E-print Network

    for low risk feeders. This effort is being done in partnership with the Electric Power Research Institute systems on the distribution grid. A significant improvement in the current screening process could, Inc. for the California Solar Initiative Project to screen distribution feeders to develop

  4. A single nucleotide change in the prolidase gene in fibroblasts from two patients with polypeptide positive prolidase deficiency. Expression of the mutant enzyme in NIH 3T3 cells.

    PubMed Central

    Tanoue, A; Endo, F; Kitano, A; Matsuda, I

    1990-01-01

    Prolidase deficiency is an autosomal recessive disorder characterized by mental retardation and various skin lesions. Cultured skin fibroblasts were obtained from two independent patients with abnormal prolidase. Using the polymerase chain reaction, we amplified the entire coding region of human prolidase mRNA derived from patients' fibroblasts. Nucleotide sequence analysis of amplified cDNA products revealed a G to A substitution at position 826 in exon 12, where aspartic acid was replaced by asparagine at the amino acid residue 276, in cells from both patients. An analysis of the DNA showed that the substitution was homozygous. An expression plasmid clone containing a normal human prolidase cDNA (pEPD-W) or mutant prolidase cDNA (pEPD-M) was prepared, transfected, and tested for expression in NIH 3T3 cells. Incorporation of pEPD-W and pEPD-M resulted in the synthesis of an immunological polypeptide that corresponded to human prolidase. Active human enzyme was detected in cells transfected with pEPD-W, but not in those transfected with pEPD-M. These results were compatible with our observation of fibroblasts and confirmed that the substitution was responsible for the enzyme deficiency. As active prolidase was recovered in prolidase-deficient fibroblasts transfected with pEPD-W, this restoration of prolidase activity after transfection means that gene replacement therapy for individuals with this human disorder can be given due consideration. Images PMID:2365824

  5. Limonin, a Component of Dictamni Radicis Cortex, Inhibits Eugenol-Induced Calcium and cAMP Levels and PKA/CREB Signaling Pathway in Non-Neuronal 3T3-L1 Cells.

    PubMed

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

    2015-01-01

    Limonin, one of the major components in dictamni radicis cortex (DRC), has been shown to play various biological roles in cancer, inflammation, and obesity in many different cell types and tissues. Recently, the odorant-induced signal transduction pathway (OST) has gained attention not only because of its function in the perception of smell but also because of its numerous physiological functions in non-neuronal cells. However, little is known about the effects of limonin and DRC on the OST pathway in non-neuronal cells. We investigated odorant-stimulated increases in Ca(2+) and cAMP, major second messengers in the OST pathway, in non-neuronal 3T3-L1 cells pretreated with limonin and ethanol extracts of DRC. Limonin and the extracts significantly decreased eugenol-induced Ca(2+) and cAMP levels and upregulated phosphorylation of CREB and PKA. Our results demonstrated that limonin and DRC extract inhibit the OST pathway in non-neuronal cells by modulating Ca(2+) and cAMP levels and phosphorylation of CREB. PMID:26690397

  6. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  7. N-acetyl cysteine inhibits H2O2-mediated reduction in the mineralization of MC3T3-E1 cells by down-regulating Nrf2/HO-1 pathway.

    PubMed

    Lee, Daewoo; Kook, Sung-Ho; Ji, Hyeok; Lee, Seung-Ah; Choi, Ki-Choon; Lee, Kyung-Yeol; Lee, Jeong-Chae

    2015-11-01

    There are controversial findings regarding the roles of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway on bone metabolism under oxidative stress. We investigated how Nrf2/HO-1 pathway affects osteoblast differentiation of MC3T3-E1 cells in response to hydrogen peroxide (H2O2), N-acetyl cysteine (NAC), or both. Exposing the cells to H2O2 decreased the alkaline phosphatase activity, calcium accumulation, and expression of osteoblast markers, such as osteocalcin and runt-related transcription factor-2. In contrast, H2O2 treatment increased the expression of Nrf2 and HO-1 in the cells. Treatment with hemin, a chemical HO-1 inducer, mimicked the inhibitory effect of H2O2 on osteoblast differentiation by increasing the HO-1 expression and decreasing the osteogenic marker genes. Pretreatment with NAC restored all changes induced by H2O2 to near normal levels in the cells. Collectively, our findings suggest that H2O2-mediated activation of Nrf2/HO-1 pathway negatively regulates the osteoblast differentiation, which is inhibited by NAC. [BMB Reports 2015; 48(11): 636-641]. PMID:26303969

  8. Insulin-Mimetic Action of Rhoifolin and Cosmosiin Isolated from Citrus grandis (L.) Osbeck Leaves: Enhanced Adiponectin Secretion and Insulin Receptor Phosphorylation in 3T3-L1 Cells

    PubMed Central

    Rao, Yerra Koteswara; Lee, Meng-Jen; Chen, Keru; Lee, Yi-Ching; Wu, Wen-Shi; Tzeng, Yew-Min

    2011-01-01

    Citrus grandis (L.) Osbeck (red wendun) leaves have been used in traditional Chinese medicine to treat several illnesses including diabetes. However, there is no scientific evidence supporting these actions and its active compounds. Two flavone glycosides, rhoifolin and cosmosiin were isolated for the first time from red wendun leaves and, identified these leaves are rich source for rhoifolin (1.1%, w/w). In differentiated 3T3-L1 adipocytes, rhoifolin and cosmosiin showed dose-dependent response in concentration range of o.oo1–5??M and 1–20??M, respectively, in biological studies beneficial to diabetes. Particularly, rhoifolin and cosmosiin at 0.5 and 20??M, respectively showed nearly similar response to that 10?nM of insulin, on adiponectin secretion level. Furthermore, 5??M of rhoifolin and 20??M of cosmosiin showed equal potential with 10?nM of insulin to increase the phosphorylation of insulin receptor-?, in addition to their positive effect on GLUT4 translocation. These findings indicate that rhoifolin and cosmosiin from red wendun leaves may be beneficial for diabetic complications through their enhanced adiponectin secretion, tyrosine phosphorylation of insulin receptor-? and GLUT4 translocation. PMID:20008903

  9. Trans-Cinnamic Acid Increases Adiponectin and the Phosphorylation of AMP-Activated Protein Kinase through G-Protein-Coupled Receptor Signaling in 3T3-L1 Adipocytes

    PubMed Central

    Kopp, Christina; Singh, Shiva P.; Regenhard, Petra; Müller, Ute; Sauerwein, Helga; Mielenz, Manfred

    2014-01-01

    Adiponectin and intracellular 5?adenosine monophosphate-activated protein kinase (AMPK) are important modulators of glucose and fat metabolism. Cinnamon exerts beneficial effects by improving insulin sensitivity and blood lipids, e.g., through increasing adiponectin concentrations and AMPK activation. The underlying mechanism is unknown. The Gi/Go-protein-coupled receptor (GPR) 109A stimulates adiponectin secretion after binding its ligand niacin. Trans-cinnamic acid (tCA), a compound of cinnamon is another ligand. We hypothesize whether AMPK activation and adiponectin secretion by tCA is transmitted by GPR signaling. Differentiated 3T3-L1 cells were incubated with pertussis toxin (PTX), an inhibitor of Gi/Go-protein-coupling, and treated with different tCA concentrations. Treatment with tCA increased adiponectin and the pAMPK/AMPK ratio (p ? 0.001). PTX incubation abolished the increased pAMPK/AMPK ratio and adiponectin secretion. The latter remained increased compared to controls (p ? 0.002). tCA treatment stimulated adiponectin secretion and AMPK activation; the inhibitory effect of PTX suggests GPR is involved in tCA stimulated signaling. PMID:24557583

  10. Design, synthesis and characterization of novel binary V(V)-Schiff base materials linked with insulin-mimetic vanadium-induced differentiation of 3T3-L1 fibroblasts to adipocytes. Structure-function correlations at the molecular level.

    PubMed

    Halevas, E; Tsave, O; Yavropoulou, M P; Hatzidimitriou, A; Yovos, J G; Psycharis, V; Gabriel, C; Salifoglou, A

    2015-06-01

    Among the various roles of vanadium in the regulation of intracellular signaling, energy metabolism and insulin mimesis, its exogenous activity stands as a contemporary challenge currently under investigation and a goal to pursue as a metallodrug against Diabetes mellitus II. In this regard, the lipogenic activity of vanadium linked to the development of well-defined anti-diabetic vanadodrugs has been investigated through: a) specifically designing and synthesizing Schiff base organic ligands L, bearing a variable number of terminal alcohols, b) a series of well-defined soluble binary V(V)-L compounds synthesized and physicochemically characterized, c) a study of their cytotoxic effect and establishment of adipogenic activity in 3T3-L1 fibroblasts toward mature adipocytes, and d) biomarker examination of a closely-linked molecular target involving or influenced by the specific V(V) forms, cumulatively delineating factors involved in potential pathways linked to V(V)-induced insulin-like activity. Collectively, the results a) project the importance of specific structural features in Schiff ligands bound to V(V), thereby influencing the emergence of its (a)toxicity and for the first time its insulin-like activity in pre-adipocyte differentiation, b) contribute to the discovery of molecular targets influenced by the specific vanadoforms seeking to induce glucose uptake, and c) indicate an interplay of V(V) structural speciation and cell-differentiation biological activity, thereby gaining insight into vanadium's potential as a future metallodrug in Diabetes mellitus. PMID:25920352

  11. Doses of Quercetin in the Range of Serum Concentrations Exert Delipidating Effects in 3T3-L1 Preadipocytes by Acting on Different Stages of Adipogenesis, but Not in Mature Adipocytes

    PubMed Central

    Eseberri, Itziar; Miranda, Jonatan; Lasa, Arrate; Churruca, Itziar; Portillo, María P.

    2015-01-01

    Scope. To determine whether doses of quercetin in the range of serum concentrations exert any effect on triacylglycerol accumulation in maturing preadipocytes and mature adipocytes. The influence on the expression of adipogenic markers as well as on gene expression and activity of enzymes involved in triacylglycerol metabolism were assessed. Methods and Results. 3T3-L1 preadipocytes were treated during differentiation and mature adipocytes for 24 hours with low doses (0.1–10?µM) of quercetin. Triacylglycerol content in both cell types and free fatty acid and glycerol in the incubation medium of mature adipocytes were measured spectrophotometrically. Gene and protein expression were assessed by RT-PCR and Western blot. LPL and FAS activities were quantified. During differentiation quercetin reduced triacylglycerol content at doses from 0.5 to 10?µM. 1?µM of quercetin reduced C/EBP? gene expression, SREBP1 mature protein levels, and PPAR? gene expression. 10?µM of quercetin reduced LPL gene expression and PPAR? and SREBP1c expression. In mature adipocytes, only 10?µM of quercetin reduced triacylglycerol content. Lipogenic FAS expression and activity were reduced at this dose. Conclusion. Quercetin, in the range of serum concentrations, is able to inhibit adipogenesis, but higher doses, at least 10?µM, are needed to reduce fat accumulation in mature adipocytes. PMID:26180590

  12. Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

    SciTech Connect

    Chang, Young-Chae; Cho, Hyun-Ji

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Ascofuranone increases expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Inhibitors for MEK and JNK increased the expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPAR{gamma}, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPAR{gamma} agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPAR{gamma}, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPAR{gamma} through the modulation of MAP kinase family members.

  13. Biomixing generated by benthic filter feeders: a diffusion model for near-bottom phytoplankton depletion

    NASA Astrophysics Data System (ADS)

    Larsena, Poul S.; Riisgård, Hans Ulrik

    1997-05-01

    Transient concentration distributions of flagellate cells ( Rhodomonas sp.) previously measured by Riisgård and co-workers in laboratory experiments have been examined to develop a diffusion model for the process of phytoplankton depletion in stagnant seawater above populations of benthic filter feeders, the polychaete Nereis diversicolor and the ascidian Ciona intestinalis, respectively. The model is based on sinks located at inhalant openings and Fick's law with an effective diffusivity that decreases with distance above the bottom due to the biomixing generated by exhalant and inhalant feeding currents. For N. diversicolor, having inhalant and exhalant openings flush with the sediment surface and a moderate exhalant jet velocity of ˜0.01 m s -1, concentration boundary layer growth is retarded and limited by the low values of diffusivity prevailing at heights greater >˜0.05 m above the bottom. For C. intestinalis, having inhalant and exhalant openings situated ˜0.05-0.1 m above the bottom and a higher and inclined exhalant jet velocity of ˜0.1-0.2 m s -1, the concentration distributions show a nearly uniform depletion over a layer reaching a thickness of 0.2-0.3 m above the bottom due to high biomixing in this layer. Numerical predictions of concentration distributions reproduce essential features of experiments, and suggest near-bottom values of effective diffusivity of 0.3 x 10 -6 and 150 x 10 -6 m 2 s -1, for N. diversicolor and C. intestinalis, respectively. It is suggested that the latter value is so large that the induced mixing should be accounted for in modelling benthic concentration boundary layers under flow conditions.

  14. A New Approach to Mitigate the Impact of Distributed Generation on the Overcurrent Protection Scheme of Radial Distribution Feeders 

    E-print Network

    Funmilayo, Hamed

    2010-01-14

    Increased Distributed Generation (DG) presence on radial distribution feeders is becoming a common trend. The existing Overcurrent Protection (OCP) scheme on such feeders consists mainly of overcurrent protection devices (OCPDs) such as fuses...

  15. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires §...

  16. 30 CFR 75.1003-1 - Other requirements for guarding of trolley wires and trolley feeder wires.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... wires and trolley feeder wires. 75.1003-1 Section 75.1003-1 Mineral Resources MINE SAFETY AND HEALTH... Trolley Wires and Trolley Feeder Wires § 75.1003-1 Other requirements for guarding of trolley wires and trolley feeder wires. Adequate precaution shall be taken to insure that equipment being moved...

  17. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires §...

  18. 30 CFR 75.1003-1 - Other requirements for guarding of trolley wires and trolley feeder wires.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... wires and trolley feeder wires. 75.1003-1 Section 75.1003-1 Mineral Resources MINE SAFETY AND HEALTH... Trolley Wires and Trolley Feeder Wires § 75.1003-1 Other requirements for guarding of trolley wires and trolley feeder wires. Adequate precaution shall be taken to insure that equipment being moved...

  19. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003... Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires...

  20. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003... Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires...

  1. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003... Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires...

  2. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...Resources 1 2012-07-01 2012-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 75.1003 Insulation of trolley wires, trolley feeder wires and bare signal...

  3. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...Resources 1 2013-07-01 2013-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare signal...

  4. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...Resources 1 2011-07-01 2011-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare signal...

  5. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...Resources 1 2010-07-01 2010-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare signal...

  6. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...Resources 1 2014-07-01 2014-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 75.1003 Insulation of trolley wires, trolley feeder wires and bare signal...

  7. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...Resources 1 2013-07-01 2013-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 75.1003 Insulation of trolley wires, trolley feeder wires and bare signal...

  8. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...Resources 1 2012-07-01 2012-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare signal...

  9. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...Resources 1 2011-07-01 2011-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 75.1003 Insulation of trolley wires, trolley feeder wires and bare signal...

  10. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...Resources 1 2010-07-01 2010-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 75.1003 Insulation of trolley wires, trolley feeder wires and bare signal...

  11. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...Resources 1 2014-07-01 2014-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare signal...

  12. 7 CFR 1230.113 - Collection and remittance of assessments for the sale of feeder pigs and market hogs.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...remittance of assessments for the sale of feeder pigs and market hogs. 1230.113 Section...remittance of assessments for the sale of feeder pigs and market hogs. Pursuant to the provisions of § 1230.71, purchasers of feeder pigs or market hogs shall collect...

  13. Minimal incorporation of Deepwater Horizon oil by estuarine filter feeders.

    PubMed

    Fry, Brian; Anderson, Laurie C

    2014-03-15

    Natural abundance carbon isotope analyses are sensitive tracers for fates and use of oil in aquatic environments. Use of oil carbon in estuarine food webs should lead to isotope values approaching those of oil itself, -27‰ for stable carbon isotopes reflecting oil origins and -1000‰ for carbon-14 reflecting oil age. To test for transfer of oil from the 2010 Deepwater Horizon spill into estuarine food webs, filter-feeding barnacles (Balanus sp.) and marsh mussels (Geukensia demissa) were collected from Louisiana estuaries near the site of the oil spill. Carbon-14 analyses of these animals from open waters and oiled marshes showed that oil use was <1% and near detection limits estimated at 0.3% oil incorporation. Respiration studies showed no evidence for enhanced microbial activity in bay waters. Results are consistent with low dietary impacts of oil for filter feeders and little overall impact on respiration in the productive Louisiana estuarine systems. PMID:24461698

  14. Microplastic in a macro filter feeder: Humpback whale Megaptera novaeangliae.

    PubMed

    Besseling, E; Foekema, E M; Van Franeker, J A; Leopold, M F; Kühn, S; Bravo Rebolledo, E L; Heße, E; Mielke, L; IJzer, J; Kamminga, P; Koelmans, A A

    2015-06-15

    Marine filter feeders are exposed to microplastic because of their selection of small particles as food source. Baleen whales feed by filtering small particles from large water volumes. Macroplastic was found in baleen whales before. This study is the first to show the presence of microplastic in intestines of a baleen whale (Megaptera novaeangliae). Contents of its gastrointestinal tract were sieved, dissolved in 10% potassium hydroxide and washed. From the remaining dried material, potential synthetic polymer particles were selected based on density and appearance, and analysed by Fourier transform infrared (FTIR) spectroscopy. Several polymer types (polyethylene, polypropylene, polyvinylchloride, polyethylene terephthalate, nylon) were found, in varying particle shapes: sheets, fragments and threads with a size of 1mm to 17cm. This diversity in polymer types and particle shapes, can be interpreted as a representation of the varying characteristics of marine plastic and the unselective way of ingestion by M. novaeangliae. PMID:25916197

  15. Development of an extruder-feeder biomass direct liquefaction process

    SciTech Connect

    White, D.H.; Wolf, D. . Dept. of Chemical Engineering)

    1991-10-01

    As an abundant, renewable, domestic energy resource, biomas